Triazolopyrimidines Target Aerobic Respiration in Mycobacterium tuberculosis.

We previously identified a series of triazolopyrimidines with antitubercular activity. We determined that Mycobacterium tuberculosis strains with mutations in QcrB, a subunit of the cytochrome bcc-aa3 supercomplex, were resistant. A cytochrome bd oxidase deletion strain was more sensitive to this series. We isolated resistant mutants with mutations in Rv1339. Compounds led to the depletion of intracellular ATP levels and were active against intracellular bacteria, but they did not inhibit human mitochondrial respiration. These data are consistent with triazolopyrimidines acting via inhibition of QcrB.

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections.

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.

8-Hydroxyquinolines are bactericidal against Mycobacterium tuberculosis.

There is an urgent need for new treatments effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. The 8-hydroxyquinoline series is a privileged scaffold with anticancer, antifungal, and antibacterial activities. We conducted a structure-activity relationship study of the series regarding its antitubercular activity using 26 analogs. The 8-hydroxyquinolines showed good activity against M. tuberculosis, with minimum inhibitory concentrations (MIC90) of <5 µM for some analogs. Small substitutions at C5 resulted in the most potent activity. Substitutions at C2 generally decreased potency, although a sub-family of 2-styryl-substituted analogs retained activity. Representative compounds demonstrated bactericidal activity against replicating M. tuberculosis with >4 log kill at 10× MIC over 14 days. The majority of the compounds demonstrated cytotoxicity (IC50 of <100 µM). Further development of this series as antitubercular agents should address the cytotoxicity liability. However, the 8-hydroxyquinoline series represents a useful tool for chemical genomics to identify novel targets in M. tuberculosis.

Synthesis and biological evaluation of aryl-oxadiazoles as inhibitors of Mycobacterium tuberculosis.

Despite increased research efforts to find new treatments for tuberculosis in recent decades, compounds with novel mechanisms of action are still required. We previously identified a series of novel aryl-oxadiazoles with anti-tubercular activity specific for bacteria using butyrate as a carbon source. We explored the structure activity relationship of this series. Structural modifications were performed in all domains to improve potency and physico-chemical properties. A number of compounds displayed sub-micromolar activity against M. tuberculosis utilizing butyrate, but not glucose as the carbon source. Compounds showed no or low cytotoxicity against eukaryotic cells. Three compounds were profiled in mouse pharmacokinetic studies. Plasma clearance was low to moderate but oral exposure suggested solubility-limited drug absorption in addition to first pass metabolism. The presence of a basic nitrogen in the linker slightly increased solubility, and salt formation optimized aqueous solubility. Our findings suggest that the 1,3,4-oxadiazoles are useful tools and warrant further investigation.

A high content microscopy assay to determine drug activity against intracellular Mycobacterium tuberculosis.

Tuberculosis is one of the infectious diseases with the greatest global burden, affecting millions of people. The rise of multi- and extensively-drug resistant forms of Mycobacterium tuberculosis over the last few decades has highlighted the urgent need for development of new drugs to treat the disease. Many drug development pipelines are based on in vitro assays examining a compound's effect on M. tuberculosis alone. These do not account for the effect of a compound on mammalian cells nor the interaction between host and pathogen. We therefore developed a live-cell fluorescence-based screen utilizing high content microscopy of mammalian macrophages infected with M. tuberculosis to screen for compounds with both substantial inhibition of M. tuberculosis growth and low cytotoxicity. Isoniazid, a first line tuberculosis drug, and staurosporine, a compound with well documented cytotoxic activity, were used to validate the assay. These and other control compounds showed results for M. tuberculosis growth consistent with the field. Together, this method of screening allows for high throughput testing of potential tuberculosis drugs while capturing more information per compound in a physiologically relevant context.

Oxadiazoles Have Butyrate-Specific Conditional Activity against Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ∼87,000 small compounds and identified compounds which demonstrated good antitubercular activity against M. tuberculosis grown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity against M. tuberculosis but which lacked cytotoxicity against mammalian cells.

The 4-aminopiperidine series has limited anti-tubercular and anti-staphylococcus aureus activity.

