150. Breaking cycles : saponification‐enhanced NMR fingerprint matching for the identification and stereochemical evaluation of cyclic lipodepsipeptides from natural sources
Penthip Muangkaew (UGent) , Durga Prasad (UGent) , Vic De Roo, Yentl Verleysen, Lu Zhou (UGent) , René De Mot, Monica Höfte (UGent) , Annemieke Madder (UGent) , Niels Geudens (UGent) and José Martins (UGent)
We previously described NMR based fingerprint matching with peptide backbone resonances as a fast and reliable structural dereplication approach for Pseudomonascyclic lipodepsipeptides (CLiPs). In combination with total synthesis of a small library of configurational CLiP congeners this also allows unambiguous determination of stereochemistry, facilitating structure‐activity relationship studies and enabling three‐dimensional structure determination. However, the on‐resin macrocycle formation in the synthetic workflow brings considerable burden and limits universal applicability. This drawback is here removed altogether by also transforming the native CLiP into a linearized analogue by controlled saponification of the ester bond. This eliminates the need for macrocycle formation, limiting the synthesis effort to linear peptide analogues. NMR fingerprints of such linear peptide analogues display a sufficiently distinctive chemical shift fingerprint to act as effective discriminators. The approach is developed using viscosin group CLiPs and subsequently demonstrated on putisolvin, leading to a structural revision, and tanniamide from Pseudomonas ekonensis COR58, a newly isolated lipododecapeptide that defines a new group characterized by a ten‐residue large macrocycle, the largest to date in the Pseudomonas CLiP portfolio. These examples demonstrate the effectiveness of the saponification‐ enhanced approach that broadens applicability of NMR fingerprint matching for the determination of the stereochemistry of CLiPs.
149. Time-resolved fluorescence of tryptophan characterizes membrane perturbation by cyclic lipopeptides
Iulia Carabadjac, Jessica Steigenberger, Niels Geudens (UGent) , Vic De Roo, Penthip Muangkaew (UGent) , Annemieke Madder (UGent) , José Martins (UGent) and Heiko Heerklotz
Viscosin is a membrane-permeabilizing, cyclic lipopeptide (CLiP) produced by Pseudomonas species. Here, we have studied four synthetic analogs (L1W, V4W, L5W, and L7W), each with one leucine (Leu; L) or valine residue exchanged for tryptophan (Trp; W) by means of time-resolved fluorescence spectroscopy of Trp. To this end, we recorded the average fluorescence lifetime, rotational correlation time and limiting anisotropy, dipolar relaxation time and limiting extent of relaxation, rate constant of acrylamide quenching, effect of H2O-D2O exchange, and time-resolved half-width of the spectrum in the absence and presence of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. Structure, localization, and hydration of the peptides were described by molecular dynamics simulations. The combination of the parameters provides a good description of the molecular environments of the Trp positions and the behavior of viscosin as a whole. Of particular value for characterizing the impact of viscosin on the membrane is the dipolar relaxation of Trp4 in V4W, which is deeply embedded in the hydrophobic core. The limiting relaxation level represents the membrane perturbation-unlike typical membrane probes-at the site of the perturbant. Fractions of Trp4 relax at different rates; the one not in contact with water upon excitation relaxes via recruitment of a water molecule on the 10-ns timescale. This rate is sensitive to the concerted membrane perturbation by more than one lipopeptide, which appears at high lipopeptide concentration and is assumed a prerequisite for the final formation of a membrane-permeabilizing defect. Trp7 relaxes primarily with respect to neighboring Ser residues. Trp5 flips between a membrane-inserted and surface-exposed orientation.
148. Sustained release of a human PD-L1 single-domain antibody using peptide-based hydrogels
Julie Heremans, Robin Maximilian Awad, Jessica Bridoux, Thomas Ertveldt, Vicky Caveliers, Annemieke Madder (UGent) , Richard Hoogenboom (UGent) , Nick Devoogdt, Steven Ballet, Sophie Hernot, Karine Breckpot and Charlotte Martin
Monoclonal antibodies (mAbs) targeting the immune checkpoint axis, which contains the programmed cell death protein-1 (PD-1) and its ligand PD-L1, revolutionized the field of oncology. Unfortunately, the large size of mAbs and the presence of an Fc fraction limit their tumor penetrative capacities and support off-target effects, potentially resulting in unresponsive patients and immune-related adverse events (irAEs) respectively. Singledomain antibodies (sdAbs) are ten times smaller than conventional mAbs and represent an emerging antibody subclass that has been proposed as next generation immune checkpoint inhibitor (ICI) therapeutics. They demonstrate favorable characteristics, such as an excellent stability, high antigen-binding affinity and an enhanced tumor penetration. Because sdAbs have a short half-life, methods to prolong their presence in the circulation and at the target site might be necessary in some cases to unfold their full therapeutic potential. In this study, we investigated a peptide-based hydrogel as an injectable biomaterial depot formulation for the sustained release of the human PD-L1 sdAb K2. We showed that a hydrogel composed of the amphipathic hexapeptide hydrogelator H-FQFQFK-NH2 prolonged the in vivo release of K2 after subcutaneous (s.c.) injection, up to at least 72 h, as monitored by SPECT/CT and fluorescence imaging. Additionally, after encapsulation in the hydrogel and s.c. administration, a significantly extended systemic presence and tumor uptake of K2 was observed in mice bearing a melanoma tumor expressing human PD-L1. Altogether, this study describes how peptide hydrogels can be exploited to provide the sustained release of sdAbs, thereby potentially enhancing its clinical and therapeutic effects.
147. Proximity-induced ligation and one-pot macrocyclization of 1,4-diketone-tagged peptides derived from 2,5-disubstituted furans upon release from the solid support
Alex Manicardi (UGent) , Atiruj Theppawong (UGent) , Marleen Van Troys (UGent) and Annemieke Madder (UGent)
1,4-Dione-containing peptides are generated during the cleavage of 2,5-disubstituted furan-containing systems. The generated electrophilic systems then react with a-effect nucleophiles, following a Paal-Knorr-like mechanism, for the generation of macrocyclic peptides, occurring after simple resuspension of the crude peptide in water. Conveniently, the in situ generation of the electrophile from a stable furan ring avoids the complications associated with the synthesis of carbonyl-containing peptides. Detailed investigation of the reaction characteristics was first performed on supramolecular coiled-coil systems.
146. Furan-modified PNA probes for covalent targeting and ligation of nucleic acids
Lessandro De Paepe (UGent) , Enrico Cadoni (UGent) , Alex Manicardi and Annemieke Madder (UGent)
While natural oligonucleotides (ONs) are increasingly used as therapeutic and diagnostic tools, they still face certain challenges such as low resistance to enzymatic degradation, potential immunogenicity, and delivery issues, which can limit their applications. Peptide Nucleic Acids (PNAs) are promising alternatives due to their high affinity for DNA and RNA, the high resistance to enzymatic degradation, and the easy introduction of a wide range of potential modifications. Chemical modifications that enable the covalent targeting of specific DNA and RNA strands offer additional advantages, including enhanced potency. The current study focuses on the utilization of furan-PNAs as pro-reactive probe systems and their applications to DNA and RNA targeting. Specifically, in this methodological paper, we provide practical insights into the design, synthesis, and application of furan-containing PNA probes for achieving efficient PNA-DNA and PNA-RNA interstrand crosslinking (ICL), as well as ON-templated PNA-PNA ligation systems. Furthermore, we discuss the applications of these probes in targeting DNA secondary structures, such as G-quadruplexes and i-motifs, target pull-down assays, and on-surface detection.
145. Furan-based (photo)oxidation reactions and their application in nucleic acid and protein targeting
Jack Barr (UGent) , Gertjan Colpaert (UGent) , Enrico Cadoni (UGent) and Annemieke Madder (UGent)
Oligonucleotides (ODNs) find applications as diagnostic and therapeutic tools due to their unique ability to interact, thanks to Watson-Crick base pairing, with a specific DNA or RNA target strand. Although most of the tools available today rely on mere hydrogen bond formation, chemical modifications to enable covalent interstrand-crosslinking (ICL) have been reported, and are gaining a place under the spotlight as they potentially offer a series of advantages over the state of the art, including a higher potency and selectivity. This methodological paper focuses on the use of a pro-reactive furan moiety and its subsequent oxidation for applications in ODN targeting. The design of effective capture and targeting probes to ensure high ICL yields is discussed and the mechanisms underlying the (photo)chemical oxidation of furan are explained. Furthermore, examples of furan-containing DNAs designed for different applications, including DNA-DNA or DNA-RNA ICL and DNA-peptide/protein targeting, are provided. The paper highlights the advantages of using different oxidative chemical triggers, such as N-bromosuccinimide or singlet oxygen, to offer additional selectivity control over the ICL reaction.
144. Development of teixobactin analogues containing hydrophobic, non-proteogenic amino acids that are highly potent against multidrug-resistant bacteria and biofilms
Anish Parmar, Rajamani Lakshminarayanan, Abhishek Iyer (UGent) , Eunice Tze Leng Goh, Tsz Ying To, Joey Kuok Hoong Yam, Liang Yang, Enas Newire, Maria C. Robertson, Stephen H. Prior, Eefjan Breukink, Annemieke Madder (UGent) and Ishwar Singh
Teixobactin is a cyclic undecadepsipeptide that has shown excellent potency against multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). In this article, we present the design, synthesis, and antibacterial evaluations of 16 different teixobactin analogues. These simplified analogues contain commercially available hydrophobic, non-proteogenic amino acid residues instead of synthetically challenging expensive L-allo-enduracididine amino acid residue at position 10 together with different combinations of arginines at positions 3, 4 and 9. The new teixobactin analogues showed potent antibacterial activity against a broad panel of Gram-positive bacteria, including MRSA and VRE strains. Our work also presents the first demonstration of the potent antibiofilm activity of teixobactin analogoues against Staphylococcus species associated with serious chronic infections. Our results suggest that the use of hydrophobic, non-proteogenic amino acids at position 10 in combination with arginine at positions 3, 4 and 9 holds the key to synthesising a new generation of highly potent teixobactin analogues to tackle resistant bacterial infections and biofilms.
143. Smart hydrogels delivered by high pressure aerosolization can prevent peritoneal adhesions
Helena Braet (UGent) , Peter-Paul Fransen, Yong Chen (UGent) , Simon Van Herck (UGent) , Remco Mariën (UGent) , Valérie Vanhoorne (UGent) , Wim Ceelen (UGent) , Annemieke Madder (UGent) , Steven Ballet, Richard Hoogenboom (UGent) , et al.
Postoperative peritoneal adhesions occur in the majority of patients undergoing intra-abdominal surgery and are one of the leading causes of hospital re-admission. There is an unmet clinical need for effective anti-adhesive biomaterials, which can be applied evenly across the damaged tissues. We examined three different respon-sive hydrogel types, i.e. a thermosensitive PLGA-PEG-PLGA, a pH responsive UPy-PEG and a shear-thinning hexapeptide for this purpose. More specifically, their potential to be homogeneously distributed in the perito-neal cavity by high pressure nebulization and prevent peritoneal adhesions was evaluated. Solutions of each polymer type could be successfully nebulized while retaining their responsive gelation behavior in vitro and in vivo. Furthermore, none of the polymers caused in vitro toxicity on SKOV3-IP2 cells. Following intraperitoneal administration, both the PLGA-PEG-PLGA and the hexapeptide hydrogels resulted in local inflammation and fibrosis and failed in preventing peritoneal adhesions 7 days after adhesion induction. In contrast, the pH sensitive UPy-PEG formulation was well tolerated and could significantly reduce the formation of peritoneal adhesions, even outperforming the commercially available Hyalobarrier (R) as positive control. To conclude, local nebulization of the bioresponsive UPy-PEG hydrogel can be considered as a promising approach to prevent postsurgical peritoneal adhesions.
142. Stereomeric Lipopeptides from a Single Non-Ribosomal Peptide Synthetase as an Additional Source of Structural and Functional Diversification in Pseudomonas Lipopeptide Biosynthesis
Penthip Muangkaew (UGent) , Vic De Roo (UGent) , Lu Zhou (UGent) , Léa Girard, Catherine Cesa-Luna, Monica Höfte (UGent) , René De Mot, Annemieke Madder (UGent) , Niels Geudens (UGent) and José Martins (UGent)
In Pseudomonas lipopeptides, the D-configuration of amino acids is generated by dedicated, dual-function epimerization/condensation (E/C) domains. The increasing attention to stereochemistry in lipopeptide structure elucidation efforts has revealed multiple examples where epimerization does not occur, even though an E/C-type domain is present. While the origin of the idle epimerization in those E/C-domains remains elusive, epimerization activity has so far shown a binary profile: it is either ‘on’ (active) or ‘off’ (inactive). Here, we report the unprecedented observation of an E/C-domain that acts ‘on and off’, giving rise to the production of two diastereoisomeric lipopeptides by a single non-ribosomal peptide synthetase system. Using dereplication based on solid-phase peptide synthesis and NMR fingerprinting, we first show that the two cyclic lipopeptides produced by Pseudomonas entomophila COR5 correspond to entolysin A and B originally described for P. entomophila L48. Next, we prove that both are diastereoisomeric homologues differing only in the configuration of a single amino acid. This configurational variability is maintained in multiple Pseudomonas strains and typically occurs in a 3:2 ratio. Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains. In permeabilization assays, using propidium iodide entolysin B has a higher antifungal activity compared to entolysin A against Botrytis cinerea and Pyricularia oryzae spores. The fact that configurational homologues are produced by the same NRPS system in a Pseudomonas strain adds a new level of structural and functional diversification to those already known from substrate flexibility during the recruitment of the amino acids and fatty acids and underscores the importance of complete stereochemical elucidation of non-ribosomal lipopeptide structures
141. Photo-induced crosslinking uncovers an antiparallel strand orientation in heterodimeric (EIAALEK)(3)/(KIAALKE)(3) and (EIAALEK)(3)/(RIAALRE)(3) coiled-coil systems
Dorien Aerssens (UGent) , Laia Miret Casals (UGent) , D. Gomez, D. Sousa-Neves, Y. Levy, M. De Vleesschouwer, Alex Manicardi (UGent) and Annemieke Madder (UGent)
We describe for the first time the co-existence of the parallel and antiparallel conformation of the heterodimeric E3/K3 and E3/R3 coiled-coil systems in solution. The introduction of a furanylated amino acid in the (EIAALEK)(3) sequence allowed, upon photo-induced covalent crosslinking, freezing of the respective coiled-coil complexes present in solution. The occurrence of both parallel and antiparallel conformations in solution was supported by computational simulations and further confirmed by fluorescence experiments based on pyrene-pyrene stacking.
