Development Of New Chemotherapeutics For Tuberculosis

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

Currently this project focuses on five key areas: (1) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mycobacterium tuberculosis (MTb) under in vivo relevant conditions, (2) pre-clinical candidate development of gyrase B inhibitor with optimized activity against MTb, (3) identification of environmental organisms that produce anti-tubercular secondary metabolites, (4) unraveling the mechanism of action of hits of interest as well as the mechanisms by which the pathogen adapts to the xenobiotic stress either through modulation of compound uptake, compound metabolism or mutations in the target pathway and in (5) we are exploring the physiological function of important mycobacterial enzymes and microbial biochemistry underlying host pathogenesis. In Project (1) in which we are screening compound libraries obtained from global collaborators including pharmaceutical companies to identify inhibitors of MTb growth under in vivo relevant conditions, performing dose-titration follow-up of hits and synthesizing or purchasing chemically similar compounds. These series are evaluated using secondary screens with a battery of conditions that are thought to be relevant during in vivo growth of MTb. We are using a 4-tiered hit prioritization approach to bin compounds into major mechanistic classes, in particular highlighting those compounds that hit well-known targets in cell wall synthesis or respiration and excluding generally cytotoxic compounds. Hit series with multiple members showing activity for the scaffold with low-complexity, acceptable solubility and promising physicochemical properties for profiling are prioritized for follow-up to determine if the desirable balance of potency and ADME (absorption, distribution, metabolism and excretion) properties could be achieved in Lead Optimization. In contrast, series with structural alerts suggesting toxicophores are deprioritized. Preliminary SAR to determine the key pharmacophore through the synthesis and testing of selected compounds that interrogate each structural moiety of a hit was completed for a half dozen selected series since October 2022. Kinetic and thermodynamic solubility determinations and microsomal stability assays are also done to further develop the information that will be essential to facilitate go / no-go progression into lead optimization. In project 2, after promising results in human single-ascending-dose safety testing of our lead oxazolidinone developed jointly with Merck, we are working with the same Merck team on developing a gyrase B inhibitor with an increased potency against MTb compared to other bacteria to decrease adverse events associated with disruption of the human microbiome. The compounds bind to the ATPase site of the gyrase B subunit. Genome-wide CRISPRi studies have revealed that the open reading frames encoding the two subunits of the gyrase enzyme, are amongst the most vulnerable genes during transcriptional interference leading to rapid loss of viability. We are developing two lead series that have nanomolar potency against the pathogen by optimizing PK/PD properties. Animal toxicity studies are in progress on the lead compound which will be followed by in vivo efficacy studies to provide the proof-of-concept results needed to progress these series for candidate selection. In parallel, we are confirming on-target activity of the compounds by sequencing of resistant mutants of MTb raised against the compounds as well as evaluation of their inhibition of the ATPase activity of the gyrase enzyme. In project 3, we are interrogating different sources of natural products to identify novel inhibitors of MTb metabolism. We are setting up multiplexed screens of different hypomorph strains of MTb that have been labeled with a panel of non-overlapping fluorescent proteins, to identify not only the panel of hits that inhibit a diversity of targets in the organism but also to identify those that selectively target genes of interest in the hypomorph strains. The NCI natural product collection, the largest collection of natural chemical diversity in the world consisting of crude and partially purified fractions, will be screened in these assays. In addition, we have identified sphagnum peat bogs as environmental reservoirs that are rich in mycobacteria that compete with other environmental bacteria for limited nutrients under conditions of hypoxia and acidic stress which mimic aspects of the human granuloma. We have processed fungal isolates for production of antitubercular compounds by selecting those for which production of these growth inhibitory metabolites is specifically induced by co-culture with MTb. We have screened extracts from these organisms not only against MTb but also other important bacterial pathogens. Comparison of whole genome sequencing information from the fungal isolates with RNAseq data of these organisms obtained under inducing co-culture as well as non-inducing conditions, allowed us to identify the biosynthetic gene clusters in the genome that are induced by MTb. The probable identity of the secondary metabolite was predicted by functional genomics based on the known enzymatic activities of the protein products of the induced genes and confirmed by chemical synthesis, analytical characterization and bioassay testing of the synthetic product. In project 4, target identification for prioritized series is initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, and metabolomics analyses. For top hits of interest where SAR indicates that certain positions on the molecule can be modified while retaining anti-tubercular activity, we have chemically modified the compounds by addition of a linker that can be UV-crosslinked onto the putative targets, as well as a linker moiety that provides a handle allowing purification of the resultant ligand-protein complexes. This chemical biology approach is guiding our efforts in target identification. Recombinant expression of predicted target proteins and enzymatic analysis in the presence or absence of the hit of interest has enabled on-target confirmation for these compounds. One of our hit series currently in lead-optimization is a pro-drug that likely produces a radical intermediate. We have synthesized radiolabeled analogs of this compound to identify protein and non-protein targets in MTb. In project 5 we are continuing work to explore the importance of respiratory pathways, the biosynthesis of various cofactors, as well as MTb-specific metabolites such as mycocyclosin in maintaining viability under replicating, non-replicating and during pathogenesis of the host.