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International audience; Antimicrobial resistance (AMR) is a major public health issue, causing 5 million deaths per year. Without any action plan, AMR will be in a near future the leading cause of death ahead of cancer. AMR comes from the ability of bacteria to rapidly develop and share resistance mechanisms towards current antibiotics, rendering them less effective. To circumvent this issue and avoid the phenomenon of cross-resistance, new antibiotics acting on novel targets or with new modes of action are required. Today, the pipeline of potential new treatments with these characteristics includes promising compounds such as gepotidacin, zoliflodacin, ibezapolstat, MGB-BP-3, CRS-3123, afabicin and TXA-709, which are currently in clinical trials, and lefamulin, which has been recently approved by FDA and EMA. In this review, we report the chemical synthesis, mode of action, structure-activity relationships, in vitro and in vivo activities as well as clinical data of these eight small molecules listed above.

International audience; It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.

The sensitivity of Mycobacterium tuberculosis , the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis , which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

Chemical biology and drug discovery are two scientific activities that pursue different goals but complement each other. The former is an interventional science that aims at understanding living systems through the modulation of its molecular components with compounds designed for this purpose. The latter is the art of designing drug candidates, i.e., molecules that act on selected molecular components of human beings and display, as a candidate treatment, the best reachable risk benefit ratio. In chemical biology, the compound is the means to understand biology, whereas in drug discovery, the compound is the goal. The toolbox they share includes biological and chemical analytic technologies, cell and whole-body imaging, and exploring the chemical space through state-of-the-art design and synthesis tools. In this article, we examine several tools shared by drug discovery and chemical biology through selected examples taken from research projects conducted in our institute in the last decade. These examples illustrate the design of chemical probes and tools to identify and validate new targets, to quantify target engagement in vitro and in vivo, to discover hits and to optimize pharmacokinetic properties with the control of compound concentration both spatially and temporally in the various biophases of a biological system.

Killing more than one million people each year, tuberculosis remains the leading cause of death from a single infectious agent. The growing threat of multidrug-resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. EthR, a mycobacterial transcriptional regulator, is involved in the control of the bioactivation of the second-line drug ethionamide. We have previously reported the discovery of in vitro nanomolar boosters of ethionamide through fragment-based approaches. In this study, we have further explored the structure-activity and structure-property relationships in this chemical family. By combining structure-based drug design and in vitro evaluation of the compounds, we identified a new oxadiazole compound as the first fragment-based ethionamide booster which proved to be active in vivo, in an acute model of tuberculosis infection., info:eu-repo/semantics/published

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments., info:eu-repo/semantics/published

Killing more than one million people each year, tuberculosis remains the leading cause of death from a single infectious agent. The growing threat of multidrug-resistant strains of

Countering TB prodrug resistance The arsenal of antibiotics for treating tuberculosis (TB) contains many prodrugs, such as ethionamide, which need activation by normal metabolism to release their toxic effects. Ethionamide is potentiated by small molecules. Blondiaux et al. screened for more potent analogs and identified a lead compound called SMARt-420. This small molecule inactivates a TetR-like repressor, EthR2, and boosts ethionamide activation. SMARt-420 successfully promoted clearance of multidrug-resistant strains of Mycobacterium tuberculosis from the lungs of mice. Science , this issue p. 1206

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-β-cyclodextrins (pβCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pβCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pβCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pβCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pβCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pβCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB., SCOPUS: ar.j, info:eu-repo/semantics/published

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis, represents one of the most challenging threat to public health worldwide, and with the increasing resistance to approved TB drugs, it is needed to develop new strategies to address this issue. Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant TB. It is a prodrug that requires activation by mycobacterial monooxygenases to inhibit the enoyl-ACP reductase InhA, which is involved in mycolic acid biosynthesis. Very recently, we identified that inhibition of a transcriptional repressor, termed EthR2, derepresses a new bioactivation pathway that results in the boosting of ethionamide activation. Herein, we describe the identification of potent EthR2 inhibitors using fragment-based screening and structure-based optimization. A target-based screening of a fragment library using thermal shift assay followed by X-ray crystallography identified 5 hits. Rapid optimization of the tropinone chemical series led to compounds with improved in vitro potency., SCOPUS: ar.j, info:eu-repo/semantics/published

