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Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 β-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of β-lactams screened were active against Mtb, many without a β-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-β-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E. coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20 mg kg

Herein, we describe the myxobacterial natural product Corramycin (1) isolated from Corallococcus coralloides (C. coralloides). Corramycin is a linear peptide containing an unprecedented (2R,3S)  N-methyl-β-hydroxy histidine moiety and exhibiting anti-Gram-negative activity against Escherichia coli. Moreover, we describe the Corramycin biosynthetic gene cluster (BGC) and propose a biosynthesis model involving a 12-modular non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS). Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule’s absolute configuration. Furthermore, we show that the uptake of Corramycin in E. coli depends on two transporter systems, SbmA and YejABEF. Importantly, intravenous administration of 30 mg kg 1 of Corramycin in an E. coli mouse infection model resulted in significantly reduced colony forming units (CFU) and in 60 % survival of animals, with no toxic effects observed in vitro or in vivo. Corramycin is thus an intriguing innovative starting point to develop a potent antibacterial drug against hospital acquired infections.

Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA (‘poisoning’) in an analogous fashion to fluoroquinolones, but without the requirement for the water–metal–ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase–DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.

Antimycobacterial agents might shorten the course of treatment by reducing the number of phenotypically tolerant bacteria if they could kill M. tuberculosis in diverse metabolic states. Here we report two chemically disparate classes of agents that kill M. tuberculosis both when it is replicating and when it is not. Under replicating conditions, the tricyclic 4-hydroxyquinolines and a barbituric acid analogue deplete intrabacterial magnesium as a mechanism of action, and for both compounds, mutations in CorA, a putative Mg2+/Co2+ transporter, conferred resistance to the compounds when M. tuberculosis was under replicating conditions but not under nonreplicating conditions, illustrating that a given compound can kill M. tuberculosis in different metabolic states by disparate mechanisms. Targeting magnesium metallostasis represents a previously undescribed antimycobacterial mode of action that might cripple M. tuberculosis in a Mg2+-deficient intraphagosomal environment of macrophages., A defining characteristic of treating tuberculosis is the need for prolonged administration of multiple drugs. This may be due in part to subpopulations of slowly replicating or nonreplicating Mycobacterium tuberculosis bacilli exhibiting phenotypic tolerance to most antibiotics in the standard treatment regimen. Confounding this problem is the increasing incidence of heritable multidrug-resistant M. tuberculosis. A search for new antimycobacterial chemical scaffolds that can kill phenotypically drug-tolerant mycobacteria uncovered tricyclic 4-hydroxyquinolines and a barbituric acid derivative with mycobactericidal activity against both replicating and nonreplicating M. tuberculosis. Both families of compounds depleted M. tuberculosis of intrabacterial magnesium. Complete or partial resistance to both chemotypes arose from mutations in the putative mycobacterial Mg2+/Co2+ ion channel, CorA. Excess extracellular Mg2+, but not other divalent cations, diminished the compounds’ cidality against replicating M. tuberculosis. These findings establish depletion of intrabacterial magnesium as an antimicrobial mechanism of action and show that M. tuberculosis magnesium homeostasis is vulnerable to disruption by structurally diverse, nonchelating, drug-like compounds.

The HGF/MET pathway is frequently activated in a variety of cancer types. Several selective small molecule inhibitors of the MET kinase are currently in clinical evaluation, in particular for NSCLC, liver, and gastric cancer patients. We report herein the discovery of a series of triazolopyridazines that are selective inhibitors of wild-type (WT) MET kinase and several clinically relevant mutants. We provide insight into their mode of binding and report unprecedented crystal structures of the Y1230H variant. A multiparametric chemical optimization approach allowed the identification of compound 12 (SAR125844) as a development candidate. In this chemical series, absence of CYP3A4 inhibition was obtained at the expense of satisfactory oral absorption. Compound 12, a promising parenteral agent for the treatment of MET-dependent cancers, promoted sustained target engagement at tolerated doses in a human xenograft tumor model. Preclinical pharmacokinetics conducted in several species were predictive for the observed pharmacokinetic behavior of 12 in cancer patients.