BACKGROUND: Tuberculosis (TB) caused by Mycobacterium tuberculosis is the leading cause of death from a bacterial infection. The 4-aminopiperidine (PIP) series has been reported as having anti-bacterial activity against M. tuberculosis. We explored this series for its potential to inhibit aerobic growth of M. tuberculosis. We examined substitution at the N-1 position and C-4 position of the piperidine and modifications of the piperidine moiety systematically to delineate structure-activity relationships influencing potency. Compounds were tested for growth-inhibitory activity against virulent M. tuberculosis. A selected set of compounds were also tested for its activity against Staphylococcus aureus. RESULTS: The compound with a norbornenylmethyl substituent at the N-1 position and N-benzyl-N-phenethylamine at the C-4 position of the piperidine (1) was the only active compound with a minimum inhibitory concentration (MIC) of 10 µM against M. tuberculosis. Compounds were not active against S. aureus. CONCLUSIONS: We were unable to derive any other analogs with MIC < 20 µM against M. tuberculosis. Therefore we conclude that the lack of activity is a liability in this series precluding it from further development.

Corrigendum: A highly potent anti-VISTA antibody KVA12123 - a new immune checkpoint inhibitor and a promising therapy against poorly immunogenic tumors.

[This corrects the article DOI: 10.3389/fimmu.2023.1311658.].

Identification of 2-Amino Benzothiazoles with Bactericidal Activity against Mycobacterium tuberculosis.

We identified an amino-benzothiazole scaffold from a whole-cell screen against recombinant Mycobacterium tuberculosis under expressing the essential signal peptidase LepB. The seed molecule had 2-fold higher activity against the LepB hypomorph. Through a combination of purchase and chemical synthesis, we explored the structure-activity relationship for this series; 34 analogs were tested for antitubercular activity and for cytotoxicity against eukaryotic cells. We identified molecules with improved potency and reduced cytotoxicity. However, molecules did not appear to target LepB directly and did not inhibit protein secretion. Key compounds showed good permeability, low protein binding, and lack of CYP inhibition, but metabolic stability was poor with short half-lives. The seed molecule showed good bactericidal activity against both replicating and nonreplicating bacteria, as well as potency against intracellular M. tuberculosis in murine macrophages. Overall, the microbiological properties of the series are attractive if metabolic stability can be improved, and identification of the target could assist in the development of this series. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis, is a serious global health problem requiring the development of new therapeutics. We previously ran a high-throughput screen and identified a series of compounds with antitubercular activity. In this paper, we test analogs of our hit molecules for activity against M. tuberculosis, as well as for activity against eukaryotic cells. We identified molecules with improved selectivity. Our molecules killed both replicating and nonreplicating bacteria but did not work by targeting protein secretion.

A highly potent anti-VISTA antibody KVA12123 - a new immune checkpoint inhibitor and a promising therapy against poorly immunogenic tumors.

Background: Immune checkpoint therapies have led to significant breakthroughs in cancer patient treatment in recent years. However, their efficiency is variable, and resistance to immunotherapies is common. VISTA is an immune-suppressive checkpoint inhibitor of T cell response belonging to the B7 family and a promising novel therapeutic target. VISTA is expressed in the immuno-suppressive tumor microenvironment, primarily by myeloid lineage cells, and its genetic knockout or antibody blockade restores an efficient antitumor immune response. Methods: Fully human monoclonal antibodies directed against VISTA were produced after immunizing humanized Trianni mice and single B cell sequencing. Anti-VISTA antibodies were evaluated for specificity, cross-reactivity, monocyte and T cell activation, Fc-effector functions, and antitumor efficacy using in vitro and in vivo models to select the KVA12123 antibody lead candidate. The pharmacokinetics and safety profiles of KVA12123 were evaluated in cynomolgus monkeys. Results: Here, we report the development of a clinical candidate anti-VISTA monoclonal antibody, KVA12123. KVA12123 showed high affinity binding to VISTA through a unique epitope distinct from other clinical-stage anti-VISTA monoclonal antibodies. This clinical candidate demonstrated high specificity against VISTA with no cross-reactivity detected against other members of the B7 family. KVA12123 blocked VISTA binding to its binding partners. KVA12123 induced T cell activation and demonstrated NK-mediated monocyte activation. KVA12123 treatment mediated strong single-agent antitumor activity in several syngeneic tumor models and showed enhanced efficacy in combination with anti-PD-1 treatment. This clinical candidate was engineered to improve its pharmacokinetic characteristics and reduce Fc-effector functions. It was well-tolerated in preclinical toxicology studies in cynomolgus monkeys, where hematology, clinical chemistry evaluations, and clinical observations revealed no indicators of toxicity. No cytokines associated with cytokine release syndrome were elevated. Conclusion: These results establish that KVA12123 is a promising drug candidate with a distinct but complementary mechanism of action of the first generation of immune checkpoint inhibitors. This antibody is currently evaluated alone and in combination with pembrolizumab in a Phase 1/2 open-label clinical trial in patients with advanced solid tumors.