Since few years, tuberculosis (TB) rivals AIDS as the leading cause of death worldwide. In addition, multidrug-resistant-TB (MDR-TB) has emerged as a major concern. Therapeutic solutions to stem this scourge are based on a 2–4 year treatment regimen of second-line drug cocktails, often associated with serious side effects, which does not facilitate patients compliance. In parallel to the search for new class of antibiotics, the development of alternative strategies to improve the efficacy of drugs already used in the clinic has been proposed. One of these strategies takes advantage of the fact that many anti-TB drugs are bioactivated by specific mycobacterial enzymes. Bioactivation of ethionamide or prothionamide, leading to inhibition of mycolic acids biosynthesis, involves a flavin-dependent Baeyer-Villiger monooxygenase called EthA, which expression is under the control of the transcriptional repressor EthR. A new therapeutic concept emerged from this observation and the development of EthR inhibitors was proposed as a solution to improve ethionamide efficacy. In this chapter, we outline the most recent efforts of different research groups, using modern drug discovery strategies that led to the design, discovery and optimization of the first inhibitors of EthR and boosters of ETH bioactivation in vitro and in vivo. We finally discuss the limitations of this approach and we highlight the breakthroughs in the identification of new chemical series and alternative bioactivation pathways. This discovery paves the way for the clinical development of a combination allowing thioamides to return fully active against all sensitive or resistant clinical isolates.

The Mycobacterium tuberculosis EthR is a member of the TetR family of repressors, controlling the expression of EthA, a mono-oxygenase responsible for the bioactivation of the prodrug ethionamide. This protein was established as a promising therapeutic target against tuberculosis, allowing, when inhibited by a drug-like molecule, to boost the action of ethionamide. Dozens of EthR crystal structures have been solved in complex with ligands. Herein, we disclose EthR structures in complex with 18 different small molecules and then performed in-depth analysis on the complete set of EthR structures that provides insights on EthR-ligand interactions. The 81 molecules solved in complex with EthR show a large diversity of chemical structures that were split up into several chemical clusters. Two of the most striking common points of EthR-ligand interactions are the quasi-omnipresence of a hydrogen bond bridging compounds with Asn179 and the high occurrence of π-π interactions involving Phe110. A systematic analysis of the protein-ligand contacts identified eight hot spot residues that defined the basic structural features governing the binding mode of small molecules to EthR. Implications for the design of new potent inhibitors are discussed., SCOPUS: ar.j, info:eu-repo/semantics/published

Tuberculosis (TB) is a leading infectious cause of death worldwide. The use of ethionamide (ETH), a main second line anti-TB drug, is hampered by its severe side effects. Recently discovered "booster" molecules strongly increase the ETH efficacy, opening new perspectives to improve the current clinical outcome of drug-resistant TB. To investigate the simultaneous delivery of ETH and its booster BDM41906 in the lungs, we co-encapsulated these compounds in biodegradable polymeric nanoparticles (NPs), overcoming the bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of this Booster. The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform, showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, "green" β-cyclodextrin (pCD) based NPs displayed the best physico-chemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mouse lungs using a Microsprayer®, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations as compared to untreated mice. This study paves the way for a future use of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair in TB chemotherapy.

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP+-bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP+-bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

Erratum in : Publisher Correction: Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles. [Sci Rep. 2018]; International audience; Tuberculosis (TB) is a leading infectious cause of death worldwide. The use of ethionamide (ETH), a main second line anti-TB drug, is hampered by its severe side effects. Recently discovered "booster" molecules strongly increase the ETH efficacy, opening new perspectives to improve the current clinical outcome of drug-resistant TB. To investigate the simultaneous delivery of ETH and its booster BDM41906 in the lungs, we co-encapsulated these compounds in biodegradable polymeric nanoparticles (NPs), overcoming the bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of this Booster. The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform, showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, "green" β-cyclodextrin (pCD) based NPs displayed the best physico-chemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mouse lungs using a Microsprayer®, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations as compared to untreated mice. This study paves the way for a future use of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair in TB chemotherapy.

