We recently reported C48, a potent and orally effective inhibitor targeting biotin biosynthesis in Mycobacterium tuberculosis (Mtb). Notably, C48 exhibited a favorable pharmacokinetic profile and suppressed Mtb growth in a mouse model that recapitulates human biotin physiology, demonstrating that biotin is a validated target for antibacterial agents. This paper details the previously undisclosed lead optimization studies, which employed rational drug design by the strategic introduction of fluorine, reduction of rotatable bonds, and incorporation of nitrogen atom to enhance π-π stacking. A series of novel BioA inhibitors were designed, synthesized, and evaluated with respect to their biochemical properties, in vitro ADME, and pharmacokinetic profiles. Key analogues were tested against isogenic Mtb strains (BioA underexpressed or overexpressed) to confirm on-target engagement. C48 emerged as the most potent candidate with minimum inhibitory concentrations (MICs) ranging from 0.012 to 0.093 μM against a panel of drug-sensitive and drug-resistant Mtb strains.

Structure-activity relationship studies of previously reported 3-(4,4-dimethyl-1,4-azasilinane)methylpyrazoles with potent anti-tuberculosis activity were conducted to identify leads with drug-like properties by optimizing the lipophilicity of the compounds. Removal of phenyl substituents at 1 or 5 positions of the pyrazole ring or introducing polar substituents on the 5-phenyl ring identified potent compounds with lower logD and improved solubility. Compounds with a C5-cyclopentyl substituent showed improved stability in human microsomes. In vitro and in vivo metabolite identification studies were conducted to facilitate further compound optimization. Compounds are bactericidal in vitro against replicating Mycobacterium tuberculosis (Mtb) and retain activity against drug-resistant Mtb. Profiling against the MmpL3 TetON and tet-inducible over-expression (OE) mutants confirmed direct inhibition of the MtbMmpL3 transporter as a mode-of-action of the compounds.

Abstract Despite the approval of two first-in-class anti-tuberculars over the past two decades, the global burden of tuberculosis (TB) remains unacceptably high, in part due to the emergence and spread of drug-resistant strains of Mycobacterium tuberculosis (Mtb). This review summarizes advances and ongoing challenges in anti-TB drug discovery, focusing on identifying and validating novel targets. Highlighted is a framework developed by the TB Drug Accelerator (TBDA) consortium for target validation in Mtb. Two computational platforms, DAIKON and PARSNIP, allow the systematic evaluation of targets across multiple dimensions, including chemical validation, genetic essentiality, vulnerability, and the feasibility to identify drug-like molecules for a target of interest. Case studies of Pks13 and NadE illustrate how these parameters guide target prioritization and risk assessment. By integrating these metrics, the framework enables dynamic, transparent target ranking, supporting development of both pan-TB and treatment-shortening regimens. This paradigm is adaptable to other bacterial pathogens and is designed to improve evidence-based decision-making in antibacterial drug discovery.

Globally, Mycobacterium tuberculosis remains a significant burden. Although effective treatment regimens exist, drug resistance has continued to emerge. This clinical resistance, combined with side effects and protracted treatment times from the current front-line therapies, means that there is a need to identify novel agents to combat this disease. Here, we report on a new chemical series, identified by whole-cell phenotypic growth inhibition screening, that demonstrates significant activity across multiple media. Mode of action studies indicate that this series targets the same biological pathway as ethambutol (EMB), a drug used in the current front-line treatment of tuberculosis. Screening selected analogues against clinical isolates, resistant to EMB, demonstrated differential sensitivity both across the molecules and against the different specific resistant mutations. The data obtained suggest that this series has potential to be developed into a viable alternative to EMB.

The intrinsic drug resistance of Mycobacterium tuberculosis (Mtb) is a major barrier to effective tuberculosis (TB) treatment and is largely due to its complex, impermeable cell envelope. We identified a periplasmic protein complex comprising FecB and Rv3035 that is essential for maintaining envelope integrity and mediating intrinsic multidrug resistance in Mtb. FecB interacts with Rv3035, forming a stable heterodimer that associates with the cell envelope biosynthesis protein AftB. We report the structures of Rv3035 alone and in complex with FecB and identify critical residues for complex formation and function. Coessentiality and genetic interaction analyses support a functional link between FecB, Rv3035, and AftB, an arabinofuranosyltransferase that synthesizes arabinogalactan and lipoarabinomannan. Loss of FecB or Rv3035 disrupted AftB-mediated arabinan synthesis, suggesting that these proteins support AftB's enzymatic activity. FecB is required for Mtb virulence in mice, underscoring its physiological relevance. These findings highlight FecB, Rv3035, and AftB as promising therapeutic targets.

