Development Of New Chemotherapeutics For Tuberculosis

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Tuberculosis has recently re-emerged as a public health concern in the United States. Current treatments require a minimum of 4 months of treatment which becomes cumulatively expensive not only due to the cost of the drugs but also due to the requirement of therapeutic monitoring to ensure adherence during the extensive treatment. To address the need for faster and safer therapies, 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, we are screening compound libraries obtained from 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. We completed the screens and dose response confirmations of a 250,000 compound AstraZeneca library and a 30,000 Global Health Chemical diversity library. 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 dozen selected series since October 2024. 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, we completed studies to explore the mechanistic underpinning of the anti-tubercular selectivity of an oxazolidinone that has succeeded in phase I clinical testing at the Gates Medical Research Institute. Cryo-EM studies with stalled mycobacterial ribosomes bound to the inhibitor indicated that key stabilizing interactions were missing from the human mitochondrial ribosome explaining the selectivity of these for the mycobacterial ribosome. The successful outcome of the phase I clinical trials this project developed jointly with Merck, prompted further collaboration with the same team in developing a gyrase B inhibitor with 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. The lead series which had nanomolar potency against the pathogen, associated with a very low frequency of resistance and excellent in vivo efficacy as demonstrated by TRS was further optimized for improved PK/PD properties. The optimized lead will progress is being tested for in vivo efficacy in combination with a panel of other anti-tubercular drugs to demonstrate its ability to reduce the duration of chemotherapy and is expected to progress to phase I safety studies soon thereafter. In project 3, we are interrogating different sources of natural products to identify novel inhibitors of MTb metabolism. We have developed 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. Our future goal is to complete screening of the NCI natural product collection, the largest collection of natural chemical diversity in the world consisting of crude and partially purified fractions. Our previous work on environmental reservoirs of mycobacteria competing with other environmental organisms uncovered a range of fungi that produced highly selective antitubercular metabolites upon coincubation with MTb. This work exposed unique metabolic vulnerabilities of the pathogen that we are currently exploiting for drug design. In project 4, target identification for prioritized drug candidate series is initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, transcriptional profiling by RNAseq, 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 that is entering selection phase for phase I safety testing, is a pro-drug that catastrophically disrupts thiol homeostatic mechanisms. We have uncovered the metabolic fate of the pro-drug during its activation in MTb and how the enzymatic conversion differs from in vitro chemical oxidation of the molecule. We have developed additional reporter strains of MTb that inform on the engagement of target pathways including disruption of thiol homeostasis, intracellular ATP levels and respiratory redox homeostasis. In project 5 we are continuing work to explore the importance of respiratory pathways, the function of novel toxin/anti-toxin systems in bacterial pathogenesis, the biosynthesis and metabolic role of various cofactors including alternative electron carriers, proteins expressed by MTb that modulate host cell respiration, as well as MTb-specific metabolites such as mycocyclosin in maintaining viability under replicating, non-replicating and during pathogenesis of the host.