The design of new sp 3 and spiro-enriched fragments has been achieved from 1,3-dipolar cycloaddition between alkenes and chloro-oximes. The selection of reagents was performed to afford a panel of 2-isoxazoline-containing fragments that show desirable three dimensional (3D) characteristics to allow the probing of biologically-relevant chemical space. Principal moments of inertia (PMI) were calculated to evaluate the 3D diversity. The resulting 3D fragments with suitable physicochemical properties, especially a good solubility, will be used to improve the hit rate of our fragment-based screening.

Oxytocin (OT) is a cyclic nonapeptide containing one internal disulfide bond between its Cys(1) and Cys(6) residues. Although OT is one of the most commonly used peptidic drugs, the mechanism of its degradation in aqueous solution and the identity of its degradants have not been fully elucidated. To investigate the pathways and products of OT degradation in slightly acidic to neutral solutions, we prepared the peptides: OT, [D-Cys(1)]OT, a series of N-alkylated OT analogues, [[(13)C3,(15) N]Cys(1)]OT, and OT where each sulfur atom was systematically replaced by either methylene, (34)S, or Se. The peptides were incubated at 40°C and the degradation products studied by HPLC, LCMS, and (13)C-NMR. Our findings suggest that the degradation begins with β-elimination of the disulfide linkage to form a putative intermediate linear peptide containing an S-thiocysteine (a persulfide) in position 6 and a dehydroalanine in position 1. This intermediate persulfide appears to donate a sulfur atom to an intact OT molecule to form OT trisulfide and higher monomeric polysulfides, while the dehydroalanine residue is hydrolyzed with loss of the N-terminal amino group to yield a linear N-pyruvoylated octapeptide containing a reduced Cys(6). Based on the MS and (13)C-NMR data of the products from degradation of [[(13)C3,(15)N]Cys(1)]OT, we postulate that the ultimate degradation products of OT are dimers composed of two pyruvoylated octapeptides held together by one disulfide bridge between the two Cys(6) residues and by one more, non-reducible, linkage resulting from an aldol-type condensation between the two N-terminal pyruvoyl groups.

Fragment-based drug design has been successfully applied to a large set of proteins, however in order to expand this concept to the most demanding targets, such as protein–protein interactions, it is required to enrich current fragment libraries with new and original 3D privileged fragments. Our goal was to develop a rapid microwave-assisted synthesis of 27 new privileged spirohydantoin fragments. Among them 24 compounds showed a high water solubility. These molecules were plotted according to the normalized principal moments of inertia of their minimized conformers, and most of the compounds were prone to occupy under-populated regions of the triangular plot. Finally we demonstrated that the hydantoin ring can be selectively N-monoalkylated providing the access to rapid functionalization for further elaboration.

Fragment-based drug design has been successfully applied to a large set of proteins, however in order to expand this concept to the most demanding targets, such as protein–protein interactions, it is required to enrich current fragment libraries with new and original 3D privileged fragments. Our goal was to develop a rapid microwave-assisted synthesis of 27 new privileged spirohydantoin fragments. Among them 24 compounds showed a high water solubility. These molecules were plotted according to the normalized principal moments of inertia of their minimized conformers, and most of the compounds were prone to occupy under-populated regions of the triangular plot. Finally we demonstrated that the hydantoin ring can be selectively N-monoalkylated providing the access to rapid functionalization for further elaboration.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant

In situ generated nitrile oxides undergo 1,3-dipolar cycloaddition with alkenes to give 2-isoxazoline derivatives.

International audience; Tuberculosis remains a major cause of mortality and morbidity, killing each year more than one million people. Although the combined use of first line antibiotics (isoniazid, rifampicin, pyrazinamide and ethambutol) is efficient to treat most patients, the rapid emergence of multidrug resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. Mycobacterial transcriptional repressor EthR is a key player in the control of second-line drugs bioactivation such as ethionamide, and has been shown to impair the sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. As a way to identify new potent ligands of this protein we have developed fragment-based approaches. In the current study we combined surface plasmon resonance assay, X-ray crystallography, ligand efficiency driven design for the rapid discovery and optimization of new chemotypes of EthR ligands starting from a fragment. The design, synthesis, in vitro and ex vivo activities of these compounds will be discussed.

