A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance.

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop clinical, mostly pulmonary, TB. To dissect mechanisms of susceptibility in immunocompetent individuals, we developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: development of necrotic lung lesions after infection with virulent Mtb. In this study, we explored the connectivity of the sst1-regulated pathways during prolonged macrophage activation with TNF. We determined that the aberrant response of the sst1-susceptible macrophages to TNF was primarily driven by conflicting Myc and antioxidant response pathways that resulted in a coordinated failure to properly sequester intracellular iron and activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNß superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. Our findings suggest a novel link between metabolic dysregulation in macrophages and susceptibility to TB, offering insights into potential therapeutic targets aimed at modulating macrophage function and improving TB control.

Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP.

IMPORTANCE: In this paper, we describe novel inhibitors of cyclic dinucleotide phosphodiesterase enzymes from Mycobacterium tuberculosis (M.tb) (CdnP) and mammals (ENPP1). The phosphodiesterase enzymes hydrolyze cyclic dinucleotides, such as 2',3'-cyclic GMP-AMP and c-di-AMP, which are stimulator of interferon gene (STING) agonists. By blocking the hydrolysis of STING agonists, the cyclic GMP-AMP synthase (cGAS)-STING-IRF3 pathway is potentiated. There is strong evidence in tuberculosis and in cancer biology that potentiation of the cGAS-STING-IRF3 pathway leads to improved M.tb clearance and also improved antitumor responses in cancer. In addition to the identification of novel inhibitors and their biochemical characterization, we provide proof-of-concept evidence that our E-3 inhibitor potentiates the cGAS-STING-IRF3 pathway in both macrophage cell lines and also in primary human monocyte-derived macrophages.

Inhibition of host PARP1 contributes to the anti-inflammatory and antitubercular activity of pyrazinamide.

The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens treatment durations by several months despite being only weakly bactericidal. Intriguingly, PZA is also an anti-inflammatory molecule shown to specifically reduce inflammatory cytokine signaling and lesion activity in TB patients. However, the target and clinical importance of PZA's host-directed activity during TB therapy remain unclear. Here, we identify the host enzyme Poly(ADP-ribose) Polymerase 1 (PARP1), a pro-inflammatory master regulator strongly activated in TB, as a functionally relevant host target of PZA. We show that PZA inhibits PARP1 enzymatic activity in macrophages and in mice where it reverses TB-induced PARP1 activity in lungs to uninfected levels. Utilizing a PZA-resistant mutant, we demonstrate that PZA's immune-modulatory effects are PARP1-dependent but independent of its bactericidal activity. Importantly, PZA's bactericidal efficacy is impaired in PARP1-deficient mice, suggesting that immune modulation may be an integral component of PZA's antitubercular activity. In addition, adjunctive PARP1 inhibition dramatically reduces inflammation and lesion size in mice and may be a means to reduce lung damage and shorten TB treatment duration. Together, these findings provide insight into PZA's mechanism of action and the therapeutic potential of PARP1 inhibition in the treatment of TB.

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis.

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomic analysis of lungs from JHU083-treated Mtb-infected mice reveals citrulline accumulation, suggesting elevated nitric oxide (NO) synthesis, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. JHU083-treated macrophages also produce more NO potentiating their antibacterial activity. When tested in an immunocompromised mouse model of Mtb infection, JHU083 loses its therapeutic efficacy suggesting the drug's host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains.

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

The Mycobacterium tuberculosis genome at 25 years: lessons and lingering questions.

First achieved in 1998 by Cole et al., the complete genome sequence of Mycobacterium tuberculosis continues to provide an invaluable resource to understand tuberculosis (TB), the leading cause of global infectious disease mortality. At the 25-year anniversary of this accomplishment, we describe how insights gleaned from the M. tuberculosis genome have led to vital tools for TB research, epidemiology, and clinical practice. The increasing accessibility of whole-genome sequencing across research and clinical settings has improved our ability to predict antibacterial susceptibility, to track epidemics at the level of individual outbreaks and wider historical trends, to query the efficacy of the bacille Calmette-Guérin (BCG) vaccine, and to uncover targets for novel antitubercular therapeutics. Likewise, we discuss several recent efforts to extract further discoveries from this powerful resource.

Sex and Gender Differences in Tuberculosis Pathogenesis and Treatment Outcomes.