140. Acid‐Resistant BODIPY Amino Acids for Peptide‐Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets
Lorena Mendive-Tapia, Laia Miret Casals (UGent) , Nicole Barth, Jinling Wang, Anne de Bray, Massimiliano Beltramo, Vincent Robert, Christophe Ampe (UGent) , David Hodson, Annemieke Madder (UGent) and Marc Vendrell
The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.
139. A red light-triggered chemical tool for sequence-specific alkylation of G-quadruplex and I-motif DNA
Enrico Cadoni (UGent) , Lessandro De Paepe (UGent) , Gertjan Colpaert (UGent) , Ruben Tack (UGent) , Dries Waegeman, Alex Manicardi (UGent) and Annemieke Madder (UGent)
The importance of non-canonical DNA structures such as G-quadruplexes (G4) and intercalating-motifs (iMs) in the fine regulation of a variety of cellular processes has been recently demonstrated. As the crucial roles of these structures are being unravelled, it is becoming more and more important to develop tools that allow targeting these structures with the highest possible specificity. While targeting methodologies have been reported for G4s, this is not the case for iMs, as evidenced by the limited number of specific ligands able to bind the latter and the total absence of selective alkylating agents for their covalent targeting. Furthermore, strategies for the sequence-specific covalent targeting of G4s and iMs have not been reported thus far. Herein, we describe a simple methodology to achieve sequence-specific covalent targeting of G4 and iM DNA structures based on the combination of (i) a peptide nucleic acid (PNA) recognizing a specific sequence of interest, (ii) a pro-reactive moiety enabling a controlled alkylation reaction, and (iii) a G4 or iM ligand orienting the alkylating warhead to the reactive residues. This multi-component system allows for the targeting of specific G4 or iM sequences of interest in the presence of competing DNA sequences and under biologically relevant conditions.
MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, have received increasing attention as potential biomarkers of different diseases, including viral infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the disease it is causing, coronavirus disease (COVID-19), has affected health, society and life worldwide since its pandemic spread. Differential expression of miRNAs in COVID-19 patients compared to healthy controls and also between different severity grades of COVID-19 has been described in several recent studies. In this review, we discuss in detail studies that investigated miRNA expression in body fluids of COVID-19 patients. Several studies found a different miRNA expression profile in COVID-19 patients compared to controls but also in different severity grades of the disease. We compared the main findings of the studies in order to identify miRNAs that have been identified as differentially expressed by more than one study and could serve as diagnostic or prognostic biomarkers of COVID-19. Finally, we highlight the challenges and perspectives associated to the use of miRNAs as biomarkers of COVID-19.
137. Complex electrostatic effects on the selectivity of membrane-permeabilizing cyclic lipopeptides
Jessica Steigenberger, Yentl Verleysen, Niels Geudens (UGent) , Annemieke Madder (UGent) , José Martins (UGent) and Heiko Heerklotz
Cyclic lipopeptides (CLiPs) have many biological functions, including the selective permeabilization of target membranes, and technical and medical applications. We studied the anionic CLiP viscosin from Pseudomonas along with a neutral analog, pseudodesmin A, and the cationic viscosin-E2K to better understand electrostatic effects on target selectivity. Calcein leakage from liposomes of anionic phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) is measured in comparison with net-neutral phosphatidylcholine by time-resolved fluorescence. By contrast to the typical selectivity of cationic peptides against anionic membranes, we find viscosin more active against PG/PE at 30 μM lipid than viscosin-E2K. At very low lipid concentration, the selectivity is reversed. An equi-activity analysis reveals the reciprocal partition coefficients, 1/K, and the CLiP-to-lipid mole ratio within the membrane as leakage after 1 h reaches 50%, Re50. As expected, 1/K to PG/PE is much lower (higher affinity) for viscosin-E2K (3 μM) than viscosin (15 μM). However, the local damage to the PG/PE membrane caused by a viscosin molecule is much stronger than that of viscosin-E2K. This can be explained by the strong membrane expansion due to PG/viscosin repulsion inducing asymmetry stress between the two leaflets and, ultimately, transient limited leakage at Re50 = 0.08. PG/viscosin-E2K attraction opposes expansion and leakage starts only as the PG charges in the outer leaflet are essentially compensated by the cationic peptide (Re50 = 0.32). In the high-lipid regime (at lipid concentrations cL ≫ 1/K), virtually all CLiP is membrane bound anyway and Re50 governs selectivity, favoring viscosin. In the low-lipid regime at cL ≪ 1/K, virtually all CLiP is in solution, 1/K becomes important and the “cation attacks anionic membrane” selectivity gets restored. Overall, activity and selectivity data can only properly be interpreted if the lipid regime is known and predictions for other lipid concentrations or cell counts require knowledge of 1/K and Re50
136. Impact of doubling peptide length on in vivo hydrogel stability and sustained drug release
Julie Heremans, Lucie Chevillard, Morgane Mannes, Jessica Mangialetto, Kaat Leroy, Jacinta F. White, Arthur Lamouroux, Mathieu Vinken, James Gardiner, Bruno Van Mele, Niko Van den Brande, Richard Hoogenboom (UGent) , Annemieke Madder (UGent) , Vicky Caveliers, Bruno Megarbane, Sophie Hernot, Steven Ballet and Charlotte Martin
Peptide-based hydrogels represent promising systems for the sustained release of different types of drugs, ranging from small molecules to biologicals. Aiming at subcutaneous injection, which is a desirable parenteral administration route, especially for biologicals, we herein focus on physically crosslinked systems possessing thixotropic behaviour. The purpose of this study was to evaluate the in vitro and in vivo properties of hydrogels based on the amphipathic hexapeptide H-FQFQFK-NH2, which served as the lead sequence. Upon doubling the length of this peptide, the dodecapeptide H-FQFQFKFQFQFK-NH2 gave a significant improvement in terms of in vivo stability of the hydrogel post-injection, as monitored by nuclear SPECT/CT imaging. This increased hydrogel stability also led to a more prolonged in vivo release of encapsulated peptide cargoes. Even though no direct link with the mechanical properties of the hydrogels before injection could be made, an important effect of the subcutaneous medium was noticed on the rheological properties of the hydrogels in post in vivo injection measurements. The results were validated in vivo for a therapeutically relevant analgesic peptide using the hot-plate test as an acute pain model. It was confirmed that elongation of the hydrogelator sequence induced more extended antinociceptive effects. Altogether, this simple structural modification of the hydrogelating peptide could provide a basis for reaching longer durations of action upon use of these soft biomaterials.
135. Synthesis and structure–activity relationship of peptide nucleic acid probes with improved interstrand-crosslinking abilities: application to biotin-mediated RNA-pulldown.
Enrico Cadoni (UGent), Francesca Pennati, Penthip Muangkaew (UGent), Joke Elskens (UGent), Annemieke Madder (UGent) and Alex Manicardi (UGent)
The development of interstrand-crosslinking (ICL) probes for the covalent targeting of DNA and RNA sequences of interest has been extensively reported in the past decade. However, most of the reactions reported so far induce the formation of a stable adduct that cannot be reverted, thus rendering these chemistries less useful in applications where the reversibility of the reaction is needed for further downstream processing of the targeted and isolated sequences, such as enzymatic amplification steps. In this work, we report on the reversibility of the furan-mediated ICL reaction. ICL formation can be conveniently triggered by either chemical (N-bromo succinimide, NBS) or luminous stimuli (visible light irradiation in presence of a photosensitizer) and quantitative reversion can be achieved by heating the crosslinked sample at 95 °C, while maintaining the structure of the DNA/RNA targets intact. As a proof-of-concept and showing the benefits of the ICL reversibility, we apply furan-mediated ICL to the pulldown of a target RNA strand from cell lysate.
134. An nuclear magnetic resonance fingerprint matching approach for the identification and structural re-evaluation of Pseudomonas lipopeptides
Vic De Roo (UGent) , Yentl Verleysen (UGent) , Benjámin Kovács (UGent) , Matthias De Vleeschouwer (UGent) , Penthip Muangkaew (UGent) , Léa Girard, Monica Höfte (UGent) , René De Mot, Annemieke Madder (UGent) , Niels Geudens (UGent) and José C. Martins (UGent)
Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular, display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity, and stereochemistry (e.g., d- versus l-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly, however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide, and Xantholysin groups as test cases, we demonstrate and validate that the combined 1H and 13C Nuclear Magnetic Resonance (NMR) chemical shifts of CLiPs constitute a spectral fingerprint that is sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single d/l configuration. Rapid screening, involving simple matching of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, its spectral fingerprint allows to quickly verify whether a newly isolated CLiP is novel or already present in the reference collection. We show NMR fingerprinting leads to a simple approach for early on dereplication which should become more effective as more fingerprints are collected. To benefit research involving CLiPs, we have made a publicly available data repository accompanied by a ‘knowledge base’ at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures.
133. Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy
Klaas W. Decoene (UGent), Kamil Ünal (UGent) , An Staes (UGent) , Olivier Zwaenepoel (UGent) , Jan Gettemans (UGent) , Kris Gevaert (UGent) , Annemieke Madder (UGent) and Johan Winne (UGent)
Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur via a ‘tyrosine-click’ reaction with triazolinedione reagents (TAD). However, we here demonstrate that TAD reagents are actually not selective for tyrosine and that tryptophan residues are in fact also labelled with these reagents. This off-target labelling remained under the radar as it is challenging to detect these physiologically stable but thermally labile modifications with the commonly used HCD and CID MS/MS techniques. We show that selectivity of tryptophan over tyrosine can be achieved by lowering the pH of the aqueous buffer to effect selective Trp-labelling. Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins.
132. Novel electrochemiluminescent assay for the aptamer-based detection of testosterone
Rocio Canovas, Elise Daems, Rui Campos, Sofie Schellinck (UGent) , Annemieke Madder (UGent) , José Martins (UGent) , Frank Sobott and Karolien De Wael
This work presents a proof-of-concept assay for the detection and quantification of small molecules based on aptamer recognition and electrochemiluminescence (ECL) readout. The testosterone-binding (TESS.1) aptamer was used to demonstrate the novel methodology. Upon binding of the target, the TESS.1 aptamer is released from its complementary capture probe – previously immobilized at the surface of the electrode – producing a decrease in the ECL signal after a washing step removing the released (labeled) TESS.1 aptamer. The analytical capability of the ECL assay towards testosterone detection was investigated displaying a linear range from 0.39 to 1.56 μM with a limit of detection of 0.29 μM. The selectivity of the proposed assay was assessed by performing two different negative control experiments; i) detection of testosterone with a randomized ssDNA sequence and ii) detection of two other steroids, i.e. deoxycholic acid and hydrocortisone with the TESS.1 aptamer. In parallel, complementary analytical techniques were employed to confirm the suggested mechanism: i) native nano-electrospray ionization mass spectrometry (native nESI-MS) was used to determine the stoichiometry of the binding, and to characterize aptamer-target interactions; and, ii) isothermal titration calorimetry (ITC) was carried out to elucidate the dissociation constant (Kd) of the complex of testosterone and the TESS.1 aptamer. The combination of these techniques provided a complete understanding of the aptamer performance, the binding mechanism, affinity and selectivity. Furthermore, this important characterization carried out in parallel validates the real functionality of the aptamer (TESS.1) ensuring its use towards selective testosterone binding in further biosensors. This research will pave the way for the development of new aptamer-based assays coupled with ECL sensing for the detection of relevant small molecules.
Laia Miret Casals (UGent) , Sander Van De Putte, Dorien Aerssens (UGent) , Julien Diharce, Pascal Bonnet and Annemieke Madder (UGent)
Using a coiled-coil peptide dimer as a model system to explore furan reactivity, we describe novel cross-link partners of furan warheads for site-specific cross-linking. We demonstrate that replacement of weak interhelical ionic contacts with a furan moiety and its potential cross-link partner affords covalently connected coiled-coil motifs upon furan activation. We describe for the first time the reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported lysine, thus enhancing the versatility of the furan cross-link methodology by the possibility to target different amino acids. The present in vitro validation of “furan-armed” α-helices provides further grounds for exploiting furan technology in the development of furan-modified ligands/proteins to target proteins in a covalent way through various amino acid side chains.
130. A photosensitized singlet oxygen ( 1O2) toolbox for bio-organic applications : tailoring 1O2 generation for DNA and protein labelling, targeting and biosensing
Singlet oxygen (1O2) is the excited state of ground, triplet state, molecular oxygen (O2). Photosensitized 1O2 has been extensively studied as one of the reactive oxygen species (ROS), responsible for damage of cellular components (protein, DNA, lipids). On the other hand, its generation has been exploited in organic synthesis, as well as in photodynamic therapy for the treatment of various forms of cancer. The aim of this review is to highlight the versatility of 1O2 discussing the main bioorganic applications reported over the past decades, which rely on its production. After a brief introduction on the photosensitized production of 1O2, we will describe the main aspects involving the biologically relevant damage that can accompany an uncontrolled, aspecific generation of this ROS. We then discuss in more detail a series of biological applications featuring 1O2 generation, including protein and DNA labelling, cross-linking and biosensing. Finally, we will highlight the methodologies available to tailor 1O2 generation, in order to accomplish the proposed bioorganic transformations while avoiding, at the same time, collateral damage related to an untamed production of this reactive species.
Karel Thooft (UGent) , Wander Van Breedam (UGent) , Francis Santens (UGent) , Elise Wyseure (UGent) , Sandrine Vanmarcke (UGent) , Simon Devos (UGent) , Nico Callewaert (UGent) and Annemieke Madder (UGent)
Recently, the GlyConnect-oxime (GC) protein conjugation strategy was developed to provide a site-selective glycan-based conjugation strategy as an extension to the in-house developed GlycoDelete (GD) technology. GD gives access to glycoproteins with single GlcNAc, LacNAc, or LacNAc-Sia type glycans on their N-glycosylation sites. We have previously shown that these glycans provide a unique handle for site-selective conjugation as they provide a short, homogeneous and hydrophilic link to the protein backbone. GC focused on the use of chemical and chemo-enzymatic pathways for conjugation of a single molecule of interest via oxime formation or reductive amination. In the current work, we explore multicomponent reactions (MCR), namely Ugi and Passerini reactions, for GlycoDelete glycan directed, site-specific protein conjugation (MC-GC). The use of the Ugi and Passerini multicomponent reactions holds the potential of introducing multiple groups of interest in a single reaction step while creating a hydrophilic peptide-like linker.