Insulin degrading enzyme (IDE) is a highly conserved zinc metalloprotease that is involved in the clearance of various physiologically peptides like amyloid-beta and insulin. This enzyme has been involved in the physiopathology of diabetes and Alzheimer's disease. We describe here a series of small molecules discovered by screening. Co-crystallization of the compounds with IDE revealed a binding both at the permanent exosite and at the discontinuous, conformational catalytic site. Preliminary structure-activity relationships are described. Selective inhibition of amyloid-beta degradation over insulin hydrolysis was possible. Neuroblastoma cells treated with the optimized compound display a dose-dependent increase in amyloid-beta levels.

Oxytocin (OT) is a cyclic nonapeptide containing one internal disulfide bond between its Cys(1) and Cys(6) residues. Although OT is one of the most commonly used peptidic drugs, the mechanism of its degradation in aqueous solution and the identity of its degradants have not been fully elucidated. To investigate the pathways and products of OT degradation in slightly acidic to neutral solutions, we prepared the peptides: OT, [D-Cys(1)]OT, a series of N-alkylated OT analogues, [[(13)C3,(15) N]Cys(1)]OT, and OT where each sulfur atom was systematically replaced by either methylene, (34)S, or Se. The peptides were incubated at 40°C and the degradation products studied by HPLC, LCMS, and (13)C-NMR. Our findings suggest that the degradation begins with β-elimination of the disulfide linkage to form a putative intermediate linear peptide containing an S-thiocysteine (a persulfide) in position 6 and a dehydroalanine in position 1. This intermediate persulfide appears to donate a sulfur atom to an intact OT molecule to form OT trisulfide and higher monomeric polysulfides, while the dehydroalanine residue is hydrolyzed with loss of the N-terminal amino group to yield a linear N-pyruvoylated octapeptide containing a reduced Cys(6). Based on the MS and (13)C-NMR data of the products from degradation of [[(13)C3,(15)N]Cys(1)]OT, we postulate that the ultimate degradation products of OT are dimers composed of two pyruvoylated octapeptides held together by one disulfide bridge between the two Cys(6) residues and by one more, non-reducible, linkage resulting from an aldol-type condensation between the two N-terminal pyruvoyl groups.

International audience; Malaria is a severe infectious disease that causes between 655 000 and 1.2 million deaths annually. To overcome the resistance to current drugs, new biological targets are needed for drug development. Aminopeptidase M1 (PfAM1), a zinc metalloprotease, has been proposed as a new drug target to fight malaria. Herein, we disclosed the structure−activity relationships of a selective family of hydroxamate PfAM1 inhibitors based on the malonic template. In particular, we performed a "fluoro-scanning" around hit 1 that enlightened the key positions of the halogen for activity. The docking of the best inhibitor 2 is consistent with in vitro results. The stability of 2 was evaluated in microsomes, in plasma, and toward glutathione. The in vivo distribution study performed with the nanomolar hydroxamate inhibitor 2 (BDM14471) revealed that it reaches its site of action. However, it fails to kill the parasite at concentrations relevant to the enzymatic inhibitory potency, suggesting that killing the parasite remains a challenge for potent and druglike catalytic-site binding PfAM1 inhibitors. In all, this study provides important insights for the design of inhibitors of PfAM1 and the validity of this target.

In this paper, we describe the screening of a 14640-compound library using a novel whole mycobacteria phenotypic assay to discover inhibitors of EthR, a transcriptional repressor implicated in the innate resistance of Mycobacterium tuberculosis to the second-line antituberculosis drug ethionamide. From this screening a new chemical family of EthR inhibitors bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent ethionamide boosters on M. tuberculosis-infected macrophages. Finally, the cocrystallization of the best optimized analogue with EthR revealed an unexpected reorientation of the ligand in the binding pocket. © 2012 American Chemical Society.

