Zinc-selective inhibition of the promiscuous bacterial amide-hydrolase DapE: implications of metal heterogeneity for evolution and antibiotic drug design

The development of resistance to virtually all current antibiotics makes the discovery of new antimicrobial compounds with novel protein targets an urgent challenge. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is an essential metallo-enzyme for growth and proliferation in many bacteria, acting in the desuccinylation of N-succinyl-l,l-diaminopimelic acid (SDAP) in a late stage of the anabolic pathway towards both lysine and a crucial building block of the peptidoglycan cell wall. l-Captopril, which has been shown to exhibit very promising inhibitory activity in vitro against DapE and has attractive drug-like properties, nevertheless does not target DapE in bacteria effectively. Here we show that l-captopril targets only the Zn(2+)-metallo-isoform of the enzyme, whereas the Mn(2+)-enzyme, which is also a physiologically relevant isoform in bacteria, is not inhibited. Our finding provides a rationale for the failure of this promising lead-compound to exhibit any significant antibiotic activity in bacteria and underlines the importance of addressing metallo-isoform heterogeneity in future drug design. Moreover, to our knowledge, this is the first example of metallo-isoform heterogeneity in vivo that provides an evolutionary advantage to bacteria upon drug-challenge

Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge

Synthèse et interaction de PNA ciblant l'ARN TAR du VIH-1

Les médicaments utilisés en polythérapie anti-VIH ciblent des protéines virales (transcriptase inverse, protéase, go41). Ces thérapies ont permis de réduire le taux de mortalité mais génèrent de nombreux effets secondaires et conduisent à l émergence, parfois très rapide, de souches résistantes aux médicaments utilisés. La recherche de molécules qui agissent sur de nouvelles cibles virales reste donc nécessaire. Une piste consiste à cibler des fragments de l ARN du VIH qui sont impliqués dans es processus biologiques essentiels à la multiplication virale et dont les séquences sont conservées dans la plupart des souches virales. Dans ce cadre, le fragment d ARN TAR du VIH-1 constitue une cible privilégiée. Au cours de ce travail, nous avons développé deux stratégies en vue de cibler et d inhiber les fonctions biologiques de l ARN TAR. Une première stratégie a consisté à élaborer des PNA cycliques antisens (Peptide Nucleic Acids : analogues d ARN/ADN), susceptibles d établir un complexe très stable et spécifique avec la boucle de l ARN TAR. La seconde stratégie a consisté en l élaboration d un conjugué d arginine et de PNA, ciblant simultanément le renflement et la boucle de l ARN TAR. Dans une premier temps, nous avons donc mis au point la synthèse, en phase solide, de ces différentes molécules. Puis leur interaction avec l ARN TAR a été évaluée par dénaturation thermique, suivie par spectroscopie UV. Ces études ont montré que les PNA cycliques forment un complexe très stable et spécifique avec l ARN TAR. De même, une interaction entre le conjugué d arginine et de PNA et l ARN TAR a été mise en évidence.The drugs used in anti-HIV polytherapy target viral proteins (reverse transcriptase, protease, gp41). These therapies made it possible to reduce the death rate but generate many side effects and lead to emergence, sometimes very fast, of resistant strains to the drugs used. The research for molecules which act new viral targets thus remains necessary. A track consists in targeting fragments of the RNA of the HIV which is implied in biological processes essential with the viral multiplication and whose sequences are preserved in the majority of the viral strains. Within this framework, the HIV-1 TAR RNA fragment constitutes a privileged target. During this work, we developed two strategies in order to target and to inhibit the biological functions of TAR RNA. A first strategy consisted in working out cyclic PNA antisense (Peptide Nucleic Acids : RNA/DNA analogues), likely to establish a very stable and specific complex with the TAR RNA loop. The second strategy consisted of the development of one conjugate of arginine and PNA, simultaneously targeting the TAR RNA bugle and loop. Initially, we thus developed the synthesis, on solid phase, of these various molecules. Then their interaction with TAR RNA was evaluated by thermal denaturation, followed by UV spectroscopy. These studies showed that the cyclic PNA form a very stable and specific complex with TAR RNA. In the same way, an interaction enters combined arginine and PNA and TAR RNA were highlighted.NICE-BU Sciences (060882101) / SudocSudocFranceF

Nanoliter plates—versatile tools for the screening of split-and-mix libraries on-bead and off-bead

Nanoliter plates are versatile tools for the screening of split-and-mix libraries both on-bead and off-bead. The method was evaluated for the identification of hydrolytically active compounds and enzyme inhibitors

Acidosis Increases MHC Class II-Restricted Presentation of a Protein Endowed with a pH-Dependent Heparan Sulfate-Binding Ability.