128. Beyond small molecules : targeting G-quadruplex structures with oligonucleotides and their analogues
Enrico Cadoni (UGent) , Lessandro De Paepe, Alex Manicardi (UGent) and Annemieke Madder (UGent)
G-Quadruplexes (G4s) are widely studied secondary DNA/RNA structures, naturally occurring when G-rich sequences are present. The strategic localization of G4s in genome areas of crucial importance, such as proto-oncogenes and telomeres, entails fundamental implications in terms of gene expression regulation and other important biological processes. Although thousands of small molecules capable to induce G4 stabilization have been reported over the past 20 years, approaches based on the hybridization of a synthetic probe, allowing sequence-specific G4-recognition and targeting are still rather limited. In this review, after introducing important general notions about G4s, we aim to list, explain and critically analyse in more detail the principal approaches available to target G4s by using oligonucleotides and synthetic analogues such as Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs), reporting on the most relevant examples described in literature to date.
127. Bacillus cereus food intoxication and toxicoinfection
Jelena Jovanovic (UGent) , Vincent Ornelis (UGent) , Annemieke Madder (UGent) and Andreja Rajkovic (UGent)
Bacillus cereus is one of the leading etiological agents of toxin-induced foodborne diseases. Its omnipresence in different environments, spore formation, and its ability to adapt to varying conditions and produce harmful toxins make this pathogen a health hazard that should not be underestimated. Food poisoning by B. cereus can manifest itself as an emetic or diarrheal syndrome. The former is caused by the release of the potent peptide toxin cereulide, whereas the latter is the result of proteinaceous enterotoxins (e.g., hemolysin BL, nonhemolytic enterotoxin, and cytotoxin K). The final harmful effect is not only toxin and strain dependent, but is also affected by the stress responses, accessory virulence factors, and phenotypic properties under extrinsic, intrinsic, and explicit food conditions and host-related environment. Infamous portrait of B. cereus as a foodborne pathogen, as well as a causative agent of nongastrointestinal infections and even nosocomial complications, has inspired vast volumes of multidisciplinary research in food and clinical domains. As a result, extensive original data became available asking for a new, both broad and deep, multifaceted look into the current state-of-the art regarding the role of B. cereus in food safety. In this review, we first provide an overview of the latest knowledge on B. cereus toxins and accessory virulence factors. Second, we describe the novel taxonomy and some of the most pertinent phenotypic characteristics of B. cereus related to food safety. We link these aspects to toxin production, overall pathogenesis, and interactions with its human host. Then we reflect on the prevalence of different toxinotypes in foods opening the scene for epidemiological aspects of B. cereus foodborne diseases and methods available to prevent food poisoning including overview of the different available methods to detect B. cereus and its toxins.
Recent dramatic expansion in potential uses of protein conjugates has fueled the development of a wide range of protein modification methods; however, the desirable single-site multi-functionalization of proteins has remained a particularly intransigent challenge. Herein, we present the application of 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2Os) as advantageous alternatives to widely used maleimides for the chemo- and site-selective labeling of cysteine residues within proteins. A variety of 5HP2O building blocks have been synthesized using a one-pot photooxidation reaction starting from simple and readily accessible furans and using visible light and oxygen. These novel reagents display excellent cysteine selectivity and also yield thiol conjugates with superior stability. 5HP2O building blocks offer a unique opportunity to introduce multiple new functionalities into a protein at a single site and in a single step, thus, significantly enhancing the resultant conjugate's properties.
125. Teaching photosensitizers a new trick : red light-triggered G-quadruplex alkylation by ligand co-localization
Enrico Cadoni (UGent) , Alex Manicardi (UGent) , Mathieu Fossepre, Kaat Heirwegh, Mathieu Surin and Annemieke Madder (UGent)
We propose a bimolecular approach for G-quadruplex alkylation, using a pro-reactive furan-containing ligand, activated by red-light irradiation of a proximate G4-binding photosensitizer. G4- over dsDNA alkylation can be achieved selectively and proves high-yielding at low ligand excess. HPLC and modelling studies allowed identifying potential residues involved in the alkylation.
124. Crosslinker-modified nucleic acid probes for improved target identification and biomarker detection
Joke Elskens (UGent) and Annemieke Madder (UGent)
Understanding the intricate interaction pattern of nucleic acids with other molecules is essential to gain further insight in biological processes and disease mechanisms. To this end, a multitude of hybridization-based assays have been designed that rely on the non-covalent recognition between complementary nucleic acid sequences. However, the ephemeral nature of these interactions complicates straightforward analysis as low efficiency and specificity are rule rather than exception. By covalently locking nucleic acid interactions by means of a crosslinking agent, the overall efficiency, specificity and selectivity of hybridization-based assays could be increased. In this mini-review we highlight methodologies that exploit the use of crosslinker-modified nucleic acid probes for interstrand nucleic acid crosslinking with the objective to study, detect and identify important targets as well as nucleic acid sequences that can be considered relevant biomarkers. We emphasize on the usefulness and advantages of crosslinking agents and elaborate on the chemistry behind the crosslinking reactions they induce.
123. Furan warheads for covalent trapping of weak protein-protein interactions : cross-linking of thymosin β4 to actin
Laia Miret Casals (UGent) , Willem Vannecke (UGent) , Kurt Hoogewijs (UGent) , Gianluca Arauz-Garofalo, Marina Gay, Mireia Diaz-Lobo, Marta Vilaseca, Christophe Ampe (UGent) , Marleen Van Troys (UGent) and Annemieke Madder (UGent)
We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin beta 4 (T beta 4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' T beta 4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.
122. Hydrolysis of 5-methylfuran-2-yl to 2,5-dioxopentanyl allows for stable bio-orthogonal proximity-induced ligation
Alex Manicardi (UGent) , Enrico Cadoni (UGent) and Annemieke Madder (UGent)
Proximity-based ligations commonly require an external stimulus such as a catalyst or irradiation, or highly reactive functional groups. Here the reaction of alpha effect nucleophiles and 2,5-dioxopentanyl derivatives allows direct proximity-based ligation while avoiding highly reactive moieties. Ligation methodologies featuring bio-orthogonal units and leading to the formation of a stable adduct are the ideal candidates for being applied in a biological context. However, most of the available strategies rely on highly reactive species that require careful handling, or on the activation of pro-reactive functional groups. We here report on a proximity-induced ligation reaction that relies on a stable 2,5-dione, that can be conveniently generated under acidic conditions from a 2,5-dialkylfuran building block, and hydrazine nucleophiles. This bio-orthogonal ligation, which proceeds under physiological conditions, does not require any stimulus or trigger and leads to the formation of a pyridazinium adduct that demonstrates excellent stability under harsh conditions (24 h at 90 degrees C). The reaction was applied to the formation of PNA-PNA adducts, DNA- and RNA-templated ligations, and for the formation of peptide-peptide adducts in solution. This convenient methodology was further implemented on plastic and glass surfaces to realize self-addressable covalent constructs.
121. Towards the understanding of halogenation in peptide hydrogels : a quantum chemical approach
Tom Bettens, Valentin Lacanau, Ruben Van Lommel, Tess De Maeseneer, Wouter Vandeplassche, Jolien Bertouille, Joost Brancart, Thomas M. A. Barlow, Tatiana Woller, Niko Van den Brande, et al.
Non-covalent interactions involving aromatic rings play a central role in many areas of modern chemistry. In medicinal and bioorganic chemistry, the intermolecular interactions between the aromatic side chains of amino acids, such as phenylalanine and tyrosine, are of great interest. To enhance the affinity between such aromatic side chains, halogenation is a promising modification strategy. In the current work, the nature and strength of halogenated pi-pi stacked phenylalanine (Phe) dimers have been investigated using density functional theory, energy decomposition analyses and the non-covalent interaction (NCI) method. Our analysis shows that increasing the degree of halogenation enhances the strength of the stacking interactions and, moreover, the heavier halides (Cl, Br and I) lead to stronger interactions compared to the lighter F. This effect was traced back to local secondary interactions of the halide with the aliphatic C-H bonds of the phenylalanine side chain. Based on the computational findings, a set of peptide hydrogelators was synthesized, and the resulting hydrogel properties were further investigated via dynamic rheometry. Experimental observations can be correlated to the trends found in the theoretical analysis, suggesting that local interactions indeed play a noticeable role in enhancing peptide-based hydrogel strength.
120. Exploiting double exchange Diels-Alder cycloadditions for immobilization of peptide nucleic acids on gold nanoparticles
Enrico Cadoni (UGent) , Daniele Rosa-Gastaldo, Alex Manicardi (UGent) , Fabrizio Mancin and Annemieke Madder (UGent)
The generation of PNA-decorated gold nanoparticles (AuNPs) has revealed to be more difficult as compared to the generation of DNA-functionalized ones. The less polar nature of this artificial nucleic acid system and the associated tendency of the neutral poly-amidic backbone to aspecifically adsorb onto the gold surface rather than forming a covalent bond through gold-thiol interaction, combined with the low solubility of PNAs itself, form the main limiting factors in the functionalization of AuNP. Here, we provide a convenient methodology that allows to easily conjugate PNAs to AuNP. Positively charged PNAs containing a masked furan moiety were immobilized via a double exchange Diels-Alder cycloaddition onto masked maleimide-functionalized AuNPs in a one-pot fashion. Conjugated PNA strands retain their ability to selectively hybridize with target DNA strands. Moreover, the duplexes resulting from hybridization can be detached through a retro-Diels-Alder reaction, thus allowing straightforward catch-and-release of specific nucleic acid targets.
119. GOLVEN peptide signalling through RGI receptors and MPK6 restricts asymmetric cell division during lateral root initiation
Ana Fernandez Salina (UGent) , Nick Vangheluwe (UGent) , Ke Xu (UGent) , Joris Jourquin (UGent) , Lucas Alves Neubus Claus (UGent) , Stefania Morales (UGent) , Boris Parizot (UGent) , dr. Hugues De Gernier (UGent) , Qiaozhi Yu, Andrzej Drozdzecki (UGent) , et al.
During lateral root initiation, lateral root founder cells undergo asymmetric cell divisions that generate daughter cells with different sizes and fates, a prerequisite for correct primordium organogenesis. An excess of the GLV6/RGF8 peptide disrupts these initial asymmetric cell divisions, resulting in more symmetric divisions and the failure to achieve lateral root organogenesis. Here, we show that loss-of-function GLV6 and its homologue GLV10 increase asymmetric cell divisions during lateral root initiation, and we identified three members of the RGF1 INSENSITIVE/RGF1 receptor subfamily as likely GLV receptors in this process. Through a suppressor screen, we found that MITOGEN-ACTIVATED PROTEIN KINASE6 is a downstream regulator of the GLV pathway. Our data indicate that GLV6 and GLV10 act as inhibitors of asymmetric cell divisions and signal through RGF1 INSENSITIVE receptors and MITOGEN-ACTIVATED PROTEIN KINASE6 to restrict the number of initial asymmetric cell divisions that take place during lateral root initiation. The authors demonstrate the negative role of GOLVEN peptides during lateral root initiation in Arabidopsis, at the very early stage of the first asymmetric cell division of lateral root founder cells, and identify the receptors for these peptides.
118. Nanobody click chemistry for convenient site-specific fluorescent labelling, single step immunocytochemistry and delivery into living cells by photoporation and live cell imaging
Tim Hebbrecht, Jing Liu, Olivier Zwaenepoel (UGent) , Gaelle Boddin (UGent) , Chloé Van Leene (UGent) , Klaas Decoene (UGent) , Annemieke Madder (UGent) , Kevin Braeckmans (UGent) and Jan Gettemans (UGent)
While conventional antibodies have been an instrument of choice in immunocytochemistry for some time, their small counterparts known as nanobodies have been much less frequently used for this purpose. In this study we took advantage of the availability of nanobody cDNAs to site-specifically introduce a non-standard amino acid carrying an azide/alkyne moiety, allowing subsequent Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC). This generated a fluorescently labelled nanobody that can be used in single step immunocytochemistry as compared to conventional two step immunocytochemistry. Two strategies were explored to label nanobodies with Alexa Fluor 488. The first involved enzymatic addition of an alkyne-containing peptide to nanobodies using sortase A, while the second consisted of incorporating para-azido phenylalanine at the nanobody C-terminus. Through these approaches, the fluorophore was covalently and site-specifically attached. It was demonstrated that cortactin and beta-catenin, cytoskeletal and adherens junction proteins respectively, can be imaged in cells in this manner through single step immunocytochemistry. However, fixation and permeabilization of cells can alter native protein structure and form a dense cross-linked protein network, encumbering antibody binding. It was shown that photoporation prior to fixation not only allowed delivery of nanobodies into living cells, but also facilitated j3-catenin nanobody Nb86 imaging of its target, which was not possible in fixed cells. Pharmacological inhibitors are lacking for many non-enzymatic proteins, and it is therefore expected that new biological information will be obtained through photoporation of fluorescent nanobodies, which allows the study of short term effects, independent of gene-dependent (intrabody) expression.
117. Visible-light triggered templated ligation on surface using furan-modified PNAs
Alex Manicardi (UGent) , Enrico Cadoni (UGent) and Annemieke Madder (UGent)
Oligonucleotide-templated reactions are frequently exploited for target detection in biosensors and for the construction of DNA-based materials and probes in nanotechnology. However, the translation of the specifically used template chemistry from solution to surfaces, with the final aim of achieving highly selective high-throughput systems, has been difficult to reach and therefore, poorly explored. Here, we show the first example of a visible light-triggered templated ligation on a surface, employing furan-modified peptide nucleic acids (PNAs). Tailored photo-oxidation of the pro-reactive furan moiety is ensured by the simultaneous introduction of a weak photosensitizer as well as a nucleophilic moiety in the reacting PNA strand. This allows one to ensure a localized production of singlet oxygen for furan activation, which is not affected by probe dilution or reducing conditions. Simple white light irradiation in combination with target-induced proximity between reactive functionalities upon recognition of a short 22mer DNA or RNA sequence that functions as a template, allows sensitive detection of nucleic acid targets in a 96 well plate format.