INTRODUCTION Mycobacterium tuberculosis (Mtb) is the leading global cause of lethal infection in humans and accounts for the largest number of drug-resistant infections by a single bacterial pathogen. Resistance is particularly high against the most widely prescribed tuberculosis (TB) drug, isoniazid. Isoniazid blocks synthesis of mycolates, ultralong-chain fatty acids that provide structure to the waxy coat that surrounds Mtb cells and are incorporated into some of its virulence lipids. There is currently no known method to block the synthesis of both mycolates and nonmycolate-containing virulence lipids of Mtb at a single point of control. One such control point is phosphopantetheinyl transferase (PptT). PptT transfers 4′-phosphopantetheine (Ppt) from coenzyme A (CoA) to acyl carrier proteins (ACPs) that synthesize the lipids critical to Mtb structural integrity and virulence. RATIONALE TB drug discovery often begins with whole-cell, high-throughput screens that yield compounds that kill Mtb by unknown means. Selection of Mtb mutants resistant to these compounds can indicate candidate targets of the active compound, but experimental validation is required to confirm the functionally relevant target, which is often an enzyme. A suitable target must be essential in vivo, such that its inhibition precludes development of TB in animal models, but also “vulnerable,” meaning that a pharmacologically attainable level of inhibition should be lethal to Mtb within a patient. The inhibitor should act only on Mtb, and resistance should be rare. RESULTS Screening a chemical library revealed an amidino-urea compound called “8918” that kills Mtb, including drug-resistant clinical isolates. 8918 inhibits Mtb in mice and spares other bacteria, yeast, and mammalian cells. Rare Mtb mutants resistant to 8918 bore a point mutation in the PptT gene rv2794c, altering an amino acid residue overlying the Ppt-binding pocket of PptT. When Mtb carried the mutant allele as an extra copy of rv2794c, the Mtb was protected from 8918. 8918 inhibited recombinant PptT, albeit noncompetitively and incompletely. The impact of 8918 on the Mtb metabolome and lipids was consistent with inhibition of PptT in the intact cell. A crystal structure of the PptT-8918 complex at 1.8-Å resolution confirmed that 8918 binds within the Ppt binding pocket, adjacent to the phosphoadenosine phosphate portion of CoA. Intact CoA remained in the PptT-8918 complex, but the Ppt arm was displaced, decreasing but not abolishing PptT’s catalytic activity. Strains of Mtb producing reduced amounts of PptT became hypersensitive to 8918. It was puzzling that even partial inhibition of PptT killed Mtb. We observed that mutants with disruption of rv2795c, a gene encoding a hypothetical protein, were also highly resistant to 8918. Recombinant Rv2795c protein hydrolyzed Ppt from a mycolate-building holo-ACP that is a substrate for PptT. The action of this Ppt hydrolase (PptH) resembled that of nonhomologous enzymes called ACP hydrolases that remove Ppt from ACPs in vitro but whose physiological function is unknown. CONCLUSION We identified a small molecule that kills Mtb by inhibiting PptT, demonstrating that a key enzyme in CoA metabolism is a viable target for TB drug development. Even partial inhibition of PptT is toxic to Mtb, likely because PptH synergizes with the inhibitor by undoing the PptT reaction. PptT and PptH are co-regulated by translation from the same operon, and thus Mtb cannot respond to inhibition of PptT by making more PptT without also generating more PptH. The joint functioning of PptT and PptH suggests that Mtb closely regulates the activation of ACPs. The transcriptional co-regulation and constitutive function of both members of the PptT-PptH couple suggests that a posttranslational signal that impairs PptT more than PptH could allow Mtb to rapidly reverse a prior commitment to synthesis of its metabolically most costly lipids.