Tuberculosis remains a daunting public health concern in many countries of the world. A consistent observation in the global epidemiology of tuberculosis is an excess of cases of active pulmonary tuberculosis among males compared with females. Data from both humans and animals also suggest that males are more susceptible than females to develop active pulmonary disease. Similarly, male sex has been associated with poor treatment outcomes. Despite this growing body of evidence, little is known about the mechanisms driving sex bias in tuberculosis disease. Two dominant hypotheses have been proposed to explain the predominance of active pulmonary tuberculosis among males. The first is based on the contribution of biological factors, such as sex hormones and genetic factors, on host immunity during tuberculosis. The second is focused on non-biological factors such as smoking, professional exposure, and health-seeking behaviors, known to be influenced by gender. In this chapter, we review the literature regarding these two prevailing hypotheses by presenting human but also experimental animal studies. In addition, we presented studies aiming at examining the impact of sex and gender on other clinical forms of tuberculosis such as latent tuberculosis infection and extrapulmonary tuberculosis, which both appear to have their own specificities in relation to sex. We also highlighted potential intersections between sex and gender in the context of tuberculosis and shared future directions that could guide in elucidating mechanisms of sex-based differences in tuberculosis pathogenesis and treatment outcomes.

MDA5 RNA-sensing pathway activation by Mycobacterium tuberculosis promotes innate immune subversion and pathogen survival.

Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I-like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M. tuberculosis pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M. tuberculosis pathogenesis, we evaluated M. tuberculosis intracellular growth and innate immune responses in WT and Mda5-/- macrophages. Transfection of M. tuberculosis RNA strongly induced proinflammatory cytokine production in WT macrophages, which was abrogated in Mda5-/- macrophages. M. tuberculosis infection in macrophages induced MDA5 protein expression, accompanied by an increase in MDA5 activation as assessed by multimer formation. IFN-γ-primed Mda5-/- macrophages effectively contained intracellular M. tuberculosis proliferation to a markedly greater degree than WT macrophages. Further comparisons of WT versus Mda5-/- macrophages revealed that during M. tuberculosis infection MDA5 contributed to IL-1ß production and inflammasome activation and that loss of MDA5 led to a substantial increase in autophagy. In the mouse TB model, loss of MDA5 conferred host survival benefits with a concomitant reduction in M. tuberculosis bacillary burden. These data reveal that loss of MDA5 is host protective during M. tuberculosis infection in vitro and in vivo, suggesting that M. tuberculosis exploits MDA5 to subvert immune containment.

Intravenous BCG vaccination reduces SARS-CoV-2 severity and promotes extensive reprogramming of lung immune cells.

Bacillus Calmette-Guérin (BCG) confers heterologous immune protection against viral infections and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here, we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model. BCG vaccination conferred a modest reduction on lung SCV2 viral load, bronchopneumonia scores, and weight loss, accompanied by a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. BCG uniquely recruited immunoglobulin-producing plasma cells to the lung suggesting accelerated local antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, with a transcriptional shift away from exhaustion markers and toward antigen presentation and repair. Similarly, BCG enhanced recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, that show reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

Relationship between patient sex and anatomical sites of extrapulmonary tuberculosis in Mali.

Background: Contribution of host factors in mediating susceptibility to extrapulmonary tuberculosis is not well understood. Objective: To examine the influence of patient sex on anatomical localization of extrapulmonary tuberculosis. Methods: We conducted a retrospective cross-sectional study in Mali, West Africa. Hospital records of 1,304 suspected cases of extrapulmonary tuberculosis, available in TB Registry of a tertiary tuberculosis referral center from 2019 to 2021, were examined. Results: A total of 1,012 (77.6%) were confirmed to have extrapulmonary tuberculosis with a male to female ratio of 1.59:1. Four clinical forms of EPTB predominated, namely pleural (40.4%), osteoarticular (29.8%), lymph node (12.5%), and abdominal TB (10.3%). We found sex-based differences in anatomical localization of extrapulmonary tuberculosis, with males more likely than females to have pleural TB (OR: 1.51; 95% CI [1.16 to 1.98]). Conversely, being male was associated with 43% and 41% lower odds of having lymph node and abdominal TB, respectively (OR: 0.57 and 0.59). Conclusion: Anatomical sites of extrapulmonary tuberculosis differ by sex with pleural TB being associated with male sex while lymph node and abdominal TB are predominately associated with female sex. Future studies are warranted to understand the role of sex in mediating anatomical site preference of tuberculosis.