116. Do aptamers always bind? : the need for a multifaceted analytical approach when remonstrating binding affinity between aptamer and low molecular weight compounds
Fabio Bottari, Elise Daems, Anne-Mare de Vries (UGent) , Pieter Van Wielendaele, Stanislav Trashin, Ronny Blust, Frank Sobott, Annemieke Madder (UGent) , José Martins (UGent) and Karolien De Wael
In this manuscript, we compare different analytical methodologies to validate or disprove the binding capabilities of aptamer sequences. This was prompted by the lack of a universally accepted and robust quality control protocol for the characterization of aptamer performances coupled with the observation of independent yet inconsistent data sets in the literature. As an example, we chose three aptamers with a reported affinity in the nanomolar range for ampicillin, a beta-lactam antibiotic, used as biorecognition elements in several detection strategies described in the literature. Application of a well-known colorimetric assay based on aggregation of gold nanoparticles (AuNPs) yielded conflicting results with respect to the original report. Therefore, ampicillin binding was evaluated in solution using isothermal titration calorimetry (ITC), native nano-electrospray ionization mass spectrometry (native nESI-MS), and 1H-nuclear magnetic resonance spectroscopy (1H NMR). By coupling the thermodynamic data obtained with ITC with the structural information on the binding event given by native nESI-MS and 1H NMR we could verify that none of the ampicillin aptamers show any specific binding with their intended target. The effect of AuNPs on the binding event was studied by both ITC and 1H NMR, again without providing positive evidence of ampicillin binding. To validate the performance of our analytical approach, we investigated two well-characterized aptamers for cocaine/quinine (MN4), chosen for its nanomolar range affinity, and L-argininamide (1OLD) to show the versatility of our approach. The results clearly indicate the need for a multifaceted analytical approach, to unequivocally establish the actual detection potential and performance of aptamers aimed at small organic molecules.
115. Detection of toxins involved in foodborne diseases caused by Gram-positive bacteria
Andreja Rajkovic (UGent) , Jelena Jovanovic (UGent) , Silvia Monteiro, Marlies Decleer (UGent) , Mirjana Andjelkovic, Astrid Foubert, Natalia Beloglazova (UGent) , Varvara Tsilla, Benedikt Sas (UGent) , Annemieke Madder (UGent) , et al.
Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram‐positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose‐response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.
Enrico Cadoni (UGent) , Alex Manicardi (UGent) and Annemieke Madder (UGent)
MicroRNAs (miRNAs or miRs) are small noncoding RNAs involved in the fine regulation of post-transcriptional processes in the cell. The physiological levels of these short (20-22-mer) oligonucleotides are important for the homeostasis of the organism, and therefore dysregulation can lead to the onset of cancer and other pathologies. Their importance as biomarkers is constantly growing and, in this context, detection methods based on the hybridization to peptide nucleic acids (PNAs) are gaining their place in the spotlight. After a brief overview of their biogenesis, this review will discuss the significance of targeting miR, providing a wide range of PNA-based approaches to detect them at biologically significant concentrations, based on electrochemical, fluorescence and colorimetric assays.
Handling of the individual fragments remains a bottleneck in the convergent assembly of peptides. Overlooked since the emergence of ligation chemistries during the past two decades, so-called resin-to-resin transfer reactions (RRTR) are here reported as a strategic shortcut in this context. Condensation of the involved moieties at an acceptor resin is facilitated by shuttling peptide segments directly from a donor resin in a one-pot fashion. The straightforward synthesis of a sterically constrained 13-mer peptidosteroid model illustrates the utility of this approach. The first successful application of the RRTR methodology in the field of multivalent design and bioconjugation is here reported. Relying on established procedures to generate, monitor and isolate intermediates and products, the solid-phase nature of our entire strategy allows for the fast construction of polypeptide adducts and libraries thereof. As such, a rejuvenated use and new opportunities for RRTR is reported.
112. Identification of the molecular determinants involved in antimicrobial activity of pseudodesmin A, a cyclic lipopeptide from the viscosin group
Matthias De Vleeschouwer (UGent) , Tim Van Kersavond (UGent) , Yentl Verleysen (UGent) , Davy Sinnaeve (UGent) , Tom Coenye (UGent) , José Martins (UGent) and Annemieke Madder (UGent)
Cyclic lipo(depsi)peptides (CLiPs) from Pseudomonas constitute a class of natural products involved in a broad range of biological functions for their producers. They also display interesting antimicrobial potential including activity against Gram-positive bacteria. Literature has indicated that these compounds can induce membrane permeabilization, possibly through pore-formation, leading to the general view that the cellular membrane constitutes the primary target in their mode of action. In support of this view, we previously demonstrated that the enantiomer of pseudodesmin A, a member of the viscosin group of CLiPs, shows identical activity against a test panel of six Gram-positive bacterial strains. Here, a previously developed total organic synthesis route is used and partly adapted to generate 20 novel pseudodesmin A analogs in an effort to derive links between molecular constitution, structure and activity. From these, the importance of a macrocycle closed by an ester bond as well as a critical length of beta-OH fatty acid chain capping the N-terminus is conclusively demonstrated, providing further evidence for the importance of peptide-membrane interactions in the mode of action. Moreover, an alanine scan is used to unearth the contribution of specific amino acid residues to biological activity. Subsequent interpretation in terms of a structural model describing the location and orientation of pseudodesmin A in a membrane environment, allows first insight in the peptide-membrane interactions involved. The biological screening also identified residue positions that appear less sensitive to conservative modifications, allowing the introduction of a non-perturbing tryptophan residue which will pave the way toward biophysical studies using fluorescence spectroscopy.
111. Disruption of endocytosis through chemical inhibition of clathrin heavy chain function
Wim Dejonghe (UGent) , Isha Sharma (UGent) , Bram Denoo, Steven De Munck (UGent) , Qing Lu (UGent) , Kiril Mishev (UGent) , Haydar Bulut, Evelien Mylle (UGent) , Riet De Rycke (UGent) , Mina Vasileva, et al.
Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.
110. Thermoresponsive DNA by intercalation of dsDNA with oligoethylene-glycol-functionalized small-molecule intercalators
Debaditya Bera (UGent) , Lars Verdonck, Mathias Glassner (UGent) , Annemieke Madder (UGent) and Richard Hoogenboom (UGent)
Thermoresponsive polymeric materials are important building blocks for smart materials. In this work, the transformation of dsDNA into a thermoresponsive polymer is reported by intercalation of short, oligoethylene-glycol-modified proflavine intercalators. The thermoresponsiveness of the dsDNA-intercalator complex originates from the heating-induced dehydration of the ethylene glycol side chains, which leads to aggregation of the intercalated dsDNA. This work demonstrates the possibility of designing small-molecule intercalators to prepare thermoresponsive dsDNA complexes with tunable lower critical solution temperature behavior
109. Counteracting in vitro toxicity of the ionophoric mycotoxin beauvericin : synthetic receptors to the rescue
Vincent Ornelis (UGent) , Andreja Rajkovic (UGent) , Marlies Decleer (UGent) , Benedikt Sas (UGent) , Sarah De Saeger (UGent) and Annemieke Madder (UGent)
Beauvericin (BEA) and enniatins are toxic ionophoric cyclodepsipeptides that mainly occur in grains. As such, their presence in food commodities poses a concern for public health. To date, despite recent European Food Safety Authority emphasis on the need for more data to evaluate long-term toxicity effects, no suitable affinity reagents are available to detect the presence of BEA and derivatives in food samples. We here report on the synthesis of a small library of artificial receptors with varying cavity sizes and different hydrophobic building blocks. Immobilization of one of the receptors on solid support resulted in a strong retention of beauvericin, thus revealing promising properties as solid-phase extraction material for sample pretreatment. Furthermore, treatment of HepG2 cells with the most promising receptor markedly reduced beauvericin-induced cytotoxicity, hinting toward the possibility of using synthetic receptors as antidotes against ionophoric toxins.
108. Oxygen consumption rate analysis of mitochondrial dysfunction caused by Bacillus cereus cereulide in Caco-2 and HepG2 cells
Marlies Decleer (UGent) , Jelena Jovanovic (UGent) , Anita Vakula, Bozidar Udovicki, Rock-Seth EK Agoua, Annemieke Madder (UGent) , Sarah De Saeger (UGent) and Andreja Rajkovic (UGent)
The emetic syndrome of Bacillus cereus is a food intoxication caused by cereulide (CER) and manifested by emesis, nausea and in most severe cases with liver failure. While acute effects have been studied in the aftermath of food intoxication, an exposure to low doses of cereulide might cause unnoticed damages to the intestines and liver. The toxicity which relies on the mitochondrial dysfunction was assessed on Caco-2 and HepG2 cells after exposure of one, three and ten days to a range of low doses of cereulide. Oxygen consumption rate analyses were used to study the impact of low doses of CER on the bioenergetics functions of undifferentiated Caco-2 and HepG2 cells using Seahorse XF extracellular flux analyzer. Both Caco-2 and HepG2 cells experienced measurable mitochondrial impairment after prolonged exposure of 10 days to 0.25 nM of cereulide. Observed mitochondrial dysfunction was greatly reflected in reduction of maximal cell respiration. At 0.50 nM CER, mitochondrial respiration was almost completely shut down, especially in HepG2 cells. These results corresponded with a severe reduction in the amount of cells and an altered morphology, observed by microscopic examination of the cells. Accurate and robust quantification of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration allowed better understanding of the effects of cereulide in underlying respiratory malfunctions in low-dose exposure
107. Tethered imidazole mediated duplex stabilization and its potential for aptamer stabilization
Lars Verdonck, Dieter Buyst (UGent) , Anne-Mare de Vries (UGent) , Vicky Gheerardijn (UGent) , Annemieke Madder (UGent) and José Martins (UGent)
Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pK(a). Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and theHoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pK(a)-motif responsible for these duplex-stabilizing and pK(a)-modulating properties. Here, the robustness and utility of this pK(a)-motif was explored by introducing multiple imidazoletethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pK(a)-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pK(a) values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazoliumtethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the L-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers.
106. Templated DNA cross-linking : towards a non-invasive singlet-oxygen-based triggering method
Nathalie De Laet, Eva Marina Llamas Garcinuño (UGent) and Annemieke Madder (UGent)
Singlet-oxygen-induced DNA interstrand cross-linking was achieved at intracellularly relevant concentrations using a templated reaction setup.
The target oligonucleotide serves as a template ensuring proximity between a "furan warhead"-containing probe and an activator probe containing a photosensitizer able to generate 1O2.
Visible light irradiation of this self-activating assembly allows us to achieve highly selective interstrand crosslinking at concentration levels relevant for intracellular applications.
105. Biodegradable amphipathic peptide hydrogels as extended-release system for opioid peptides
Charlotte Martin, Maria Dumitrascuta, Morgane Mannes, Aquilino Lantero, Dominik Bucher, Katja Walker, Yannick Van Wanseele, Edith Oyen, Sophie Hernot, Ann Van Eeckhaut, et al.
Chronic pain is currently treated with opioids that offer unsatisfactory long-term analgesia and produce serious side effects. There is a clear need for alternative therapies. Herein, peptide-based hydrogels are used as extended-release drug delivery carriers. Two different formulations were developed: the drug is coformulated within the hydrogen the drug is an integral part of the hydrogelator. Both strategies afford a prolonged and significant antinociception up to 72 h after subcutaneous administration in mice.
104. Development of a synthetic receptor for the food toxin beauvericin : a tale of carbazole and steroids
Vincent Ornelis (UGent) , Andreja Rajkovic (UGent) , Benedikt Sas (UGent) , Sarah De Saeger (UGent) and Annemieke Madder (UGent)
The synthesis of the first synthetic receptor showing high affinity for the toxic ionophoric cyclodepsipeptide beauvericin is described. Binding results in a pronounced increase in fluorescence intensity of the receptor, while this increase is not observed for a very similar ionophore such as valinomycin. Experiments that shed light on the nature of this selectivity are discussed.
103. Pyrrole-mediated peptide cyclization identified through genetically reprogrammed peptide synthesis
Klaas Decoene (UGent) , Willem Vannecke (UGent) , Toby Passioura, Hiroaki Suga and Annemieke Madder (UGent)
Flexible in vitro translation (FIT) was used as a screening method to uncover a new methodology for peptide constraining based on the attack of a nucleophilic side-chain functionality onto an oxidized furylalanine side chain. A set of template peptides, each containing furylalanine as furan-modified amino acid and a nucleophilic residue (Cys, His, Lys, Arg, Ser, or Tyr), was produced through FIT. The translation mixtures were treated with N-bromosuccinimide (NBS) to achieve selective furan oxidation and subsequent MALDI analysis demonstrated Lys and Ser as promising residues for cyclisation. Solid-phase peptide synthesis (SPPS) was used to synthesize suitable amounts of material for further in-depth analysis and characterisation. It was found that in the case of the peptide containing lysine next to a furylalanine residue, a one-pot oxidation and reduction reaction leads to the generation of a cyclic peptide featuring a pyrrole moiety as cyclisation motif, resulting from the attack of the lysine side chain onto the oxidized furylalanine side chain. Structural evidence was provided via NMR and the generality of the methodology was explored. We hereby expand the scope of our previously developed furan-based peptide labeling and crosslinking strategy.
102. Reviving old protecting group chemistry for site-selective peptide-protein conjugation
Smita Gunnoo (UGent) , Abhishek Iyer (UGent) , Willem Vannecke (UGent) , Klaas Decoene (UGent) , Tim Hebbrecht, Jan Gettemans (UGent) , Mathias Laga, Stefan Loverix, Ignace Lasters and Annemieke Madder (UGent)
Methodologies to conjugate proteins to property-enhancing entities are highly sought after. We report a remarkably simple strategy for conjugating proteins bearing accessible cysteines to unprotected peptides containing a Cys(Scm) protecting group, which is introduced on-resin via a Cys(Acm) building block. The peptides employed for this proof of principle study are highly varied and structurally diverse, and undergo multiple on-resin decoration steps prior to conjugation. The methodology was applied to three different proteins, and proved to be efficient and site-selective. This twist on protecting group chemistry has led to a novel and generally applicable strategy for crossed-disulfide formation between proteins and peptides.
101. Nonselective chemical inhibition of Sec7 domain-containing ARF GTPase exchange factors
Kiril Mishev (UGent) , Qing Lu (UGent) , Bram Denoo, Francois Peurois, Wim Dejonghe (UGent) , Jan Hullaert, Riet De Rycke (UGent) , Sjef Boeren, Marine Bretou, Steven De Munck (UGent) , et al.
Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.
100. Design and syntheses of highly potent teixobactin analogues against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) in vitro and in vivo
Anish Parmar, Rajamani Lakshminarayanan, Abhishek Iyer (UGent) , Venkatesh Mayandi, Eunice Tze Leng Goh, Daniel G Lloyd, Madhavi Latha S Chalasani, Navin Kumar Verma, Stephen H Prior, Roger W Beuerman, et al.