International audience; Tuberculosis is a major disease causing every year 1.8 million deaths worldwide and represents the leading cause of mortality resulting from a bacterial infection. Introduction in the 60's of first-line drug regimen resulted in the control of the disease and TB was perceived as defeating. However, since the progression of HIV leading to co-infection with AIDS and the emergence of drug resistant strains, the need of new anti-tuberculosis drugs was not overstated. However in the past 40 years any new molecule did succeed in reaching the market. Today, the pipeline of potential new treatments has been fulfilled with several compounds in clinical trials or preclinical development with promising activities against sensitive and resistant Mycobacterium tuberculosis strains. Compounds as gatifloxacin, moxifloxacin, metronidazole or linezolid already used against other bacterial infections are currently evaluated in clinical phases 2 or 3 for treating tuberculosis. In addition, analogues of known TB drugs (PA-824, OPC-67683, PNU-100480, AZD5847, SQ609, SQ109, DC-159a) and new chemical entities (TMC207, BTZ043, DNB1, BDM31343) are under development. In this review, we report the chemical synthesis, mode of action when known, in vitro and in vivo activities and clinical data of all current small molecules targeting tuberculosis.

Mycobacterial transcriptional repressor EthR controls the expression of EthA, the bacterial monooxygenase activating ethionamide, and is thus largely responsible for the low sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. We recently reported structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors leading to the discovery of potent ethionamide boosters. Despite high metabolic stability, pharmacokinetic evaluation revealed poor mice exposure; therefore, a second phase of optimization was required. Herein a structure-property relationship study is reported according to the replacement of the two aromatic heterocycles: 2-thienyl and 1,2,4-oxadiazolyl moieties. This work was done using a combination of structure-based drug design and in vitro/ex vivo evaluations of ethionamide boosters on the targeted protein EthR and on the human pathogen Mycobacterium tuberculosis. Thanks to this process, we identified compound 42 (BDM41906), which displays improved efficacy in addition to high exposure to mice after oral administration.

We report in this article an extensive structure-activity relationships (SAR) study with 58 thiophen-2-yl-1,2,4-oxadiazoles as inhibitors of EthR, a transcriptional regulator controling ethionamide bioactivation in Mycobacterium tuberculosis. We explored the replacement of two key fragments of the starting lead BDM31343. We investigated the potency of all analogues to boost subactive doses of ethionamide on a phenotypic assay involving M. tuberculosis infected macrophages and then ascertained the mode of action of the most active compounds using a functional target-based surface plasmon resonance assay. This process revealed that introduction of 4,4,4-trifluorobutyryl chain instead of cyanoacetyl group was crucial for intracellular activity. Replacement of 1,4-piperidyl by (R)-1,3-pyrrolidyl scaffold did not enhance activity but led to improved pharmacokinetic properties. Furthermore, the crystal structures of ligand-EthR complexes were consistent with the observed SAR. In conclusion, we identified EthR inhibitors that boost antibacterial activity of ethionamide with nanomolar potency while improving solubility and metabolic stability.

International audience; Hydroxamates are valuable tools for chemical biology as well as interesting leads for medicinal chemistry. Though many hydroxamates display nanomolar activities against metalloproteases, only three hydroxamates have reached the market, among which is the HDAC inhibitor vorinostat. Failures in development are generally attributed to lack of selectivity, toxicity or poor stability. To help medicinal chemists with respect to plasma stability, we have performed the first and preliminary study on structure-plasma stability for hydroxamates. We define some structural rules to predict or improve the plasma stability in the preclinical stage.

International audience; Proteases that are expressed during the erythocytic stage of Plasmodium falciparum are newly explored drug targets for the treatment of malaria. We report here the discovery of potent inhibitors of PfA-M1, a metallo-aminopeptidase of the parasite. These compounds are based on a malonic hydroxamic template and present a very good selectivity toward neutral aminopeptidase (APN-CD13), a related protease in mammals. Structure-activity relationships in these series are described. Further optimization of the best inhibitor yielded a nanomolar, selective inhibitor of PfA-M1. This inhibitor displays good physicochemical and pharmacokinetic properties and a promising antimalarial activity.

International audience; We report here the design and parallel synthesis of 217 compounds based on a malonic-hydroxamic acid template. These compounds are obtained via a two-step solution-phase procedure. The set of diverse building-blocks used makes this strategy suitable for the search of inhibitors of various metallo-proteases and for the investigation of the biological role of new metallo-proteases. As a proof of concept, we screened this library on Neutral Aminopeptidase (APN; EC 3.4.11.2), the prototypal enzyme of the M1 family. Several submicromolar inhibitors were identified.