In our quest for new antibiotics able to address the growing threat of multidrug resistant infections caused by Gram-negative bacteria, we have investigated an unprecedented series of non-quinolone bacterial topoisomerase inhibitors from the Sanofi patrimony, named IPYs for imidazopyrazinones, as part of the Innovative Medicines Initiative (IMI) European Gram Negative Antibacterial Engine (ENABLE) organization. Hybridization of these historical compounds with the quinazolinediones, a known series of topoisomerase inhibitors, led us to a novel series of tricyclic IPYs that demonstrated potential for broad spectrum activity, in vivo efficacy, and a good developability profile, although later profiling revealed a genotoxicity risk. Resistance studies revealed partial cross-resistance with fluoroquinolones (FQs) suggesting that IPYs bind to the same region of bacterial topoisomerases as FQs and interact with at least some of the keys residues involved in FQ binding.

A series of imidazo[1,2-a]indeno[1,2-e]pyrazin-4-ones that potently inhibit M. tuberculosis glutamine synthetase (GlnA1) has been identified by high throughput screening. Exploration of this series was performed owing to a short chemistry program. Despite possibly nanomolar inhibitions, none of these compounds was active on whole cell Mtb, suggesting that GlnA1 may not be a suitable target to find new anti-tubercular drugs.

The total synthesis of the naturally occurring antibiotic GE81112A, a densely functionalized tetrapeptide, is reported. Comparison of spectral data with those of the natural product and the lack of biological activity of the synthesized compound led us to revise the published configuration of the 3‐hydroxypipecolic acid moiety. This hypothesis was fully validated by the synthesis of the corresponding epimer.

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase–DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed “Structure Intracellular Concentration Activity Relationship” (SICAR) concept.

A high throughput phenotypic screening against Mycobacterium smegmatis led us to the discovery of a new class of bacteriostatic, highly hydrophobic antitubercular quinazolinones that potently inhibited the in vitro growth of either extracellular or intramacrophagic M. tuberculosis (Mtb), via modulation of an unidentified but yet novel target. Optimization of the initial hit compound culminated in the identification of potent but poorly soluble Mtb growth inhibitors, three of which were progressed to in vivo efficacy studies. Despite nanomolar in vitro potency and attractive PK properties, none of these compounds was convincingly potent in our in vivo mouse tuberculosis models. This lack of efficacy may be linked to the poor drug-likeness of the test molecules and/or to the properties of the target.

The potentially through-bond “dihomoaromatic” title compound has been elaborated by means of a tandem acyloin-ring expansion sequence and shown by spectroscopic means not to possess significant ground-state polarization.

The tyrosine kinase MET is a membrane receptor that is essential for embryonic development and wound healing in normal cells. Stimulation of MET by its natural ligand, the hepatocyte growth factor (HGF), induces cell proliferation, migration, and invasion. Abnormal MET activation (over-expression of MET protein, amplification or mutations of the MET gene) has been observed in multiple human cancer types. We report herein the discovery of a potent and selective small molecule inhibitor (SAR125844) with potential therapeutic application in cancer patients with deregulated MET-dependent malignancies. Our initial hit identification approach was based on the biochemical screen of an in-house kinase inhibitor biased library where a series of benzimidazole sulfonate derivatives were identified with sub-micromolar MET inhibition. In particular, the initial hit exhibited an IC50 of 140 nM vs MET but it also had strong affinity for CDK9 (IC50= 6 nM), a CDK isoform involved in gene transcription. Chemical modifications of the series to dial out CDK9 affinity and remove potential normal cell cytotoxicity led to a more selective derivative with IC50's of 80nM and 1355nM vs MET and CDK9 respectively. Further sub-structural exploration allowed us to identify a heteorocyclic moiety which was shown by X-ray data to specifically interact with Tyr1230 in a non active conformation of the protein. The resulting highly favourable U-shape mode of binding in MET of representative examples from these series (e.g. IC50= 1nM) was not tolerated in CDK9 (IC50 > 10µM). Final multi-parametric medicinal chemistry optimisation led to SAR125844 with single digit nanomolar antiproliferative activity on MET-amplified cell lines. SAR125844 is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. This compound exhibits also satisfactory eADMET in vitro properties and has shown moderate total plasma clearance, large volume of distribution and moderate to long terminal elimination half-life in rats. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2911. doi:1538-7445.AM2012-2911