Controlled human infection models (CHIMs) can accelerate vaccine development for infectious diseases. Mycobacterium tuberculosis is a human-adapted pathogen that is the leading infectious cause of death worldwide. M tuberculosis infection results in a spectrum of clinical outcomes that are incompletely modelled in animals. To date, the risks of infection, prolonged treatment, and sequelae related to CHIMs with M tuberculosis have been considered ethically unacceptable. However, recent advances in bacterial engineering have resulted in safe strains that could permit M tuberculosis CHIM studies with reduced risks. In this Personal View, we address the practical considerations for conducting a pulmonary M tuberculosis CHIM study. We summarise the ethical issues of M tuberculosis CHIM studies in tuberculosis-endemic and non-endemic settings; describe safety considerations, such as optimising the challenge dose and minimising risks to third parties; and outline and prioritise clinical, microbiological, immunological, and radiological endpoints that would render such a model useful for vaccine development.

Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. Using a library comprising ~20,000 sgRNAs covering >96% of Mtb open reading frames, we identified genes required for growth within the changing immune microenvironment. Mutant depletion patterns varied across immune environments sampled at 2, 4, and 6 weeks post-infection, which revealed a weighted dependency on cell wall biosynthesis genes early and the reliance on cholesterol catabolism and iron acquisition across all time points. Functional validation of three genes-embB, fadE29, and mbtI-confirmed their temporal significance in vivo. This screen provides increased resolution of the differential metabolic vulnerabilities in Mtb in the evolving immune environments during infection, stressing the temporal nature of conditional essentiality in vivo. IMPORTANCE:Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. This novel approach enabled us to identify how different bacterial metabolic pathways were of greater importance to the bacterium at different time points post-infection. The results provide insights into how the evolving immune response to infection shapes the metabolic and replicative status of the bacterium. This information has significance in the design of therapeutic strategies toward cure.

The increasing prevalence of antimicrobial resistance is an important challenge that warrants new approaches to antibiotic development. Currently, all antibiotics inhibit biological processes. To explore whether activation of a biochemical pathway can elicit bactericidal effects we engineered variants of Mycobacterium tuberculosis ATP-phosphoribosyltransferase (ATP-PRT) that are resistant to allosteric inhibition by L-histidine, leading to supraphysiological activation of ATP-PRT and L-histidine overproduction. Upregulation of L-histidine biosynthesis significantly reduces the growth of M. tuberculosis in culture and causes a loss of fitness owing to nutrient and energy depletion. Moreover, the expression of allosteric variants in M. tuberculosis significantly reduced infections in human macrophages and in a mouse model of infection. Thus, metabolic activation represents a new mycobactericidal mechanism that could be applied to antimycobacterial drug discovery.

The human pathogen Mycobacterium tuberculosis (Mtb) thrives in lipid-rich microenvironments. A strong body of evidence demonstrated that, during infection, Mtb utilizes long-chain fatty acids (LCFA) as a preferred carbon source. However, LCFA also have antimicrobial properties. Mtb must therefore employ mechanisms to utilize LCFA while mitigating their toxicity. Using transposon sequencing (TnSeq), we defined the Mtb LCFA resistome as comprising 38 genes. Surprisingly, LCFA resistance requires diverse metabolic pathways, indicating pleiotropic effects of LCFA on Mtb physiology. We investigated the function of the TnSeq top-hit, the universal stress protein TB15.3, and demonstrate that it participates in a "metabolic brake" mechanism restricting LCFA uptake and catabolism to prevent membrane hyperpolarization. TB15.3 absence caused Mtb to lose viability during chronic infection in mice and in an in vitro caseum model. Our work highlights Mtb LCFA resistance mechanisms as an important host adaptation and a promising target space for drug development.

Despite significant advancements in drug development and discovery, tuberculosis remains one of the world's deadliest infectious diseases. The rise and global spread of multidrug-resistant strains underscore a critical need for new antibiotics with distinct mechanisms of action. Screening of a myxobacterial extract library uncovered the previously unrecognized antitubercular activity of corramycin, a natural product with potent bactericidal activity against both drug-sensitive and -resistant Mycobacterium tuberculosis. Corramycin enters the bacterial cell by exploiting multiple transport systems, and induces DNA double-strand breaks through a novel form of DNA gyrase poisoning. The cryo-electron microscopy structure of M. tuberculosis gyrase in complex with corramycin reveals that the natural product targets a site overlapping with the binding site of synthetic fluoroquinolones, thereby locking the gyrase in an inactive conformation and preventing re-ligation of DNA. Importantly, its unique mode of binding allows corramycin to overcome fluoroquinolone resistance, highlighting its promise as a novel antibiotic scaffold to address the antimicrobial resistance crisis.