Chemical Exploration of a Highly Selective Scaffold with Activity against Intracellular Mycobacterium tuberculosis.

We previously identified a phenylthiourea series with activity against intracellular Mycobacterium tuberculosis using a high-throughput, high-content assay. We conducted a catalog structure-activity relationship study with a collection of 35 analogs. We identified several thiourea derivatives with excellent potency against intracellular bacteria and good selectivity over eukaryotic cells. Compounds had much lower activity against extracellular bacteria, which was not increased by using cholesterol as the sole carbon source. Compounds were equally active against strains with mutations in QcrB or MmpL3, thereby excluding common, promiscuous targets as the mode of action. The phenylthiourea series represents a good starting point for further exploration to develop novel antitubercular agents. IMPORTANCE Mycobacterium tuberculosis is responsible for the highest number of deaths from a bacterial pathogen, with >1.5 million in 2020. M. tuberculosis is a sophisticated pathogen that can replicate inside immune cells. There is an urgent need for new drugs to combat M. tuberculosis and to shorten therapy from 6 to 24 months. We have identified a series of molecules that inhibit the growth of M. tuberculosis inside macrophages; we tested a number of derivatives to link structural features to biological activity. The compounds are likely to have novel mechanism of action and so could be developed as new agents for drug-resistant tuberculosis.

Identification of Novel Chemical Scaffolds that Inhibit the Growth of Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis is an important global pathogen for which new drugs are urgently required. The ability of the organism to survive and multiply within macrophages may contribute to the lengthy treatment regimen with multiple drugs that are required to cure the infection. We screened the MyriaScreen II diversity library of 10,000 compounds to identify novel inhibitors of M. tuberculosis growth within macrophage-like cells using high content analysis. Hits were selected which inhibited the intramacrophage growth of M. tuberculosis without significant cytotoxicity to infected macrophages. We selected and prioritized compound series based on their biological and physicochemical properties and the novelty of the chemotypes. We identified five chemical classes of interest and conducted limited catalog structure-activity relationship studies to determine their tractability. We tested activity against intracellular and extracellular M. tuberculosis, as well as cytoxicity against murine RAW264.7 and human HepG2 cells. Benzene amide ethers, thiophene carboxamides and thienopyridines were only active against intracellular bacteria, whereas the phenylthiourea series was also active against extracellular bacteria. One member of a phenyl pyrazole series was moderately active against extracellular bacteria. We identified the benzene amide ethers as an interesting series for further work. These new compound classes serve as starting points for the development of novel drugs to target intracellular M. tuberculosis.

Novel Trifluoromethyl Pyrimidinone Compounds With Activity Against Mycobacterium tuberculosis.

The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against Mycobacterium tuberculosis. Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC90 < 5 µM). We conducted a structure-activity relationship investigation for this series. We designed and synthesized an additional 44 molecules and tested all analogs for activity against M. tuberculosis and cytotoxicity against the HepG2 cell line. Substitution at the 5-position of the pyrimidinone with a wide range of groups, including branched and straight chain alkyl and benzyl groups, resulted in active molecules. Trifluoromethyl was the preferred group at the 6-position, but phenyl and benzyl groups were tolerated. The 2-pyridyl group was required for activity; substitution on the 5-position of the pyridyl ring was tolerated but not on the 6-position. Active molecules from the series demonstrated low selectivity, with cytotoxicity against eukaryotic cells being an issue. However, there were active and non-cytotoxic molecules; the most promising molecule had an MIC (IC90) of 4.9 µM with no cytotoxicity (IC50 > 100 µM). The series was inactive against Gram-negative bacteria but showed good activity against Gram-positive bacteria and yeast. A representative molecule from this series showed rapid concentration-dependent bactericidal activity against replicating M. tuberculosis bacilli with ~4 log kill in <7 days. Overall the biological properties were promising, if cytotoxicity could be reduced. There is scope for further medicinal chemistry optimization to improve the properties without major change in structural features.

MCAK associates with the tips of polymerizing microtubules.

MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.

Functional characterization of MCAK/Kif2C cancer mutations using high-throughput microscopic analysis.