CRISPR Correction of the GBA Mutation in Human-Induced Pluripotent Stem Cells Restores Normal Function to Gaucher Macrophages and Increases Their Susceptibility to Mycobacterium tuberculosis.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the ß-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.

A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan sidechains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan sidechains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics and altered spatial localization of new peptidoglycan. In cell culture experiments, training of monocytes with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, resulting in unmasking of the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. This work demonstrates the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Structure-directed identification of pyridine-2-methylamine derivatives as MmpL3 inhibitors for use as antitubercular agents.

Mycobacterial membrane protein Large 3 (MmpL3), an inner membrane protein, plays a crucial role in the transport of mycolic acids that are essential for the viability of M. tuberculosis and has been a promising therapeutic target for new anti-TB agents. Herein, we report the discovery of pyridine-2-methylamine antitubercular compounds using a structure-based drug design strategy. Compound 62 stands out as the most potent compound with high activity against M. tb strain H37Rv (MIC = 0.016 µg/mL) as well as the clinically isolated strains of MDR/XDR-TB (MIC = 0.0039-0.0625 µg/mL), low Vero cell toxicity (IC50 ≥ 16 µg/mL), and moderate liver microsomal stability (CLint = 28 µL/min/mg). Furthermore, the resistant mutant of S288T due to single nucleotide polymorphism in mmpL3 was resistant to pyridine-2-methylamine 62, demonstrating compound 62 is likely target to MmpL3.

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis.

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a novel glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomics analysis of lungs from JHU083-treated Mtb-infected mice revealed reduced glutamine levels, citrulline accumulation suggesting elevated NOS activity, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. When tested in an immunocompromised mouse model of Mtb infection, JHU083 lost its therapeutic efficacy suggesting the drug's host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

Adjunctive Integrated Stress Response Inhibition Accelerates Tuberculosis Clearance in Mice.

Despite numerous advances in tuberculosis (TB) drug development, long treatment durations have led to the emergence of multidrug resistance, which poses a major hurdle to global TB control. Shortening treatment time therefore remains a top priority. Host-directed therapies that promote bacterial clearance and/or lung health may improve the efficacy and treatment duration of tuberculosis antibiotics. We recently discovered that inhibition of the integrated stress response, which is abnormally activated in tuberculosis and associated with necrotic granuloma formation, reduced bacterial numbers and lung inflammation in mice. Here, we evaluated the impact of the integrated stress response (ISR) inhibitor ISRIB, administered as an adjunct to standard tuberculosis antibiotics, on bacterial clearance, relapse, and lung pathology in a mouse model of tuberculosis. Throughout the course of treatment, ISRIB robustly lowered bacterial burdens compared to the burdens with standard TB therapy alone and accelerated the time to sterility in mice, as demonstrated by significantly reduced relapse rates after 4 months of treatment. In addition, mice receiving adjunctive ISRIB tended to have reduced lung necrosis and inflammation. Together, our findings identify the ISR pathway as a promising therapeutic target with the potential to shorten TB treatment durations and improve lung health. IMPORTANCE Necrosis of lung lesions is a hallmark of tuberculosis (TB) that promotes bacterial growth, dissemination, and transmission. This process is driven by the persistent hyperactivation of the integrated stress response (ISR) pathway. Here, we show that adjunctive ISR inhibition during standard antibiotic therapy accelerates bacterial clearance and reduces immunopathology in a clinically relevant mouse model of TB, suggesting that host-directed therapies that de-escalate these pathological stress responses may shorten TB treatment durations. Our findings present an important conceptual advance toward overcoming the challenge of improving TB therapy and lowering the global burden of disease.

Acyldepsipeptide Antibiotics and a Bioactive Fragment Thereof Differentially Perturb Mycobacterium tuberculosis ClpXP1P2 Activity in Vitro.