We previously reported an antibiotic discovery screening platform that identifies whole-cell active compounds with high sensitivity while simultaneously providing mechanistic insight, necessary for hit prioritization. Named PROSPECT, (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), this platform measures chemical-genetic interactions between small molecules and pooled Mycobacterium tuberculosis mutants, each depleted of a different essential protein. Here, we introduce Perturbagen CLass (PCL) analysis, a computational method that infers a compound's mechanism-of-action (MOA) by comparing its chemical-genetic interaction profile to those of a curated reference set of 437 known molecules. In leave-one-out cross-validation, we correctly predict MOA with 70% sensitivity and 75% precision, and achieve comparable results (69% sensitivity, 87% precision) with a test set of 75 antitubercular compounds with known MOA previously reported by GlaxoSmithKline (GSK). From 98 additional GSK antitubercular compounds with unknown MOA, we predict 60 to act via a reference MOA and functionally validate 29 compounds predicted to target respiration. Finally, from a set of ~5,000 compounds from larger unbiased libraries, we identify a novel QcrB-targeting scaffold that initially lacked wild-type activity, experimentally confirming this prediction while chemically optimizing this scaffold. PCL analysis of PROSPECT data enables rapid MOA assignment and hit prioritization, streamlining antimicrobial discovery.

Characterizing genetic essentiality across various conditions is fundamental for understanding gene function. Transposon sequencing (TnSeq) is a powerful technique to generate genome-wide essentiality profiles in bacteria and has been extensively applied to Mycobacterium tuberculosis (Mtb). Dozens of TnSeq screens have yielded valuable insights into the biology of Mtb in vitro, inside macrophages, and in model host organisms. Despite their value, these Mtb TnSeq profiles have not been standardized or collated into a single, easily searchable database. This results in significant challenges when attempting to query and compare these resources, limiting our ability to obtain a comprehensive and consistent understanding of genetic conditional essentiality in Mtb. We address this problem by building a central repository of publicly available Mtb TnSeq screens, the Mtb transposon sequencing database (MtbTnDB). The MtbTnDB is a living resource that encompasses to date ≈150 standardized TnSeq screens, enabling open access to data, visualizations, and functional predictions through an interactive web app (www.mtbtndb.app). We conduct several statistical analyses on the complete database, such as demonstrating that (i) genes in the same genomic neighborhood have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles are enriched for genes from similar functional categories. We further analyze the performance of machine learning models trained on TnSeq profiles to predict the functional annotation of orphan genes in Mtb. By facilitating the comparison of TnSeq screens across conditions, the MtbTnDB will accelerate the exploration of conditional genetic essentiality, provide insights into the functional organization of Mtb genes, and help predict gene function in this important human pathogen.

Mycobacterium tuberculosis (Mtb) remains the world's deadliest bacterial pathogen1. There is an urgent medical need to develop new drugs that shorten the treatment duration to combat widespread multi-drug-resistant and extensive-drug-resistant Mtb. Here, we present a preclinical covalent compound, CMX410, that contains an aryl fluorosulfate (SuFEx)2 warhead and uniquely targets the acyltransferase domain of Pks13, an essential enzyme in cell-wall biosynthesis. CMX410 is equipotent against drug-sensitive and drug-resistant strains of Mtb and efficacious in multiple mouse models of infection. Inhibition by CMX410 is irreversible through a previously undescribed mechanism: CMX410 reacts with the catalytic serine of the AT domain of Pks13, rapidly and irreversibly disabling the active site by forming a β-lactam. CMX410 is highly selective for its target and thus demonstrates excellent pharmacological and safety profiles, including no adverse effects in a 14-day rat toxicity study up to 1,000 mg kg-1 per day. The distinctive mode of action from current drugs, high potency across all tested clinical isolates, oral bioavailability, favourable performance in drug combination testing and superior pharmacological and safety characteristics make CMX410 a promising first-in-class candidate to replace outdated cell-wall biosynthesis inhibitors, such as isoniazid and ethambutol, in tuberculosis regimens.

The antibacterial agent Bio-AMS is metabolized in vivo through hydrolysis of the central acyl-sulfamide linker leading to high clearance and release of a moderately cytotoxic metabolite M1. Herein, we disclose analogues designed to prevent the metabolism of the central acyl-sulfamide moiety through steric hindrance or attenuation of the acyl-sulfamide electrophilicity. Bio-9 was identified as a metabolically stable analogue with a single-digit nanomolar dissociation constant for biotin protein ligase (BPL) and minimum inhibitory concentrations (MICs) against Mycobacterium tuberculosis and Staphylococcus aureus ranging from 0.2 to 20 μM. The antibacterial activity of Bio-9 was dependent on BPL expression level and was more than 70-fold better against a strain underexpressing BPL and, conversely, more than 5-fold less effective against a strain overexpressing BPL. Pharmacokinetic and metabolic studies demonstrated that Bio-9 was metabolically stable in vivo, showing negligible hydrolysis that translated to substantially reduced clearance and concomitantly boosted drug exposure and half-life compared to Bio-AMS.