The cyclic depsipeptide, teixobactin, kills a number of Grampositive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium tuberculosis without detectable resistance. To date, teixobactin is the only molecule in its class that has shown in vivo antibacterial efficacy. In this work, we designed and synthesized 10 new in vivo ready teixobactin analogues. These analogues showed highly potent antibacterial activities against Staphylococcus aureus, MRSA, and vancomycin-resistant enterococci (VRE) in vitro. One analogue, D-Arg(4)-Leu(10)-teixobactin, 2, was found to be noncytotoxic in vitro and in vivo. Moreover, topical instillation of peptide 2 in a mouse model of S. aureus keratitis decreased the bacterial bioburden (>99.0% reduction) and corneal edema significantly as compared to untreated mouse corneas. Collectively, our results have established the high therapeutic potential of a teixobactin analogue in attenuating bacterial infections and associated severities in vivo.
99. A thiolactone strategy for straightforward synthesis of disulfide-linked side-chain-to-tail cyclic peptides featuring an N-terminal modification handle
Dorien Van Lysebetten, Stefania Felissati, Eirini Antonatou (UGent) , Lieselot Carrette (UGent) , Pieter Espeel (UGent) , Evelien Focquet, Filip Du Prez (UGent) and Annemieke Madder (UGent)
The development of straightforward and versatile peptide cyclisation methods is highly desired to meet the demand for more stable peptide-based drugs.
Herein, a new method for the synthesis of side-chain-to-tail cyclic peptides with the simultaneous introduction of an N-terminal handle, based on the introduction of an N-terminal thiolactone building block, is described.
A primary amine liberates a homocysteine analogue from the thiolactone building block, which further enables cyclisation of the peptide through disulfide-bond formation with a C-terminal cysteamine.
Postcyclisation modification can be achieved by using small bifunctional amines.
Alternatively, the synthesis of lipopeptides is demonstrated through direct thiolactone opening with long-chain alkyl amines.
98. An anticaffeine antibody-oligonucleotide conjugate for DNA-directed immobilization in environmental immunoarrays
Ana Margarida Carvalho, Cinthya Veliz Montes, Rudolf J. Schneider and Annemieke Madder (UGent)
The development of fast and cheap high-throughput platforms for the detection of environmental contaminants is of particular importance to understand the human-related impact on the environment. The application of DNA-directed immobilization (DDI) of IgG molecules is currently limited to the clinical diagnostics scenario, possibly because of the high costs of production of such addressable platforms. We here describe the efficient and specific hybridization of an antibody-oligonucleotide conjugate to a short 12-mer capture probe. The specific antibody used is a monoclonal antibody against caffeine, a stimulant and important anthropogenic marker. With this work, we hope to contribute to broadening the application potential of DDI to environmental markers in order to develop cheaper and more stable high-throughput screening platforms for standard routine analysis of pollutants in a variety of complex matrices.
97. Porphyrin-based photosensitizers and their DNA conjugates for singlet oxygen induced nucleic acid interstrand crosslinking
Eva Marina Llamas Garcinuño (UGent) , João Tomé (UGent) , João MM Rodrigues, Tomas Torres and Annemieke Madder (UGent)
The development of methods for the generation of site-selective interstrand crosslinks (ICLs) in synthetic oligonucleotides provides a platform for the study of ICL repair mechanisms and the stabilisation of DNA-based materials. Our group has previously reported on the use of a furan moiety as a masked reactive functionality for ICL generation and recently introduced the use of O-1(2) as an efficient light-induced oxidant. Here, the use of porphyrin-based photosensitizers (PSs) has been explored for ICL generation. The efficiency of the ICL reaction has been investigated using PSs added into solution as well as attached to oligonucleotide probes. Interestingly, even a highly hydrophobic phthalocyanine was able to produce ICLs. Either in solution or conjugated to an ON, chlorin e6 was the most efficient ICL generator for the current purpose.
96. Cross-linking furan-modified kisspeptin-10 to the KISS receptor
Willem Vannecke (UGent) , Christophe Ampe (UGent) , Marleen Van Troys (UGent) , Massimiliano Beltramo and Annemieke Madder (UGent)
Chemical cross-linking is well-established for investigating protein-protein interactions. Traditionally, photo cross-linking is used but is associated with problems of selectivity and UV toxicity in a biological context. We here describe, with live cells and under normal growth conditions, selective cross-linking of a furan-modified peptide ligand to its membrane-presented receptor with zero toxicity, high efficiency, and spatio-specificity. Furan-modified kisspeptin-10 is covalently coupled to its glycosylated membrane receptor, GPR54(KISS1R). This newly expands the applicability of furan-mediated cross-linking not only to proteinprotein cross-linking but also to cross-linking in situ. Moreover, in our earlier reports on nucleic acid interstrand cross-linking, furan activation required external triggers of oxidation (via addition of N-bromo succinimide or singlet oxygen). In contrast, we here show, for multiple cell lines, the spontaneous endogenous oxidation of the furan moiety with concurrent selective cross-link formation. We propose that reactive oxygen species produced by NADPH oxidase (NOX) enzymes form the cellular source establishing furan oxidation.
95. Photoinduced cross-linking of short furan-modified DNA on surfaces
Cinthya Véliz Montes, Henry Memczak, Ellen Gyssels (UGent) , Tomás Torres, Annemieke Madder (UGent) and Rudolf J Schneider
We report for the first time the formation of site specific interstrand cross p-linked (ICL) surface -immobilized furanmodified DNA duplexes-via singlet oxygen. 102, necessary for effecting furan-mediated ICL formation, was produced in situ using methylene blue or a zinc phthalocyanine derivative (TT1) as a photosensitizer. Via surface plasmon resonance spectroscopy, we show that surface ICL was achieved, and a robust link formed that enhances the stability of the 12-mer duplex even after surface regeneration. The described method represents a novel platform technology based on surfaces with addressable and stable DNA duplexes requiring only short oligonudeotides.
94. Synthesis of N-methylated pseudodesmin A analogues : on the structural importance of N-H hydrogen bonds
Matthias De Vleeschouwer (UGent) , Davy Sinnaeve (UGent) , Nele Matthijs (UGent) , Tom Coenye (UGent) , Annemieke Madder (UGent) and José Martins (UGent)
The structural importance of backbone amide protons in the bacterial cyclic lipopeptide pseudodesmin A, a viscosin group member, is studied by selective introduction of N-methylated amino acids. Site-selective N-methylation was conveniently achieved by introduction of N-Me-L-Leu at positions 1 and 7 during solid phase peptide synthesis. The impact of this modification on conformation, antibiotic activity and supramolecular behaviour allowed to establish the significance of intra- and intermolecular hydrogen bonds.
93. Defining the molecular structure of teixobactin analogues and understanding their role in antibacterial activities
Anish Parmar, Stephen H Prior, Abhishek Iyer (UGent) , Charlotte S Vincent, Dorien Van Lysebetten, Eefjan Breukink, Annemieke Madder (UGent) , Edward J Taylor and Ishwar Singh
The discovery of the highly potent antibiotic teixobactin, which kills the bacteria without any detectable resistance, has stimulated interest in its structure-activity relationship. However, a molecular structure-activity relationship has not been established so far for teixobactin. Moreover, the importance of the individual amino acids in terms of their (L/D) configuration and their contribution to the molecular structure and biological activity are still unknown. For the first time, we have defined the molecular structure of seven teixobactin analogues through the variation of the (D/L) configuration of its key residues, namely N-Me-(D)-Phe, (D)-Gln, (D)-allo-Ile and (D)-Thr. Furthermore, we have established the role of the individual (D) amino acids and correlated this with the molecular structure and biological activity. Through extensive NMR and structural calculations, including molecular dynamics simulations, we have revealed the residues for maintaining a reasonably unstructured teixobactin which is imperative for biological activity.
92. Injectable peptide-based hydrogel formulations for the extended in vivo release of opioids
Charlotte Martin, Edith Oyen (UGent) , Yannick Van Wanseele, Tanila Ben Haddou, Helmut Schmidhammer, Jessica Andrade, Lynne Waddington, Ann Van Eeckhaut, Bruno Van Mele, James Gardiner, et al.
To overcome drawbacks related to repeated opioid administration during the treatment of chronic pain, several controlled-drug delivery systems of opioids have been designed. In order to address some of the limitations of the existing systems, injectable peptide-based hydrogels represent a promising alternative. This work reports on the design and synthesis of short amphipathic peptide-based hydrogels as controlled-drug delivery systems for opioids. Based on the lead sequence H-FEFQFK-NH2, a new set of peptide hydrogelators was designed including β-homo and d-amino acids, mainly aiming at enhancing proteolytic resistance of the peptides, and which hypothetically allows an extension of the drug release period. After self-assembly in aqueous media, the resulting hydrogels were characterized by dynamic rheometry, cryogenic transmission electronic microscopy and their cytotoxicity was assessed. The cryoTEM images of drug loaded hydrogels show the association of microcrystals of the loaded drug along the axes of the fibres, suggesting that the peptide fibres play a key-role as nucleating site for the drug crystals. Hydrogelators devoid of cytotoxicity were considered for further in vivo evaluation. Upon encapsulation of morphine and 14-methoxymetopon, two opioid analgesics, the applicability of the peptide hydrogels as controlled-drug delivery platforms was validated in vivo using the mouse tail-flick test. A sustained antinociceptive effect was observed after subcutaneous injection of the drug loaded gels and, in comparison with the lead sequence H-FEFQFK-NH2, novel sequences revealed extension of the in vivo antinociception up to 72–96 h post injection.
91. Specific dsDNA recognition by a mimic of the DNA binding domain of the c-Myc/Max transcription factor
Yara Ruiz Garcia (UGent) , Y Vladimir Pabon-Martinez, CI Edvard Smith and Annemieke Madder (UGent)
We here report on the synthesis of the first mimic of the DNA binding domain of the c-Myc/Max-bHLH-ZIP transcription factor able to selectively recognize its cognate E-box sequence 50'-CACGTG-3' through the major groove of the double-stranded DNA. The designed peptidosteroid conjugate was shown to be effective as DNA binder in the presence of excess competitor DNA.
We here describe a furan oxidation based site-specific chemical ligation approach using unprotected peptide segments. This approach involves two steps: after photooxidation of a furan-containing peptide, ligation is achieved by reaction of the unmasked keto-enal with C- or N-terminal alpha-nucleophilic moieties of the second peptide such as hydrazine or hydrazide to form a pyridazinium or pyrrolidinone linkage respectively.
89. Syntheses of potent teixobactin analogues against methicillin-resistant Staphylococcus aureus (MRSA) through the replacement of L-allo-enduracididine with its isosteres
Anish Parmar, Abhishek Iyer (UGent) , Daniel G Lloyd, Charlotte S Vincent, Stephen H Prior, Annemieke Madder (UGent) , Edward J Taylor and Ishwar Singh
The recently discovered cyclic depsipeptide, teixobactin, is a highly potent antibiotic against multi-drugresistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis. It comprises of 4 D amino acids and a rare L-allo-enduracididine amino acid. The synthesis of a properly protected L-allo-enduracididine amino acid and its incorporation into teixobactin is time consuming, synthetically challenging and low yielding and is therefore a major bottleneck in the development of potent analogues of teixobactin. In this article, we have synthesised 8 analogues of teixobactin using commercially available building blocks by replacing the L-allo-enduracididine amino acid with its isosteres. Furthermore, we have tested all the compounds against a panel of Gram positive bacteria including MRSA and explained the observed trend in biological activity. Although all the analogues were active, three analogues from this work, showed very promising activity against MRSA (MIC 1 mu g mL(-1)). We can conclude that amino acids which are the closest isosteres of L-allo-enduracididine are the key to synthesising simplified potent analogues of teixobactin using rapid syntheses and improved yields.
88. Synthesis and improved cross-linking properties of C5-modified furan bearing PNAs
Joke Elskens (UGent) , Alex Manicardi (UGent) , Valentina Costi, Annemieke Madder (UGent) and Roberto Corradini
Over the past decades, peptide nucleic acid/DNA (PNA:DNA) duplex stability has been improved via backbone modification, often achieved via introducing an amino acid side chain at the - or -position in the PNA sequence. It was previously shown that interstrand cross-linking can further enhance the binding event. In this work, we combined both strategies to fine-tune PNA crosslinking towards single stranded DNA sequences using a furan oxidation-based crosslinking method; for this purpose, -l-lysine and -l-arginine furan-PNA monomers were synthesized and incorporated in PNA sequences via solid phase synthesis. It was shown that the l-lysine -modification had a beneficial effect on crosslink efficiency due to pre-organization of the PNA helix and a favorable electrostatic interaction between the positively-charged lysine and the negatively-charged DNA backbone. Moreover, the crosslink yield could be optimized by carefully choosing the type of furan PNA monomer. This work is the first to describe a selective and biocompatible furan crosslinking strategy for crosslinking of -modified PNA sequences towards single-stranded DNA.
87. Visible-light-mediated selective arylation of cysteine in batch and flow
Cecilia Bottecchia, Maarten Rubens, Smita B Gunnoo, Volker Hessel, Annemieke Madder (UGent) and Timothy Noël (UGent)
A mild visible-light-mediated strategy for cysteine arylation is presented. The method relies on the use of eosin Y as a metal-free photocatalyst and aryldiazonium salts as arylating agents. The reaction can be significantly accelerated in a microflow reactor, whilst allowing the in-situ formation of the required diazonium salts. The batch and flow protocol described herein can be applied to obtain a broad series of arylated cysteine derivatives and arylated cysteine-containing dipeptides. Moreover, the method was applied to the chemoselective arylation of a model peptide in biocompatible reaction conditions (room temperature, phosphate-buffered saline (PBS) buffer) within a short reaction time.
86. Decoration of trastuzumab with short oligonucleotides : synthesis and detailed characterization
Ana Margarida Dias Carvalho Da Rocha, Alex Manicardi (UGent) , C Véliz Montes, Smita Gunnoo (UGent) , RJ Schneider and Annemieke Madder (UGent)
Trastuzumab (Herceptin (R)) is an FDA-approved therapeutic antibody currently employed in the treatment of metastatic stages of breast cancer. Herein, we propose a simple, fast and cost-effective methodology to conjugate trastuzumab with 22-mer 5' thiol-modified oligonucleotides using a bifunctional crosslinker. The conjugates were successfully characterized by MALDI-ToF MS and SDS-PAGE, obviating the need for enzymatic digestion and difficult chromatographic separations. Furthermore, ELISA was performed to ensure that trastuzumab activity is not affected by oligonucleotide conjugation.
85. Teixobactin analogues reveal enduracididine to be non-essential for highly potent antibacterial activity and lipid II binding
Anish Parmar, Abhishek Iyer (UGent) , Stephen H Prior, Daniel G Lloyd, Eunice Tze Leng Goh, Charlotte S Vincent, Timea Palmai-Pallag, Csanad Z Bachrati, Eefjan Breukink, Annemieke Madder (UGent) , et al.
