Recent advances in Fragment-based strategies against tuberculosis.

Tuberculosis remains one of the world's leading infectious disease killers, causing more than 1.5 million of deaths each year. It is therefore a priority to discover and develop new classes of anti-tuberculosis drugs to design new treatments in order to fight the increasing burden of resistant-tuberculosis. Fragment-based drug discovery (FBDD) relies on the identification of small molecule hits, further improved to high-affinity ligands through three main approaches: fragment growing, merging and linking. The aim of this review is to highlight the recent progresses made in fragment-based approaches for the discovery and development of Mycobacterium tuberculosis inhibitors in a wide range of pathways. Hit discovery, hit-to-lead optimization, SAR and binding mode when available are discussed.

Rapid and Efficient Access to Novel Bio-Inspired 3-Dimensional Tricyclic SpiroLactams as Privileged Structures via Meyers' Lactamization.

The concept of privileged structure has been used as a fruitful approach for the discovery of novel biologically active molecules. A privileged structure is defined as a semi-rigid scaffold able to display substituents in multiple spatial directions and capable of providing potent and selective ligands for different biological targets through the modification of those substituents. On average, these backbones tend to exhibit improved drug-like properties and therefore represent attractive starting points for hit-to-lead optimization programs. This article promotes the rapid, reliable, and efficient synthesis of novel, highly 3-dimensional, and easily functionalized bio-inspired tricyclic spirolactams, as well as an analysis of their drug-like properties.

Tricyclic SpiroLactams Kill Mycobacteria In Vitro and In Vivo by Inhibiting Type II NADH Dehydrogenases.

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.

On the Hunt for Next-Generation Antimicrobial Agents: An Online Symposium Organized Jointly by the French Society for Medicinal Chemistry (Société de Chimie Thérapeutique) and the French Microbiology Society (Société Française de Microbiologie) on 9-10 December 2021.

The restrictions posed by the COVID-19 pandemic obliged the French Society for Medicinal Chemistry (Société de chimie thérapeutique) and the French Microbiology Society (Société Française de Microbiologie) to organize their joint autumn symposium (entitled "On the hunt for next-generation antimicrobial agents") online on 9-10 December 2021. The meeting attracted more than 200 researchers from France and abroad with interests in drug discovery, antimicrobial resistance, medicinal chemistry, and related disciplines. This review summarizes the 13 invited keynote lectures. The symposium generated high-level scientific dialogue on the most recent advances in combating antimicrobial resistance. The University of Lille, the Institut Pasteur de Lille, the journal Pharmaceuticals, Oxeltis, and INCATE, sponsored the event.

Molecular Design in Practice: A Review of Selected Projects in a French Research Institute That Illustrates the Link between Chemical Biology and Medicinal Chemistry.

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.

Rubrolone production by Dactylosporangium vinaceum: biosynthesis, modulation and possible biological function.

Rare actinomycetes are likely treasure troves for bioactive natural products, and it is therefore important that we enrich our understanding of biosynthetic potential of these relatively understudied bacteria. Dactylosporangium are a genus of such rare Actinobacteria that are known to produce a number of important antibacterial compounds, but for which there are still no fully assembled reference genomes, and where the extent of encoded biosynthetic capacity is not defined. Dactylosporangium vinaceum (NRRL B-16297) is known to readily produce a deep wine red-coloured diffusible pigment of unknown origin, and it was decided to define the chemical identity of this natural product pigment, and in parallel use whole genome sequencing and transcriptional analysis to lay a foundation for understanding the biosynthetic capacity of these bacteria. Results show that the produced pigment is made of various rubrolone conjugates, the spontaneous product of the reactive pre-rubrolone, produced by the bacterium. Genome and transcriptome analysis identified the highly expressed biosynthetic gene cluster (BGC) for pre-rubrolone. Further analysis of the fully assembled genome found it to carry 24 additional BGCs, of which the majority were poorly transcribed, confirming the encoded capacity of this bacterium to produce natural products but also illustrating the main bottleneck to exploiting this capacity. Finally, analysis of the potential environmental role of pre-rubrolone found it to react with a number of amine containing antibiotics, antimicrobial peptides and siderophores pointing to its potential role as a "minesweeper" of xenobiotic molecules in the bacterial environment. KEY POINTS: • D. vinaceum encodes many BGC, but the majority are transcriptionally silent. • Chemical screening identifies molecules that modulate rubrolone production. • Pre-rubrolone is efficient at binding and inactivating many natural antibiotics.

Fragment-Based Optimized EthR Inhibitors with in Vivo Ethionamide Boosting Activity.

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.

Pyrrolomycins Are Potent Natural Protonophores.

The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.

Intrinsic Antibacterial Activity of Nanoparticles Made of ß-Cyclodextrins Potentiates Their Effect as Drug Nanocarriers against Tuberculosis.

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-ß-cyclodextrins (pßCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pßCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pßCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pßCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pßCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pßCD 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.