Proteolytic complexes in Mycobacterium tuberculosis (Mtb), the deadliest bacterial pathogen, are major foci in tuberculosis drug development programs. The Clp proteases, which are essential for Mtb viability, are high-priority targets. These proteases function through the collaboration of ClpP1P2, a barrel-shaped heteromeric peptidase, with associated ATP-dependent chaperones like ClpX and ClpC1 that recognize and unfold specific substrates in an ATP-dependent fashion. The critical interaction of the peptidase and its unfoldase partners is blocked by the competitive binding of acyldepsipeptide antibiotics (ADEPs) to the interfaces of the ClpP2 subunits. The resulting inhibition of Clp protease activity is lethal to Mtb. Here, we report the surprising discovery that a fragment of the ADEPs retains anti-Mtb activity yet stimulates rather than inhibits the ClpXP1P2-catalyzed degradation of proteins. Our data further suggest that the fragment stabilizes the ClpXP1P2 complex and binds ClpP1P2 in a fashion distinct from that of the intact ADEPs. A structure-activity relationship study of the bioactive fragment defines the pharmacophore and points the way toward the development of new drug leads for the treatment of tuberculosis.

Therapeutic potential of coumestan Pks13 inhibitors for tuberculosis.

Polyketide synthase 13 (Pks13) is an important enzyme found in Mycobacterium tuberculosis (M. tuberculosis) that condenses two fatty acyl chains to produce α-alkyl ß-ketoesters, which in turn serve as the precursors for the synthesis of mycolic acids that are essential building blocks for maintaining the cell wall integrity of M. tuberculosis Coumestan derivatives have recently been identified in our group as a new chemotype that exert their antitubercular effects via targeting of Pks13. These compounds were active on both drug-susceptible and drug-resistant strains of M. tuberculosis as well as showing low cytotoxicity to healthy cells and a promising selectivity profile. No cross-resistance was found between the coumestan derivatives and first-line TB drugs. Here we report that treatment of M. tuberculosis bacilli with 15 times the MIC of compound 1, an optimized lead coumestan compound, resulted in a colony forming unit (CFU) reduction from 6.0 log10 units to below the limit of detection (1.0 log10 units) per mL culture, demonstrating a bactericidal mechanism of action. Single dose (10 mg/kg) pharmacokinetic studies revealed favorable parameters with a relative bioavailability of 19.4%. In a mouse infection and chemotherapy model, treatment with 1 showed dose-dependent mono-therapeutic activity, whereas treatment with 1 in combination with rifampin showed clear synergistic effects. Together these data suggest that coumestan derivatives are promising agents for further TB drug development.

Improved bladder cancer antitumor efficacy with a recombinant BCG that releases a STING agonist.

Despite the introduction of several new agents for the treatment of bladder cancer (BC), intravesical BCG remains a first line agent for the management of non-muscle invasive bladder cancer. In this study we evaluated the antitumor efficacy in animal models of BC of a recombinant BCG known as BCG-disA-OE that releases the small molecule STING agonist c-di-AMP. We found that compared to wild-type BCG (BCG-WT), in both the orthotopic, carcinogen-induced rat MNU model and the heterotopic syngeneic mouse MB-49 model BCG-disA-OE afforded improved antitumor efficacy. A mouse safety evaluation further revealed that BCG-disA-OE proliferated to lesser degree than BCG-WT in BALB/c mice and displayed reduced lethality in SCID mice. To probe the mechanisms that may underlie these effects, we found that BCG-disA-OE was more potent than BCG-WT in eliciting IFN-ß release by exposed macrophages, in reprogramming myeloid cell subsets towards an M1-like proinflammatory phenotypes, inducing epigenetic activation marks in proinflammatory cytokine promoters, and in shifting monocyte metabolomic profiles towards glycolysis. Many of the parameters elevated in cells exposed to BCG-disA-OE are associated with BCG-mediated trained innate immunity suggesting that STING agonist overexpression may enhance trained immunity. These results indicate that modifying BCG to release high levels of proinflammatory PAMP molecules such as the STING agonist c-di-AMP can enhance antitumor efficacy in bladder cancer.

Structure-Based Optimization of Coumestan Derivatives as Polyketide Synthase 13-Thioesterase(Pks13-TE) Inhibitors with Improved hERG Profiles for Mycobacterium tuberculosis Treatment.

Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antitubercular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC50 = 0.52 µM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039-0.0078 µg/mL) and exhibited limited hERG inhibition (IC50 ≥ 25 µM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.