The microtubule (MT)-depolymerizing activity of MCAK/Kif2C can be quantified by expressing the motor in cultured cells and measuring tubulin fluorescence levels after enough hours have passed to allow tubulin autoregulation to proceed. This method allows us to score the impact of point mutations within the motor domain. We found that, despite their distinctly different activities, many mutations that impact transport kinesins also impair MCAK/Kif2C's depolymerizing activity. We improved our workflow using CellProfiler to significantly speed up the imaging and analysis of transfected cells. This allowed us to rapidly interrogate a number of MCAK/Kif2C motor domain mutations documented in the cancer database cBioPortal. We found that a large proportion of these mutations adversely impact the motor. Using green fluorescent protein-FKBP-MCAK CRISPR cells we found that one deleterious hot-spot mutation increased chromosome instability in a wild-type (WT) background, suggesting that such mutants have the potential to promote tumor karyotype evolution. We also found that increasing WT MCAK/Kif2C protein levels over that of endogenous MCAK/Kif2C similarly increased chromosome instability. Thus, endogenous MCAK/Kif2C activity in normal cells is tuned to a mean level to achieve maximal suppression of chromosome instability.

Aurora B regulates MCAK at the mitotic centromere.

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.

K-loop insertion restores microtubule depolymerizing activity of a "neckless" MCAK mutant.

Unlike most kinesins, mitotic centromere-associated kinesin (MCAK) does not translocate along the surface of microtubules (MTs), but instead depolymerizes them. Among the motile kinesins, refinements that are unique for specific cellular functions, such as directionality and processivity, are under the control of a "neck" domain adjacent to the ATP-hydrolyzing motor domain. Despite its apparent lack of motility, MCAK also contains a neck domain. We found that deletions and alanine substitutions of highly conserved positively charged residues in the MCAK neck domain significantly reduced MT depolymerization activity. Furthermore, substitution of MCAK's neck domain with either the positively charged KIF1A K-loop or poly-lysine rescues the loss of MT-depolymerizing activity observed in the neckless MCAK mutant. We propose that the neck, analogously to the K-loop, interacts electrostatically with the tubulin COOH terminus to permit diffusional translocation of MCAK along the surface of MTs. This weak-binding interaction may also play an important role in processivity of MCAK-induced MT depolymerization.

Novel Screen to Assess Bactericidal Activity of Compounds Against Non-replicating Mycobacterium abscessus.

Mycobacterium abscessus infections are increasing worldwide. Current drug regimens are largely ineffective, yet the current development pipeline for M. abscessus is alarmingly sparse. Traditional discovery efforts for M. abscessus assess the capability of a new drug to inhibit bacterial growth under nutrient-rich growth conditions, but this does not predict the impact when used in the clinic. The disconnect between in vitro and in vivo activity is likely due to the genetic and physiological adaptation of the bacteria to the environmental conditions encountered during infection; these include low oxygen tension and nutrient starvation. We sought to fill a gap in the drug discovery pipeline by establishing an assay to identify novel compounds with bactericidal activity against M. abscessus under non-replicating conditions. We developed and validated a novel screen using nutrient starvation to generate a non-replicating state. We used alamarBlue® to measure metabolic activity and demonstrated this correlates with bacterial viability under these conditions. We optimized key parameters and demonstrated reproducibility. Using this assay, we determined that niclosamide was bactericidal against non-replicating bacilli, highlighting its potential to be included in M. abscessus regimens. In contrast, most other drugs currently used in the clinic for M. abscessus infections, were completely inactive, potentially explaining their poor efficacy. Thus, our assay allows for rapid identification of bactericidal compounds in a model using conditions that are more relevant in vivo. This screen can be used in a high-throughput way to identify novel agents with properties that promise an increase in efficacy, while also shortening treatment times.

High content, high-throughput screening for small molecule inducers of NF-κB translocation.

NF-κB is an important mediator of immune activity and its activation is essential in mounting immune response to pathogens. Here, we describe the optimization and implementation of a high-throughput screening platform that utilizes high content imaging and analysis to monitor NF-κB nuclear translocation. We screened 38,991 compounds from three different small molecule libraries and identified 103 compound as hits; 31% of these were active in a dose response assay. Several of the molecules lacked cytotoxicity or had a selectivity index of more than 2-fold. Our image-based approach provides an important first step towards identifying small molecules with immunomodulatory activity.

Identification of cyclic hexapeptides natural products with inhibitory potency against Mycobacterium tuberculosis.

OBJECTIVE: Our aim was to identify natural products with anti-tubercular activity. RESULTS: A set of ~ 500 purified natural product compounds was screened for inhibition against the human pathogen Mycobacterium tuberculosis. A series of cyclic hexapeptides with anti-tubercular activity was identified. Five analogs from a set of sixteen closely related compounds were active, with minimum inhibitory concentrations ranging from 2.3 to 8.9 µM. Eleven structural analogs had no significant activity (MIC > 20 µM) demonstrating structure activity relationship. Sequencing of resistant mutant isolates failed to identify changes accounting for the resistance phenotype.