A fragment-based approach towards the discovery of N-substituted tropinones as inhibitors of Mycobacterium tuberculosis transcriptional regulator EthR2.

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.

A comprehensive analysis of the protein-ligand interactions in crystal structures of Mycobacterium tuberculosis EthR.

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.

Cyclodextrin-based nanocarriers containing a synergic drug combination: A potential formulation for pulmonary administration of antitubercular drugs.

Tuberculosis (TB) remains a major global health problem. The use of ethionamide (ETH), a main second line drug, is associated to severe toxic side-effects due to its low therapeutic index. In this challenging context, "booster" molecules have been synthetized to increase the efficacy of ETH. However, the administration of ETH/booster pair is mostly hampered by the low solubility of these drugs and the tendency of ETH to crystallize. Here, ETH and a poorly water-soluble booster, so-called BDM43266, were simultaneously loaded in polymeric ß-cyclodextrin nanoparticles (pßCyD NPs) following a "green" protocol. The interaction of ETH and BDM43266 with pßCyD NPs was investigated by complementary techniques. Remarkably, the inclusion of ETH and BDM43266 pßCyD NPs led to an increase of their apparent solubility in water of 10- and 90-fold, respectively. Competition studies of ETH and BDM43266 for the CyD cavities of pßCyD NPs corroborated the fact that the drugs did not compete with each other, confirming the possibility to simultaneously co-incorporate them in NPs. The drug-loaded NP suspensions could be filtered through 0.22µm filters. Finally, the drug-loaded NPs were passed through a Microsprayer® to evaluate the feasibility to administer pßCyD NPs by pulmonary route. Each spray delivered a constant amount of both drugs and the NPs were totally recovered after passage through the Microsprayer®. These promising results pave the way for a future use of pßCyD NPs for the pulmonary delivery of the ETH/BDM43266 pair.

Ligand efficiency driven design of new inhibitors of Mycobacterium tuberculosis transcriptional repressor EthR using fragment growing, merging, and linking approaches.

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, and ligand efficiency driven design for the rapid discovery and optimization of new chemotypes of EthR ligands starting from a fragment. The design, synthesis, and in vitro and ex vivo activities of these compounds will be discussed.

Unconventional surface plasmon resonance signals reveal quantitative inhibition of transcriptional repressor EthR by synthetic ligands.

EthR is a mycobacterial repressor that limits the bioactivation of ethionamide, a commonly used anti-tuberculosis second-line drug. Several efforts have been deployed to identify EthR inhibitors abolishing the DNA-binding activity of the repressor. This led to the demonstration that stimulating the bioactivation of Eth through EthR inhibition could be an alternative way to fight Mycobacterium tuberculosis. We propose a new surface plasmon resonance (SPR) methodology to study the affinity between inhibitors and EthR. Interestingly, the binding between inhibitors and immobilized EthR produced a dose-dependent negative SPR signal. We demonstrate that this signal reveals the affinity of small molecules for the repressor. The affinity constants (K(D)) correlate with their capacity to inhibit the binding of EthR to DNA. We hypothesize that conformational changes in EthR during ligand interaction could be responsible for this SPR signal. Practically, this unconventional result opens perspectives onto the development of an SPR assay that would at the same time reveal structural changes in the target upon binding with an inhibitor and the binding constant of this interaction.

Structural and docking studies of potent ethionamide boosters.

Tuberculosis remains the second only to HIV as the leading cause of death by infectious disease worldwide, and was responsible for 1.4 million deaths globally in 2011. One of the essential drugs of the second-line antitubercular regimen is the prodrug ethionamide, introduced in the 1960s. Ethionamide is primarily used in cases of multi-drug resistant (MDR) and extensively drug resistant (XDR) TB due to severe adverse side effects. As a prodrug, ethionamide is bioactivated by EthA, a mono-oxygenase whose activity is repressed by EthR, a member of the TetR family of regulators. Previous studies have established that inhibition of EthR improves ethionamide potency. We report here the crystal structures of three EthR inhibitors at 0.8 Šresolution (3-oxo-3-{4-[3-(thiophen-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-1-yl}propanenitrile (BDM31343), 4,4,4-trifluoro-1-{4-[3-(6-methoxy-1,3-benzothiazol-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-1-yl}butanone (BDM41325) and 5,5,5-trifluoro-1-{4-[3-(4-methanesulfonylphenyl)-1,2,4-oxadiazol-5-yl]piperidin-1-yl}pentanone (BDM41907)), and the docking studies undertaken to investigate possible binding modes. The results revealed two distinct orientations of the three compounds in the binding channel, a direct consequence of the promiscuous nature of the largely lipophilic binding site.

Tuberculosis: the drug development pipeline at a glance.

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.

Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug design to solve pharmacokinetic issues in a series of potent 1,2,4-oxadiazole EthR inhibitors.

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.