International audienceHeparan sulfate proteoglycans (HSPGs) are ubiquitously expressed molecules that participate in numerous biological processes. We previously showed that HSPGs expressed on the surface of APCs can serve as receptors for a hybrid protein containing an HS ligand and an Ag, which leads to more efficient stimulation of Th cells. To investigate whether such behavior is shared by proteins with inherent HS-binding ability, we looked for proteins endowed with this characteristic. We found that diphtheria toxin and its nontoxic mutant, called CRM197, can interact with HS. However, we observed that their binding ability is higher at pH 6 than at pH 7.4. Therefore, as extracellular acidosis occurs during infection by various micro-organisms, we assessed whether HS-binding capacity affects MHC class II-restricted presentation at different pHs. We first observed that pH decrease allows CRM197 binding to HSPG-expressing cells, including APCs. Then, we showed that this interaction enhances Ag uptake and presentation to Th cells. Lastly, we observed that pH decrease does not affect processing and presentation abilities of the APCs. Our findings show that acidic pH causes an HSPG-mediated uptake and an enhancement of T cell stimulation of Ags with the inherent ability to bind HSPGs pH-dependently. Furthermore, they suggest that proteins from micro-organisms with this binding characteristic might be supported more efficiently by the adaptive immune system when acidosis is triggered during infection

High-throughput production of two disulphide-bridge toxins

International audienceA quick and efficient production method compatible with high-throughput screening was developed using 36 toxins belonging to four different families of two disulphide-bridge toxins. Final toxins were characterized using HPLC co-elution, CD and pharmacology studies

RgIA4 potently blocks mouse α\alpha9α\alpha10 nAChRs and provides long lasting protection against oxaliplatin-induced cold allodynia

International audienceTranscripts for $\alpha$9 and $\alpha$10 nicotinic acetylcholine receptor (nAChR) subunits are found in diverse tissues. The function of $\alpha$9$\alpha$10 nAChRs is best known in mechanosensory cochlear hair cells, but elsewhere their roles are less well-understood. $\alpha$9$\alpha$10 nAChRs have been implicated as analgesic targets and $\alpha$-conotoxins that block $\alpha$9$\alpha$10 nAChRs produce analgesia. However, some of these peptides show large potency differences between species. Additionally several studies have indicated that these conotoxins may also activate GABA$_B$ receptors (GABA$_B$Rs). To further address these issues, we cloned the cDNAs of mouse $\alpha$9 and $\alpha$10 nAChR subunits. When heterologously expressed in Xenopus oocytes, the resulting $\alpha$9$\alpha$10 nAChRs had the expected pharmacology of being activated by acetylcholine and choline but not by nicotine. A conotoxin analog, RgIA4, potently, and selectively blocked mouse $\alpha$9$\alpha$10 nAChRs with low nanomolar affinity indicating that RgIA4 may be effectively used to study murine $\alpha$9$\alpha$10 nAChR function. Previous reports indicated that RgIA4 attenuates chemotherapy-induced cold allodynia. Here we demonstrate that RgIA4 analgesic effects following oxaliplatin treatment are sustained for 21 days after last RgIA4 administration indicating that RgIA4 may provide enduring protection against nerve damage. RgIA4 lacks activity at GABA$_B$ receptors; a bioluminescence resonance energy transfer assay was used to demonstrate that two other analgesic $\alpha$-conotoxins, Vc1.1 and AuIB, also do not activate GABA$_B$Rs expressed in HEK cells. Together these findings further support the targeting of $\alpha$9$\alpha$10 nAChRs in the treatment of pain

From a Cone Snail Toxin to a Competitive MC4R Antagonist

International audienceThe melanocortin 4 receptor (MC4R) plays a role in energy homeostasis and represents a target for treating energy balance disorders. For decades, synthetic ligands have been derived from MC4R endogenous agonists and antagonists, such as setmelanotide used to treat rare forms of genetic obesity. Recently, animal venoms have demonstrated their capacity to provide melanocortin ligands with toxins from a scorpion and a spider. Here, we described a cone snail toxin, N-CTX-Ltg1a, with a nanomolar affinity for hMC4R but unrelated to any known toxins or melanocortin ligands. We then derived from the conotoxin the linear peptide HT1-0, a competitive antagonist of G s , G 15 , and β-arrestin2 pathways with a low nanomolar affinity for hMC4R. Similar to endogenous ligands, HT1-0 needs hydrophobic and basic residues to bind hMC4R. Altogether, it represents the first venom-derived peptide of high affinity on MC4R and paves the way for the development of new MC4R antagonists