Teixobactin is a highly promising antibacterial depsipeptide consisting of four D-amino acids and a rare L-allo-enduracididine amino acid. L-allo-Enduracididine is reported to be important for the highly potent antibacterial activity of teixobactin. However, it is also a key limiting factor in the development of potent teixobactin analogues due to several synthetic challenges such as it is not commercially available, requires a multistep synthesis, long and repetitive couplings (16-30 hours). Due to all these challenges, the total synthesis of teixobactin is laborious and low yielding (3.3%). In this work, we have identified a unique design and developed a rapid synthesis (10 min mu wave assisted coupling per amino acid, 30 min cyclisation) of several highly potent analogues of teixobactin with yields of 10-24% by replacing the L-allo-enduracididine with commercially available non-polar residues such as leucine and isoleucine. Most importantly, the Leu(10)-teixobactin and Ile(10)-teixobactin analogues have shown highly potent antibacterial activity against a broader panel of MRSA and Enterococcus faecalis (VRE). Furthermore, these synthetic analogues displayed identical antibacterial activity to natural teixobactin (MIC 0.25 mu g mL(-1)) against MRSA ATCC 33591 despite their simpler design and ease of synthesis. We have confirmed lipid II binding and measured the binding affinities of individual amino acid residues of Ala(10)-teixobactin towards geranyl pyrophosphate by NMR to understand the nature and strength of binding interactions. Contrary to current understanding, we have shown that a cationic amino acid at position 10 is not essential for target (lipid II) binding and potent antibacterial activity of teixobactin. We thus provide strong evidence contrary to the many assumptions made about the mechanism of action of this exciting new antibiotic. Introduction of a non-cationic residue at position 10 allows for tremendous diversification in the design and synthesis of highly potent teixobactin analogues and lays the foundations for the development of teixobactin analogues as new drug-like molecules to target MRSA and Mycobacterium tuberculosis.
84. In vivo imaging of the stability and sustained cargo release of an injectable amphipathic peptide-based hydrogel
Edith Oyen (UGent) , Charlotte Martin, Vicky Caveliers, Annemieke Madder (UGent) , Bruno Van Mele, Richard Hoogenboom (UGent) , Sophie Hemot and Steven Ballet
Hydrogels are promising materials for biomedical applications such as tissue engineering and controlled drug release. In the past two decades, the peptide hydrogel subclass has attracted an increasing level of interest from the scientific community because of its numerous advantages, such as biocompatibility, biodegradability, and, most importantly, injectability. Here, we report on a hydrogel consisting of the amphipathic hexapeptide H-FEFQFK-NH2, which has previously shown promising in vivo properties in terms of releasing morphine. In this study, the release of a small molecule, a peptide, and a protein cargo as representatives of the three major drug classes is directly visualized by in vivo fluorescence and nuclear imaging. In addition, the in vivo stability of the peptide hydrogel system is investigated through the use of a radiolabeled hydrogelator sequence. Although it is shown that the hydrogel remains present for several days, the largest decrease in volume takes place within the first 12 h of subcutaneous injection, which is also the time frame wherein the cargos are released. Compared to the situation in which the cargos are injected in solution, a prolonged release profile is observed up to 12 h, showing the potential of our hydrogel system as a scaffold for controlled drug delivery. Importantly, this study elucidates the release mechanism of the peptide hydrogel system that seems to be based on erosion of the hydrogel providing a generally applicable controlled release platform for small molecule, peptide, and protein drugs.
83. Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions
Lieselot LG Carrette, Ellen Gyssels (UGent) , Nathalie De Laet (UGent) and Annemieke Madder (UGent)
Furan mediated nucleic acid cross-linking, initially developed for DNA interstrand duplex cross-linking, has matured into a versatile tool for the study of protein and nucleic acid interactions, ready to face its applications. The methodology was initially developed for easy and clean chemical generation of DNA interstrand cross-linked duplexes, but has been further expanded for use with other probes, targets and triggers, now allowing mild biologically significant cross-linking with potential therapeutic benefit. It was shown that the methodology could be repurposed for RNA interstrand cross-linking, which is very relevant in today's antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation method's generality and mildness, especially when using red light for oxidation. A complementary antigene approach has been explored through duplex targeting with furan modified triplex forming oligonucleotides (TFOs) and DNA binding proteins. Also targeting of peptides and proteins by furan-modified DNA and peptides has been explored. Thorough methodology examination exploring variable reaction conditions in combination with a series of different furan-modified building blocks and application of different activation signals resulted in a detailed understanding of the mechanisms involved and factors influencing the yield and selectivity of the reaction. In order to draw the bigger picture of the scope and limitations of furan-oxidation cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes.
82. Efficient total syntheses and biological activities of two teixobactin analogues
Anish Parmar, Abhishek Iyer (UGent) , Charlotte S Vincent, Dorien Van Lysebetten, Stephen H Prior, Annemieke Madder (UGent) , Edward J Taylor and Ishwar Singh
The discovery of the new antibiotic teixobactin has been timely in the race for unearthing novel antibiotics wherein the emergence of drug resistant bacteria poses a serious threat worldwide. Herein, we present the total syntheses and biological activities of two teixobactin analogues. This approach is simple, efficient and has several advantages: it uses commercially available building blocks (except AllocHN-D-Thr-OH), has a single purification step and a good recovery (22%). By using this approach we have synthesised two teixobactin analogues and established that the D-amino acids are critical for the antimicrobial activity of these analogues. With continuing high expectations from teixobactin, this work can be regarded as a stepping stone towards an in depth study of teixobactin, its analogues and the quest for synthesising similar molecules.
81. Synthesis and evaluation of methylene blue oligonucleotide conjugates for DNA interstrand cross-linking
Nathalie De Laet (UGent) and Annemieke Madder (UGent)
Efficient DNA interstrand cross-linking can be achieved with furan containing oligonucleotide probes upon activation by singlet oxygen (1O2). Previously, we have described how this can be achieved by irradiation of these furan probes with visible light in the presence of photosensitizers. Now, in an effort to explore cross-linking under conditions that are representative for experiments in cellular context, the furan mediated oligonucleotide cross-linking was investigated at low oligonucleotide concentrations, ensuring a sufficiently high local concentration of singlet oxygen by attaching the sensitizing methylene blue moiety to the oligonucleotide complementary to the furan modified strand. Four methylene blue-oligonucleotide conjugates were synthesized, each with a different positioning of methylene blue with respect to the furan unit present on the complementary strand. The conjugates were evaluated for singlet oxygen generation and subsequent cross-linking ability. It was observed that not only the distance of the 1O2 source to the furan unit, but also the specific interaction of methylene blue moiety with the duplex, which is position dependent, influences cross-linking yields.
79. Controlled-release of opioids for improved pain management
Charlotte Martin, Andy De Baerdemaeker, Jan Poelaert, Annemieke Madder (UGent) , Richard Hoogenboom (UGent) and Steven Ballet
The adequate treatment of pain remains one of the major medical challenges. Morphine and other opioid drugs are most commonly used to counteract moderate to severe pain, but they are also increasingly accessed by patients with chronic non-malignant pain. To achieve long-term analgesia, opioid therapy still represents the standard treatment for chronic pain alleviation. This work presents an overview of current strategies aiming at controlled opioid release. Two important, and intrinsically linked, features are discussed in detail: the used formulations (i.e. polymer systems) and the applied drug administration routes. The different administration routes and their associated advantages and limitations are described. Links between the chemical structure of commonly used opioids and suited administration modes and formulations are made. This review can potentially give insight into new opportunities for adequate relief of chronic pain, a societal burden, by means of alternative (non)opioid analgesics and may serve as inspiration for future developments in this area.
78. Singlet oxygen-induced furan oxidation for site-specific and chemoselective peptide ligation
Eirini Antonatou (UGent) , Kurt Hoogewijs, Dimitris Kalaitzakis, Andreas Baudot, Georgios Vassilikogiannakis and Annemieke Madder (UGent)
A novel chemoselective ligation methodology has been developed for the facile construction of peptide-based fluorescent probes. Furan-containing peptides were activated by singlet oxygen and covalently engaged by nitrogen nucleophiles to yield stable conjugates. Singlet oxygen was compatible with sensitive amino acid residues within the peptides and a range of fluorophores, bearing different functionalities, were successfully incorporated, illustrating the broad scope of the developed strategy.
77. Injectable peptide hydrogels for controlled-release of opioids
Charlotte Martin, Edith Oyen, Jeroen Mangelschots, Mathieu Bibian, Tanila Ben Haddou, Jessica Andrade, James Gardiner, Bruno Van Mele, Annemieke Madder (UGent) , Richard Hoogenboom (UGent) , et al.
Herein, a family of hydrogel-forming peptides was designed starting from the short, tunable and amphipathic hexapeptide hydrogelator H-Phe-Glu-Phe-Gln-Phe-Lys-OH (1). The hydrophobic side chains as well as the nature of both N- and C-termini were modified in order to obtain suitable gelation conditions and drug release profiles for in vivo application. To potentially increase the enzymatic stability, an all-D analogue was prepared as well. After their macroscopic and microscopic characterization by rheology and transmission electron microscopy (TEM) analysis, opioid drugs were encapsulated into the hydrogels and sustained release experiments were carried out. Hydrogel toxicity was assessed in cell viability assays. Based on the physicochemical, mechanical, and noncytotoxic properties, H-Phe-Glu-Phe-Gln-Phe-Lys-NH2 (2) was further investigated for in vivo release of morphine. The antinociceptive effects following subcutaneous injection of the morphine-containing hydrogel 2 was evaluated in a model of thermal nociception using the mouse tail-flick test. Sustained antinociceptive effects over extended periods of time (up to 24 h) for morphine co-formulated with hydrogel 2, compared to morphine injection in solution (effects up to 2 h), were observed.
76. Chemical protein modification through cysteine
Smita B Gunnoo and Annemieke Madder (UGent)
The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.
75. Danger-associated peptide signaling in Arabidopsis requires clathrin
Fausto Andres Ortiz Morea (UGent) , Daniel Savatin (UGent) , Wim Dejonghe (UGent) , Rahul Kumar (UGent) , Yu Luo (UGent) , Maciej Adamowski, Jos Van den Begin (UGent) , Keini Dressano, Guilherme Oliveira (UGent) , Xiuyang Zhao, et al.
The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependentmanner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H+-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.
74. Automated synthesis of monodisperse oligomers, featuring sequence control and tailored functionalization
Steven Martens (UGent) , Jos Van den Begin (UGent) , Annemieke Madder (UGent) , Filip Du Prez (UGent) and Pieter Espeel (UGent)
Long, multifunctional sequence-defined oligomers were obtairred on solid support from a protecting-group-free two-step iterative protocol, based on the inherent reactivity of a readily available molecule containing an isocyanate and a thiolactone. Aminolysis of the latter entity with an amino alcohol liberates a thiol that reacts with an acrylate or acrylamide, present in the same medium. Subsequently, a new thiolactone can be reinstated by means of an alpha-isocyanato-gamma-thiolactone. Different acrylic compounds were used to incorporate diverse functionalities in the oligomers, which were built up to the level of decanters. The reaction conditions were closely monitored in order to fine-tune the applied strategy as well as facilitate the translation to an automated protocol.
73. Development and validation of ultra-high-performance liquid chromatography-tandem mass spectrometry methods for the simultaneous determination of beauvericin, enniatins (A, A1, B, B1) and cereulide in maize, wheat, pasta and rice
Marlies Decleer (UGent) , Andreja Rajkovic (UGent) , Benedikt Sas (UGent) , Annemieke Madder (UGent) and Sarah De Saeger (UGent)
Rapid and accurate UPLC–MS/MS methods for the simultaneous determination of beauvericin and the related enniatins (A, A1, B, B1), together with cereulide were successfully developed and validated in cereal and cereal-based food matrices such as wheat, maize, rice and pasta. Although these emerging foodborne toxins are of different microbial origin, the similar structural, toxicological and food safety features provided rationale for their concurrent detection in relevant food matrices. A Waters Acquity UPLC system coupled to a Waters Quattro Premier XE™ Mass Spectrometer operating in ESI+ mode was employed. Sample pretreatment involved a fast and simple liquid extraction of the target toxins without any further clean-up step. For all toxins the sample preparation resulted in acceptable extraction recoveries with values of 85–105% for wheat, 87–106% for maize, 84–106% for rice and 85–105% for pasta. The efficient extraction protocol, together with a fast chromatographic separation of 7 min allowed substantial saving costs and time showing its robustness and performance. The validation of the developed method was performed based on Commission Decision 2002/657/EC. The obtained limits of detection ranged from 0.1 to 1.0 μg kg−1 and the limits of quantification from 0.3 to 2.9 μg kg−1 for the targeted toxins in the selected matrices. The obtained sensitivities allow detection of relevant toxicological concentrations. All relative standard deviations for repeatability (intra-day) and intermediate precision (inter-day) were lower than 20%. Trueness, expressed as the apparent recovery varied from 80 to 107%. The highly sensitive and repeatable validated method was applied to 57 naturally contaminated samples allowing detection of sub-clinical doses of the toxins.
72. Bioconjugation of quantum dots : review & impact on future application
Astrid Foubert, Natalia Beloglazova (UGent) , Andreja Rajkovic (UGent) , Benedikt Sas (UGent) , Annemieke Madder (UGent) , Irina Yu Goryacheva and Sarah De Saeger (UGent)
Nowadays luminescent semiconductor quantum dots (QDs) are widely applied in different areas due to their unique optical properties. QDs can be used as photoluminescent labels with excellent possibilities for high-throughput detection and diagnostics. For most of such applications QDs must be coupled to biomolecules, which often represents a fundamental challenge. Although QDs have a lot of advantages over organic dyes, most of the techniques that have been developed for QD functionalization and bioconjugation, are more complicated than the corresponding techniques for organic fluorescent dyes. Here, the importance of choosing a suitable bioconjugation strategy in different applications, such as imaging and assays is described. The main goal of this review is to give a structured and detailed overview and comparison of the most widely used conjugation strategies in function of the active groups (carboxyl, amine, thiol, epoxy, hydroxyl and aldehyde groups) present on QD surface.
71. Mixed α/β-peptides as a class of short amphipathic peptide hydrogelators with enhanced proteolytic stability
Jeroen Mangelschots, Mathieu Bibian, James Gardiner, Lynne Waddington, Yannick Van Wanseele, Ann Van Eeckhaut, Maria M Diaz Acevedo, Bruno Van Mele, Annemieke Madder (UGent) , Richard Hoogenboom (UGent) , et al.