Structure-guided optimization was applied to develop a potent and selective inhibitor of the aminotransferase BioA, a key enzyme in bacterial biotin biosynthesis. Strategic modifications of a screening hit enhanced potency and pharmacokinetics, yielding C48, which binds BioA with a Ki of 200 pM and displays sub-micromolar MICs against Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria. Biochemical, structural, and genetic studies confirmed C48's mechanism of action. In vitro, C48 induced cell envelope stress and membrane remodeling, mimicking biotin starvation. Pharmacokinetic profiling revealed excellent oral bioavailability resulting in over 39,000-fold improved exposure versus the parent compound. To address the discrepancy in biotin levels between humans and mice, we developed a low-biotin mouse model that recapitulates human biotin physiology. In this model C48 reduced Mtb burden in lungs and spleen, providing the first in vivo proof-of-concept for targeting biotin biosynthesis as a therapeutic strategy against tuberculosis.

The intrinsic drug resistance of Mycobacterium tuberculosis (Mtb) is a major barrier to effective tuberculosis (TB) treatment and is largely attributed to its complex, impermeable cell envelope. We identified a periplasmic protein complex comprising FecB and Rv3035 that is essential for maintaining cell envelope integrity and mediating intrinsic multidrug resistance in Mtb. FecB interacts directly with Rv3035, forming a stable heterodimer that also associates with the cell envelope biosynthesis proteins AftB and Rv0227c. We report the structures of Rv3035 alone and in complex with FecB, and identify critical residues required for complex formation and function. Co-essentiality analysis and genetic interaction experiments further supported a functional link between FecB, Rv3035 and AftB, the enzyme responsible for the last step in the biosynthesis of the cell envelope components arabinogalactan and lipoarabinomannan. Mtb mutants of fecB , rv3035 and aftB accumulated trehalose monomycolates in the outer membrane, consistent with a perturbation of membrane assembly and/or maintenance. Importantly, we show that FecB is required for Mtb virulence in mice, underscoring its physiological relevance. Altogether, our findings uncover a previously uncharacterized periplasmic protein complex that supports cell envelope integrity and intrinsic drug resistance in Mtb, highlighting FecB and Rv3035 as promising targets for therapeutic intervention. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, https://ror.org/021nxhr62, Graduate Research Fellowship Grant No. 2139291 to TK Potts Memorial Foundation, Predoctoral Fellowship to TK

Although not currently in the infectious disease spotlight, there is still a pressing need for new agents to treat tuberculosis caused by Mycobacterium tuberculosis. As there is an ever-increasing amount of clinical resistance to the current drugs, ideally new drugs would be found against novel targets to circumvent pre-existing resistance. A phenotypic growth screen identified a novel singleton, 1, as an inhibitor of M. tuberculosis growth. Mechanism-of-action studies determined that 1 targeted Pks13, an essential enzyme in cell wall biosynthesis that, as of yet, has not been targeted by agents in the clinic. The reactive nature of the pentafluorophenyl warhead meant that the molecule was inherently metabolically unstable. A medicinal chemistry optimization program is described that resulted in the identification of a compound that was reactive enough to still inhibit Pks13 and M. tuberculosis growth while being metabolically stable enough to explore in vivo.

Amides of squaric acid are new drug candidates with activity against mycobacteria. Like the approved drug bedaquiline, these compounds achieve efficacy by inhibiting mycobacterial ATP synthase. However, squaramides have a different binding site than bedaquiline and possess the potential to inhibit bedaquiline-resistant strains. We developed an optimized synthesis for monoamino-substituted squaric acid analogues. Guided by an atomic model of a squaramide compound bound to its target, we synthesized 31 new monoamino/diamino-substituted squaric acid derivates. The efficacy of these compounds was determined in whole-cell assays against Mycobacterium tuberculosis and Mycobacterium avium. The molecular target was confirmed with measurement of inhibition of Mycobacterium smegmatis ATP synthase and by using M. tuberculosis strains that modulate the expression of ATP synthase. Compared to earlier squaramides, several analogues demonstrated micromolar activity against M. tuberculosis, improved microsomal stability in vitro, and reduced cytotoxicity. These properties contribute to the preclinical development of this class of compound.