The discovery of drug candidates is based on the identification of hits with appropriated physico-chemical properties for further development. High throughput screening and fragment-based drug discovery approaches are two strategies commonly used for this identification. These strategies were applied during my PhD research work for identifying not only new modulators of the CD81/CLDN-1 complex to prevent entry of the Hepatitis C virus (HCV) into hepatocytes but also inhibitors of the mycobacterial transcriptional repressor, called EthR2, to boost ethionamide antibacterial activity against resistant strains of M. tuberculosis.Firstly, a high throughput screening assay was developed to identify molecules bearing a thieno[2,3-c]pyrazole scaffold that modulate the CD81/CLDN-1 complex. The structure-activity relationships allowed us to design and synthesize one non-toxic compound that inhibits viral entry with an IC50 in the submicromolar range. This best analog will be used as pharmacological tool to understand the molecular mechanism involving the CD81/CLDN-1 interaction during virus entry.Secondary, we worked on the design and synthesis of a new generation of fragments called privileged fragments. We focused our interest on the 1,4-benzodiazepine-2,5-dione and spirohydantoin scaffolds and using microwave-assisted conditions 44 original privileged fragments have been synthesized. To further illustrate the potential of our privileged fragments, a virtual focused library has been generated and screened in silico on MDM2 protein. The in vitro evaluation of the identified hits will allow us to validate our approach and to show the potential of our privileged fragments for the discovery of new hits against protein-protein interactions.Finally, inhibitors of a new mycobacterial transcriptional repressor involved in the boosting of ethionamide activity have been developed. Screening of 960 fragments allowed us to identify a hit bearing a tropinone scaffold which was cocrystallized with EthR2. A rational design from this cocrystal structure led rapidly to more potent ligands., La découverte d’un candidat médicament repose sur l’identification de hits, présentant des propriétés physico-chimiques adéquates pour leur optimisation. Le criblage à haut débit et l’approche par fragments sont deux techniques couramment utilisées lors de cette étape d’identification et elles ont été mises en œuvre au cours de ma thèse dans le but de découvrir de nouveaux composés ciblant d’une part le complexe CD81/CLDN-1 pour empêcher l’entrée du virus de l’hépatite C (VHC) dans les hépatocytes et d’autre part EthR2, un régulateur transcriptionnel mycobactérien, afin de potentialiser l’activité d’un antituberculeux sur les souches résistantes de M. tuberculosis.Dans une première partie, un criblage à haut débit sur le complexe CD81/CLDN-1 a permis d’identifier des modulateurs en série thiéno[2,3-c]pyrazole. Ces composés ont été pharmacomodulés et un composé spécifique de l’étape d’entrée du VHC, non toxique et présentant une activité submicromolaire a pu être ainsi identifié. Cette sonde pharmacologique permettra de mieux comprendre les mécanismes impliqués dans le processus d’entrée virale.Dans une deuxième partie, nous nous sommes intéressés à la conception de nouveaux fragments dits privilégiés. Ainsi, le développement des voies de synthèse, sous irradiation micro-onde, de deux entités moléculaires, le noyau 1,4-benzodiazepine-2,5-dione et le noyau spirohydantoïne, nous a permis d’obtenir 34 composés originaux. Afin d’évaluer le potentiel de cette stratégie, une librairie virtuelle de fragments a été générée et son criblage in silico sur la protéine MDM2 a été effectué. La mesure in vitro de l’activité des hits identifiés permettra de valider l’intérêt de cette approche pour la découverte de nouveaux ligands ciblant les interactions protéine-protéine.Dans une troisième partie, des inhibiteurs d’un répresseur transcriptionnel mycobactérien impliqué dans la potentialisation de l’activité de l’éthionamide ont été développés. A l’issue d’un criblage de 960 fragments, l’identification d’un hit en série tropinone, et sa cocristallisation avec la protéine EthR2, a permis d’entamer une optimisation rationnelle qui a conduit à l’obtention rapide de composés présentant de meilleures activités.