Development Of New Chemotherapeutics For Tuberculosis

目前，该项目侧重于五个关键领域：(1)在体内相关条件下对整个结核分枝杆菌（MTb）进行高通量筛选的铅分子和系列的化学合成；(2)对结核分枝杆菌（MTb）具有优化抗结核活性的gyrase B抑制剂的临床前候选开发；(3)产生抗结核次级代谢物的环境生物的鉴定；(4)揭示感兴趣的hit的作用机制以及病原体通过调节化合物摄取，化合物代谢或靶途径中的突变来适应异种生物胁迫的机制；(5)我们正在探索重要分枝杆菌酶的生理功能和宿主发病机制下的微生物生物化学。

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. Since September 2021, we have progressed scaffolds of interest that were not flagged as undesirable in our hit triage strategy through formal hit assessment. Our 4-tiered hit prioritization approach bins 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. Every attempt was made to progress as many chemo-types as possible to increase the likelihood of hitting a diversity of targets. 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. To rapidly expand the SAR for the prioritized chemotypes, commercially available analogs are purchased and tested in MIC assays. In addition, selected compounds are synthesized to explore preliminary SAR. 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 successfully delivering an optimized oxazolidinone for phase I clinical testing, we are working on developing gyrase B inhibitor with an increased potency against MTb compared to other bacteria in order to decrease adverse events associated with disruption of the human microbiome. 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 have identified two lead series that have nanomolar potency against the pathogen. PK/PD analysis, mouse efficacy and animal toxicity studies are in progress to enable candidate selection. 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 pre-fractionated fractions, will be screened in these assays. In addition, have identified environmental reservoirs that are rich in mycobacteria that compete with other environmental bacteria for limited nutrients. Specifically, sphagnum peat bogs have been reported to support diverse bacterial and fungal communities including slow-growing mycobacteria closely related to MTb that compete for nutrients under conditions that recapitulate some of the defining characteristics of human granulomas including an acidic pH, hypoxia as well as nutrient limitation. We have the largest global collection of acidobacteria with this library currently being typed based on antitubercular activity. In parallel, we have processed fungal as well as lichen samples as a novel reservoir of potential antibiotic-producing organisms. We have screened extracts from these organisms not only against MTb but also other important bacterial pathogens. To enrich for organisms that produce antibiotics that are selective for MTb, we have screened the microorganisms during co-culture with MTb with subsequent anti-tubercular testing of the supernatants to identify those organisms in which antibiotic production is induced during competition with MTb. RNAseq of these organisms under inducing as well as non-inducing conditions to identify biosynthetic gene clusters in the genome that are induced by MTb coupled with whole genome sequencing is underway. 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. Whole genome sequencing data combined with compound metabolite analyses have also highlighted the extensive repertoire of xenobiotic metabolizing enzymes that MTb possesses that either inactivate or in some cases activate the hit of interest. Our MTb metabolism screens of small molecule libraries that probe the diversity of compounds transformed by the pathogen and characterize the classes of enzymatic transformations has enabled us to identify unique enzymatic reactions that are not detectable in parallel screens of human microsome metabolism assays. An understanding of these metabolic processes will help us exploit or circumvent the activity of these enzymes in our drug development process. In a complementary approach, we are biochemically characterizing a series of hits that were identified in a DNA-encoded library screen against the central metabolic enzyme fumarase in an attempt to identify ligands that modulate the activity of the enzyme by engagement of the unique allosteric sites of the MTb protein. 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.