Peptide hydrogels are a highly promising class of materials for biomedical application, albeit facing many challenges with regard to stability and tunability. Here, we report a new class of amphipathic peptide hydrogelators, namely mixed alpha/beta-peptide hydrogelators. These mixed alpha/beta-gelators possess good rheological properties (high storage moduli) and form transparent self-supporting gels with shear-thinning behavior. Infrared spectroscopy indicates the presence of beta-sheets as the underlying secondary structure. Interestingly, self-assembled nanofibers of the mixed alpha/beta-peptides display unique structural morphologies with alteration of the C-terminus (acid vs amide) playing a key role in the fiber formation and gelation properties of the resulting hydrogels. The incorporation of beta 3-homoamino acid residues within the mixed alpha/beta-peptide gelators led to an increase in proteolytic stability of the peptides under nongelating conditions (in solution) as well as gelating conditions (as hydrogel). Under diluted conditions, degradation of mixed alpha/beta-peptides in the presence of elastase was slowed down 120-fold compared to that of an alpha-peptide, thereby demonstrating beneficial enzymatic resistance for hydrogel applications in vivo. In addition, increased half-life values were obtained for the mixed alpha/beta-peptides in human blood plasma, as compared to corresponding alpha-peptides. It was also found that the mixed alpha/beta-peptides were amenable to injection via needles used for subcutaneous administrations. The preformed peptide gels could be sheared upon injection and were found to quickly reform to a state close to that of the original hydrogel. The shown properties of enhanced proteolytic stability and injectability hold great promise for the use of these novel mixed alpha/beta-peptide hydrogels for applications in the areas of tissue engineering and drug delivery.
70. Bioconjugation: using selective chemistry to enhance the properties of proteins and peptides as therapeutics and carriers
Smita Gunnoo (UGent) and Annemieke Madder (UGent)
The pharmaceutical market has largely been dominated by small molecule drugs; however, larger biomolecules have recently become important contenders. Of these biomolecules, protein and peptide therapeutics are proving useful due to their often improved pharmacokinetic properties. In many circumstances, functionalisation of the protein or peptide therapeutics results in performance enhancement, and various methodologies are applied. In addition, introducing unnatural amino acids for structural reinforcement via chemical modification is also common. These strategies are discussed in this review.
69. The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation
Sarieh Ghorbani (UGent) , Kurt Hoogewijs (UGent) , Tamara Pecenkova, Ana Fernandez Salina (UGent) , Annelies Inzé (UGent) , Dominique Eeckhout (UGent) , Dorota Kawa (UGent) , Geert De Jaeger (UGent) , Tom Beeckman (UGent) , Annemieke Madder (UGent) , et al.
Maturation of GLV signaling peptides requires two SBT6 subtilases. SBT6 proteolytic activity is further regulated by the Serpin1 inhibitor, implying a complex network that controls cell elongation in Arabidopsis.The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo. Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor.
68. Furan-PNA : a mildly inducible irreversible interstrand crosslinking system targeting single and double stranded DNA
Alex Manicardi (UGent) , Ellen Gyssels (UGent) , Roberto Corradini and Annemieke Madder (UGent)
We here report on the design and synthesis of tailor-made furan-modified peptide nucleic acid (PNA) probes for covalent targeting of single stranded DNA through a crosslinking strategy. After introducing furan-containing building blocks into a PNA sequence, hybridization and furan-oxidation based crosslinking to DNA is investigated. The structure of the crosslinked products is characterized and preliminary investigations concerning the application of these systems to double stranded DNA are shown.
67. Stapling monomeric GCN4 peptides allows for DNA binding and enhanced cellular uptake
Abhishek Iyer (UGent) , Dorien Van Lysebetten, Yara Ruiz Garcia (UGent) , Benoit Louage (UGent) , Bruno De Geest (UGent) and Annemieke Madder (UGent)
The basic DNA recognition region of the GCN4 protein comprising 23 amino acids has been modified to contain two optimally positioned cysteines which have been linked and stapled using crosslinkers of suitable lengths. This results in stapled peptides with a stabilized alpha-helical conformation which allows for DNA binding and concurrent enhancement of cellular uptake.
66. Triplex crosslinking through furan oxidation requires perturbation of the structured triple-helix
Ellen Gyssels (UGent) , Lieselot LG Carrette, Emma Vercruysse, Kristof Stevens and Annemieke Madder (UGent)
Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan-based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan-oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan-containing probes for the selective detection of single-stranded regions within nucleic acids containing a variety of structural motifs.
65. Rational design of a hexapeptide hydrogelator for controlled-release drug delivery
Mathieu Bibian, Jeroen Mangelschots, James Gardiner, Lynne Waddington, Maria M Diaz Acevedo, Bruno De Geest (UGent) , Bruno Van Mele, Annemieke Madder (UGent) , Richard Hoogenboom (UGent) and Sam Ballet
The amphiphilic peptide sequence H-Phe-Glu-Phe-Gln-Phe-Lys-OH (MBG-1) is developed as a novel hydrogelator for use in controlled-drug release administration, which is the smallest tunable ionic self-complementary hydrogelating peptide reported to date making it attractive for larger scale preparation. Hydrogelation is demonstrated to result from self-assembly of the peptide into beta-sheet nanofibers that are physically cross-linked by intertwining as well as larger bundle formation. Finally, the release of two small molecule cargos, fluorescein sodium and ciprofloxacin hydrochloride, is demonstrated revealing a two-stage zero-order sustained release profile up to 80% cumulative release over eight days.
64. Identification of a pKa-regulating motif stabilizing imidazole-modified double-stranded DNA
Dieter Buyst (UGent) , Vicky Gheerardijn (UGent) , Krisztina Fehér (UGent) , Björn Van Gasse (UGent) , Jos Van den Begin (UGent) , José Martins (UGent) and Annemieke Madder (UGent)
The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKa(H) are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6 degrees C and is key in modulating the pKa(H) of the imidazolium group. The general features, prerequisites and generic character of the new pKa(H)-regulating motif are described.
63. The GLV6/RGF8/CLEL2 peptide regulates early pericycle divisions during lateral root initiation
Ana Fernandez Salina (UGent) , Andrzej Drozdzecki (UGent) , Kurt Hoogewijs (UGent) , Valya Vassileva, Annemieke Madder (UGent) , Tom Beeckman (UGent) and Pierre Hilson (UGent)
Small peptides of the Arabidopsis GLV/RGF/CLEL family are involved in different developmental programmes, including meristem maintenance and gravitropic responses. In addition, our previous report suggested that they also participate in the formation of lateral roots. Specifically, GLV6 is transcribed during the first stages of primordium development and GLV6 overexpression results in a strong reduction of emerged lateral roots. To investigate the cause of this phenotype we analysed primordium development in gain-of-function (gof) mutants and found that GLV6 induces supernumerary pericycle divisions, hindering the formation of a dome-shaped primordium, a prerequisite for successful emergence. The GLV6 phenotype could be reproduced by ectopic expression of the gene only in xylem-pole pericycle cells. Furthermore, GLV6 seems to function at the very beginning of lateral root initiation because GLV6 excess-either gene overexpression or peptide treatment-disrupts the first asymmetric cell divisions required for proper primordium formation. Our results suggest that GLV6 acts during lateral root initiation controlling the patterning of the first pericycle divisions.
62. Hyphenation of a deoxyribonuclease I immobilized enzyme reactor with liquid chromatography for the online stability evaluation of oligonucleotides
Piotr Wiktor Alvarez Porebski (UGent) , Ellen Gyssels (UGent) , Annemieke Madder (UGent) and Frederic Lynen (UGent)
The stability of antisense oligonucleotides (ONs) toward nucleases is a key aspect for their possible implementation as therapeutic agents. Typically, ON stability studies are performed off-line, where the ONs are incubated with nucleases in solution, followed by their analysis. The problematics of off-line processing render the detailed comparison of relative ON stability quite challenging. Therefore, the development of an online platform based on an immobilized enzyme reactor (IMER) coupled to liquid chromatography (LC) was developed as an alternative for improved ON stability testing. More in detail, Deoxyribonuclease I (DNase I) was immobilized on epoxy-silica particles of different pore sizes and packed into a column for the construction of an IMER. Subsequently, the hyphenation of the IMER with ion-pair chromatography (IPC) and ion-exchange chromatography (IEC) was evaluated, leading to the successful development of two online methodologies: IMER-IPC and IMER-IEC. More specifically, natural and modified DNA and RNA oligonucleotides were used for testing the performance of the methodologies. Both methodologies proved to be simple, automatable, fast and highly reproducible for the quantitative and qualitative evaluation of ON degradation. In addition, the extended IMER life time in combination with a more straightforward control of the reaction kinetics substantiate the applicability of the IMER-LC platform for ON stability tests and its implementation in routine and research laboratories.
61. Cyclodextrin-peptide conjugates for sequence specific DNA binding
Yara Ruiz Garcia (UGent) , Jan Zelenka, Y Vladimir Pabon, Abhishek Iyer (UGent) , Miloš Buděšínský, Tomáš Kraus, CI Edvard Smith and Annemieke Madder (UGent)
Synthetic models of bZIP transcription factors have been developed with the capability of specific DNA recognition. Our design is based on the CuAAC mediated conjugation of basic region Leucine Zipper peptides to different derivatives of alpha,beta and gamma-cyclodextrins equipped with azide functionalities. Thorough optimization of reaction conditions allowed convergent and simultaneous conjugation of two long unprotected cationic peptides to cyclodextrin-bis azide derivatives. The resulting constructs were shown to specifically recognize their cognate DNA sequence with nM affinities. In comparison with previously developed TF models, the derivatives described here combine the enhanced DNA binding capabilities with an easy and convergent synthetic route.
60. Sequence-selective DNA recognition and enhanced cellular up-take by peptide–steroid conjugates
Yara Ruiz Garcia (UGent) , Abhishek Iyer (UGent) , Dorien Van Lysebetten, Y Vladimir Pabon, Benoit Louage (UGent) , Malgorzata Honcharenko, Bruno De Geest (UGent) , CI Edvard Smith, Roger Strömberg and Annemieke Madder (UGent)
Several GCN4 bZIP TF models have previously been designed and synthesized. However, the synthetic routes towards these constructs are typically tedious and difficult. We here describe the substitution of the Leucine zipper domain of the protein by a deoxycholic acid derivative appending the two GCN4 binding region peptides through an optimized double azide–alkyne cycloaddition click reaction. In addition to achieving sequence specific dsDNA binding, we have investigated the potential of these compounds to enter cells. Confocal microscopy and flow cytometry show the beneficial influence of the steroid on cell uptake. This unique synthetic model of the bZIP TF thus combines sequence specific dsDNA binding properties with enhanced cell-uptake. Given the unique properties of deoxycholic acid and the convergent nature of the synthesis, we believe this work represents a key achievement in the field of TF mimicry.
59. Versatile synthesis of amino acid functionalized nucleosides via a domino carboxamidation reaction
Vicky Gheerardijn (UGent) , Jos Van den Begin (UGent) and Annemieke Madder (UGent)
Functionalized oligonucleotides have recently gained increased attention for incorporation in modified nucleic acid structures both for the design of aptamers with enhanced binding properties as well as the construction of catalytic DNA and RNA. As a shortcut alternative to the incorporation of multiple modified residues, each bearing one extra functional group, we present here a straightforward method for direct linking of functionalized amino acids to the nucleoside base, thus equipping the nucleoside with two extra functionalities at once. As a proof of principle, we have introduced three amino acids with functional groups frequently used as key-intermediates in DNA- and RNAzymes via an efficient and straightforward domino carboxamidation reaction.
58. Cyclic lipodepsipeptides produced by Pseudomonas spp. naturally present in raw milk induce inhibitory effects on microbiological inhibitor assays for antibiotic residue screening
Wim Reybroeck, Matthias De Vleeschouwer (UGent) , Sophie Marchand, Davy Sinnaeve (UGent) , Kim Heylen (UGent) , Jan De Block, Annemieke Madder (UGent) , José Martins (UGent) and Marc Heyndrickx (UGent)
Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7 degrees C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.
57. Rapid total synthesis of cyclic lipodepsipeptides as a premise to investigate their self-assembly and biological activity
Matthias De Vleeschouwer (UGent) , Davy Sinnaeve (UGent) , Jos Van den Begin (UGent) , Tom Coenye (UGent) , José Martins (UGent) and Annemieke Madder (UGent)
A rapid and efficient total synthesis is reported for the cyclic lipodepsipeptide pseudodesmin A. This member of the Pseudomonas viscosin group is active against Gram-positive bacteria and features self-assembling properties. A conserved serine residue within the lactone macrocycle is exploited for initial immobilization on 2-chlorotrityl chloride resin through ether formation with the side-chain alcohol. Subsequent elongation proceeds through Fmoc solid-phase peptide synthesis, including automated incorporation of the enantioselectively synthesized (R)-3-hydroxydecanoic acid lipid tail. Following esterification to generate the incipient lactone bond, the macrocycle is formed by on-resin head-to-tail macrolactamization and cleaved from the resin to give the desired compound in good purity. The short and efficient synthesis route allows rapid generation of analogues by facile variation of both the peptide and lipid moieties with good control of epimerization while maximizing automation. Synthesis of the pseudodesmin A enantiomer yields identical self-assembly and biological activity to that observed for the natural compound, showing that activity is not mediated by chiral interactions. A D-Asn8 analogue developed en route retains self-assembly, but loses activity. The synthesis strategy should be generally applicable for the rapid generation of analogues from various cyclic lipodepsipeptide groups, allowing an investigation of their self-assembling properties and structure-activity relationships.
56. Straightforward synthesis of cholic acid stabilized loop mimetics
An Clemmen (UGent) , Carlo Boutton, Peter Vanlandschoot, Angela Wittelsberger, Inge Borghmans, Astrid Coppens, Peter Casteels and Annemieke Madder (UGent)
We here report on a new straightforward strategy for the synthesis of cyclic cholic acid–peptide conjugates. A solid-phase synthesis method is presented in which a selected anti-lysozyme CDR3 fragment, Asp-Ser-Thr-Ile-Tyr-Ala-Ser-Tyr-Tyr-Glu-Ser, is immobilized onto a steroidal cholic acid derived scaffold in order to yield a loop-like structure. Therefore, part of the desired sequence, that is, Ser-Tyr-Tyr-Glu-Ser, is introduced, at the C12 position of the scaffold. Subsequently, the remainder of the envisaged sequence is introduced at C3 via a Cu-catalyzed cyclo-addition reaction. Finally, amide bond formation delivers the desired cyclic peptidosteroid. This new synthetic strategy offers an easy and short access to cyclic peptidosteroids via convergent peptide ligation and macrocyclization.
55. Peptidosteroid tweezers revisited: DNA binding through an optimised design
Lieselot Carrette (UGent) , Takashi Morii and Annemieke Madder (UGent)
A dipodal peptidosteroid based on a deoxycholic acid scaffold was designed as a transcription factor model to study, and ultimately interfere in, selective protein–DNA interactions. In this paper, the selective binding of such a dipodal peptidosteroid to its DNA binding site is described for the first time, thus proving the concept and confirming the potential of the design. To achieve this, two 25-mer peptides were synthesized in parallel and in close proximity on a rigid amphiphilic scaffold, attached by flexible linkers. Peptide aggregation complicated the synthesis under conventional conditions to such an extent that optimization and the use of microwave assistance were required for a successful synthesis. As such, this synthesis is a good model to showcase the beneficial effects of microwave assistance in peptide synthesis.
An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin>15-acetyldeoxynivalenol (15-ADON)>DON, with IC50 ranging from 1.14 to 2.13 mu gml(-1). Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC50 values of 22, 15 and 34ngml(-1), respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC50 of 0.38ngml(-1). Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.
53. Impact of a stereocentre inversion in cyclic lipodepsipeptides from the viscosin group: a comparative study of the viscosinamide and pseudodesmin conformation and self-assembly
Niels Geudens (UGent) , Matthias De Vleeschouwer (UGent) , Krisztina Fehér (UGent) , Hassan Rokni-Zadeh, Maarten GK Ghequire, Annemieke Madder (UGent) , René De Mot, José Martins (UGent) and Davy Sinnaeve (UGent)
The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by Pseudomonas bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both l- and d-amino acids. Remarkably, the Leu5 amino acid-centrally located in the nonapeptide sequence-is the sole residue found to possess either an l or d configuration, depending on the producing strain. The impact of this D/L switch on the solution conformation was investigated by NMR-restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the D/L switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self-assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from Pseudomonas fluorescens DR54 were identified, and an NMR assay is proposed to assess the presence of either an L- or D-Leu5.
Copper(I)-catalyzed 1,3-dipolar cycloaddition of azides and terminal alkynes (CuAAC), better known as “click” reaction, has triggered the use of 1,2,3-triazoles in bioconjugation, drug discovery, materials science and combinatorial chemistry. Here we report a new series of water-soluble catalysts based on N-heterocyclic carbene (NHC)-Cu complexes which are additionally functionalized with a sulfonate group. The complexes show superior activity towards CuAAC reactions and display a high versatility, enabling the production of triazoles with different substitution patterns. Additionally, successful application of these complexes in bioconjugation using unprotected peptides acting as DNA binding domains was achieved for the first time. Mechanistic insight into the reaction mechanism is obtained by means of state-of-the-art first principles calculations.
51. A synthetic oligonucleotide model for evaluating the oxidation and crosslinking propensities of natural furan-modified DNA
Lieselot Carrette (UGent) and Annemieke Madder (UGent)
We have previously developed a crosslinking methodology for oligonucleotides based on the incorporation of furan moieties, which can be selectively oxidised to reactive intermediates that will quickly react with the opposite bases in DNA, forming toxic interstrand crosslinks (ICLs). Furan moieties also occur in natural DNA, as a result of oxidative stress. Moreover, the furan-containing degradation product of this modified DNA—kinetin—has been found to display beneficial anti-ageing effects. To investigate the apparent discrepancy between the effects of the synthetic and the natural furan modifications in DNA, a quick and easy postsynthetic method providing access to the natural modification in short synthetic oligonucleotides was developed. On checking for potential crosslinking propensity, we found that the furan moiety does indeed undergo oxidation, in this way functioning as an important scavenger for oxidative stress. The reactive intermediate, however, was shown to degrade without producing toxic crosslinked products.
50. A mildly inducible and selective cross-link methodology for RNA duplexes
Lieselot Carrette (UGent) , Ellen Gyssels (UGent) , Joke Loncke and Annemieke Madder (UGent)
We here report on the furan oxidation methodology for interstrand cross-linking of RNA duplexes, which have a different structure and are more stiff, reactive and labile than their DNA counterparts. Through this mildly inducible approach, natural unmodified RNA can be selectively cross-linked in high yield. The method therefore has direct applications in the increasing number of RNA based technologies.
49. Multifunctionalized sequence-defined oligomers from a single building block
Pieter Espeel (UGent) , Lieselot Carrette (UGent) , Katarzyna Bury, Sven Capenberghs (UGent) , José Martins (UGent) , Filip Du Prez (UGent) and Annemieke Madder (UGent)
Another link in the chain: A thiolactone-based approach for the preparation of multifunctionalized sequence-defined oligomeric structures on solid support was established. The iterative aminolysis/chain extension method with a single building block and a variety of commercial amines allowed for the protecting group free synthesis of various oligomeric motifs with a unique backbone and a preprogrammed organization of side chain functionalities.
48. Patchwork protein chemistry: a practitioner's treatise on the advances in synthetic peptide stitchery
Dieter Verzele (UGent) and Annemieke Madder (UGent)
With the study of peptides and proteins at the heart of many scientific endeavors, the omics era heralded a multitude of opportunities for chemists and biologists alike. Across the interface with life sciences, peptide chemistry plays an indispensable role, and progress made over the past decades now allows proteins to be treated as molecular patchworks stitched together through synthetic tailoring. The continuous elaboration of sophisticated strategies notwithstanding, Merrifield's solid-phase methodology remains a cornerstone of chemical protein design. Although the non-practitioner might misjudge peptide synthesis as trivial, routine, or dull given its long history, we comment here on its many advances, obstacles, and prospects from a practitioner's point of view. While sharing our perspectives through thematic highlights across the literature, this treatise provides an interpretive overview as a guide to novices, and a recap for specialists.
47. Synthetic progress in cMyc-Max oncoprotein miniaturization: semi-online monitoring gives solid-phase access to hydrophobic b(-HLH-)ZIP peptidosteroid tweezers
Dieter Verzele (UGent) and Annemieke Madder (UGent)
Miniature versions of basic leucine zipper (bZIP) and basic helix-loop-helix zipper (b-HLH-ZIP) transcription factors are promising tools for molecular dissection of the genetic information in a post-genomic context. Despite the opportunities of genome interfering agents based on certain oncogenic zipper proteins, structural mimicry of transcription factors is a delicate undertaking, and experimental fine-tuning through bottom-up organic chemistry could benefit from solid-phase/library approaches. Involved in a variety of human pathologies, we became interested in the miniaturization of the cMyc-Max b-HLH-ZIP oncoprotein, and herein elaborate on our synthetic progress in that direction. A bile acid scaffold was successfully employed as artificial dimerization interface in this new type of transcription factor model. Orthogonality of the applied Alloc/Boc/Fmoc chemistries allowed the synthesis of both homo-and heterodimeric peptidosteroid conjugates, covalently restricted with defined geometrical properties. Recognition peptides were assembled through standard Fmoc/tBu solid-phase peptide synthesis (SPPS) chemistry, assisted by automated procedures for consecutive chain elongation on solid support. Invaluable to monitor present strategy, a photocleavable linker allowed rapid, yet detailed analysis of side chain protected peptide intermediates, liberated from the sampled resin, by reverse-phase HPLC and MALDI-TOF-MS. By decorating each scaffold position with two basic region peptides in a 2 x 2 design, a first generation of unprecedented b(-HLH-) ZIP peptidosteroids was efficiently obtained. As such, a versatile methodology amenable to library generation is presented.
46. Toxicity inspired cross-linking for probing DNA–peptide interactions
Lieselot Carrette (UGent) , Takashi Morii and Annemieke Madder (UGent)
A cross-linking methodology for the study of DNA–protein interactions is described. The method is inspired by the metabolic activation of furans causing toxic DNA damage, including DNA–protein cross-links (DPC). The furan moiety, representing a latent functionality, is easily incorporated into oligonucleotides, and can be activated on demand to release a reactive aldehyde. Reaction with nucleophilic lysine side chains is shown to be distance-sensitive and allows for site-selective DPC formation.
45. Accurate prediction of ¹H chemical shifts in interstrand cross-linked DNA
Ewald Pauwels (UGent) , Diederica Claeys (UGent) , José Martins (UGent) , Michel Waroquier (UGent) , Giuseppe Bifulco, Veronique Van Speybroeck (UGent) and Annemieke Madder (UGent)
Structural analysis of modified DNA with NMR is becoming ever more difficult with increasingly complex compounds under scrutiny for use in medical diagnosis, therapeutics, material science and chemical synthesis. To facilitate this process, we developed a molecular modeling approach to predict proton chemical shifts in sufficient agreement with experimental NMR measurements to guide structure elucidation. It relies on a QM/MM partitioning scheme and first principle calculations to predict the spatial structure and calculate corresponding proton chemical shifts. It is shown that molecular dynamics simulations that take into account solvent and temperature effects properly are of utmost importance to sample the conformational space sufficiently. The proposed computational procedure is applicable to modified oligonucleotides and DNA, attaining a mean error for the proton chemical shifts of less than 0.2 ppm. Here, it is applied on the Drew-Dickerson d(CGCGAATTCGCG)(2) dodecamer as a benchmark system and the mispair-aligned (NT)-T-3-ethyl-(NT)-T-3 cross-linked d(CGAAAT*TTTCG)(2) undecamer, illustrating its use as computational tool to assist in structure elucidation. For the proton chemical shifts in the cross-linked system our methodology yields a strikingly superior description, surpassing the predictive power of (semi-) empirical methods. In addition, our methodology is the only one available to make an accurate prediction for the protons in the actual cross-link. To the best of our knowledge, this is the first computational study that attempts to determine the chemical shifts of oligonucleotides of this size and at this level of complexity.
44. Transcriptional and functional classification of the GOLVEN/ROOT GROWTH FACTOR/CLE-like signaling peptides reveals their role in lateral root and hair formation
Ana Fernandez Salina (UGent) , Andrzej Drozdzecki (UGent) , Kurt Hoogewijs (UGent) , Anh Thu Nguyen, Tom Beeckman (UGent) , Annemieke Madder (UGent) and Pierre Hilson (UGent)
The GOLVEN (GLV)/ROOT GROWTH FACTORS/CLE-Like small signaling peptide family is encoded by 11 genes in Arabidopsis (Arabidopsis thaliana). Some of them have already been shown to control root meristem maintenance, auxin fluxes, and gravitropic responses. As a basis for the detailed analysis of their function, we determined the expression domains for each of the 11 GLV genes with promoter-reporter lines. Although they are collectively active in all examined plant parts, GLV genes have highly specific transcription patterns, generally restricted to very few cells or cell types in the root and shoot and in vegetative and reproductive tissues. GLV functions were further investigated with the comparative analysis of root phenotypes induced by gain-and loss-of-function mutants or in treatments with GLV-derived synthetic peptides. We identified functional classes that relate to the gene expression domains in the primary root and suggest that different GLV signals trigger distinct downstream pathways. Interestingly, GLV genes transcribed at the early stages of lateral root development strongly inhibited root branching when overexpressed. Furthermore, transcription patterns together with mutant phenotypes pointed to the involvement of GLV4 and GLV8 in root hair formation. Overall, our data suggest that nine GLV genes form three subgroups according to their expression and function within the root and offer a comprehensive framework to study the role of the GLV signaling peptides in plant development.
43. The antimicrobial compound xantholysin defines a new group of Pseudomonas cyclic lipopeptides
Wen Li, Hassan Rokni-Zadeh, Matthias De Vleeschouwer (UGent) , Maarten GK Ghequire, Davy Sinnaeve (UGent) , Guan-Lin Xie, Jef Rozenski, Annemieke Madder (UGent) , José Martins (UGent) and René De Mot
The rhizosphere isolate Pseudomonas putida BW11M1 produces a mixture of cyclic lipopeptide congeners, designated xantholysins. Properties of the major compound xantholysin A, shared with several other Pseudomonas lipopeptides, include antifungal activity and toxicity to Gram-positive bacteria, a supportive role in biofilm formation, and facilitation of surface colonization through swarming. Atypical is the lipopeptide’s capacity to inhibit some Gram-negative bacteria, including several xanthomonads. The lipotetradecadepsipeptides are assembled by XtlA, XtlB and XtlC, three co-linearly operating non-ribosomal peptide synthetases (NRPSs) displaying similarity in modular architecture with the entolysin-producing enzymes of the entomopathogenic Pseudomonas entomophila L48. A shifted serine-incorporating unit in the eight-module enzyme XtlB elongating the central peptide moiety not only generates an amino acid sequence differing at several equivalent positions from entolysin, but also directs xantholysin’s macrocyclization into an octacyclic structure, distinct from the pentacyclic closure in entolysin. Relaxed fatty acid specificity during lipoinitiation by XtlA (acylation with 3-hydroxydodec-5-enoate instead of 3-hydroxydecanoate) and for incorporation of the ultimate amino acid by XtlC (valine instead of isoleucine) account for the production of the minor structural variants xantholysin C and B, respectively. Remarkably, the genetic backbones of the xantholysin and entolysin NRPS systems also bear pronounced phylogenetic similarity to those of the P. putida strains PCL1445 and RW10S2, albeit generating the seemingly structurally unrelated cyclic lipopeptides putisolvin (undecapeptide containing a cyclotetrapeptide) and WLIP (nonapeptide containing a cycloheptapeptide), respectively. This similarity includes the linked genes encoding the cognate LuxR-family regulator and tripartite export system components in addition to individual modules of the NRPS enzymes, and probably reflects a common evolutionary origin. Phylogenetic scrutiny of the modules used for selective amino acid activation by these synthetases indicates that bacteria such as pseudomonads recruit and reshuffle individual biosynthetic units and blocks thereof to engineer reorganized or novel NRPS assembly lines for diversified synthesis of lipopeptides.
42. Exploiting furan’s versatile reactivity in reversible and irreversible orthogonal peptide labeling
Kurt Hoogewijs (UGent) , Dieter Buyst (UGent) , Johan Winne (UGent) , José Martins (UGent) and Annemieke Madder (UGent)
A general method for the facile and versatile decoration of peptides is proposed exploiting furan based cycloaddition and electrophilic aromatic substitution reactions. Given the commercial availability of furylalanine derivatives for peptide synthesis, the current work significantly enlarges the toolbox of available methodologies for site specific labeling and conjugation of peptide probes.
