Gauri Shetye

The bedaquiline (B), pretomanid (Pa), and linezolid (L) (BPaL) regimen is highly effective for drug-resistant tuberculosis (TB) but is limited by linezolid-associated toxicities, including myelosuppression and peripheral neuropathy. We evaluated omadacycline as a representative tetracycline-class protein synthesis inhibitor (PSI) to assess whether it can contribute functional activity within a BPa backbone. Omadacycline exhibited a minimum inhibitory concentration (MIC) of 2–4 μg/mL against M. tuberculosis H37Rv and 10–48 μg/mL against representatives of four global clades. Daily media replacement reduced the MIC against H37Rv to 0.29 μg/mL, suggesting antibiotic instability may underestimate in vitro potency. In vivo , subcutaneous administration of omadacycline (15 mg/kg) in BALB/c mice, yielding plasma exposures within the range of those reported for the approved 300-mg oral human dose, led to a 0.7-log ₁₀ reduction in lung colony-forming units (CFU) in both acute and chronic TB models. In combination therapy, 10-week treatment with BPa plus either linezolid (BPaL) or omadacycline (BPaO) further reduced lung CFU by ∼1 log ₁₀ relative to BPa alone, demonstrating the contribution of a PSI to regimen efficacy. Notably, BPaO and BPaL reduced relapse rates after 10 weeks of treatment to 43 and 23%, respectively, compared to 100% relapse in the BPa-only group. Together, these findings provide proof-of-concept evidence that omadacycline has in vivo antitubercular activity, achieves clinically relevant exposures, and can contribute functional PSI activity in combination regimens however, these results do not establish clinical equivalence with linezolid and support further evaluation of tetracycline-class agents in TB therapy.

Disrupting protein homeostasis in Mycobacterium tuberculosis (Mtb) by targeting the ClpC1P1P2 proteolytic complex is a promising anti-TB strategy. We synthesized conformationally constrained monomeric and dimeric rufomycin 4/6 (RUF) analogs via click chemistry. While most monomeric analogs were inactive, dimeric analogs displayed potent anti-Mtb activity. Surface plasmon resonance revealed tight, slow-dissociating binding of dimers to the ClpC1 N-terminal domain (ClpC1NTD), indicating prolonged residency time. X-ray crystallography and size exclusion chromatography demonstrated that dimeric analogs induce NTD dimerization, likely shifting the equilibrium toward enzymatically active hexamers rather than inactive decamers. Dimers enhanced ATPase activity over 10-fold, surpassing that of ecumicin (8-fold) and far exceeding RUF (<2-fold). Notably, our dimers bind two NTDs per molecule (1:2), contrasting with RUF (1:1) and ecumicin (2:1), suggesting a distinct activation mechanism. These RUF-based click chemistry dimers represent potent ClpC1 modulators with extended residency and novel stoichiometry, offering promising tools for studying protein degradation.

Ganfeborole (GSK3036656) inhibits the Mycobacterium tuberculosis leucyl-tRNA-synthetase (mtLeuRS) and is in Phase 2a clinical trials for the treatment of tuberculosis. Here we show that ganfeborole is a time-dependent inhibitor of mtLeuRS (IC₅₀ 1 nM) and generates a postantibiotic effect of 77 h at 50xMIC (MIC 0.058 μM) with M. tuberculosis H37Rv, indicating that mtLeuRS is a highly vulnerable drug target and supporting the excellent in vivo efficacy of the drug. Ganfeborole is also a potent time-dependent inhibitor of Escherichia coli LeuRS (ecLeuRS, IC₅₀ 2 nM), however no antibacterial activity is observed toward E. coli up to 1 mM ganfeborole despite the observation that less potent ganfeborole analogs have antibacterial activity. To rationalize this observation, we propose that ganfeborole forms a complex with AMP that binds to the ecLeuRS editing site but does not impact aminoacylation. In support, addition of 12.5 μM norvaline generates a ganfeborole MIC of 0.4 μM since ecLeuRS is unable to hydrolyze norvaline-tRNA^Leu. Additionally, mutations that reduce the affinity and residence time of ganfeborole-AMP on ecLeuRS result in antibacterial activity. We propose that the activity of ganfeborole toward M. tuberculosis is because mtLeuRS is a highly vulnerable target so that only low levels of enzyme need to be inhibited by the ganfeborole-tRNA^Leu complex in contrast to ecLeuRS, which we previously demonstrated is a low vulnerability target.

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major global health challenge, prompting the need for new therapeutics targeting essential bacterial proteins. The caseinolytic protein C1 (ClpC1) is a promising drug target, and accurate measurement of its ATPase activity is critical for understanding drug mechanisms. We optimized a sensitive luminescence-based ATPase assay and evaluated ClpC1 constructs with various tag positions and truncations. N-terminal tagging significantly impaired enzymatic activity, whereas C-terminal tagging had no effect; truncated domains showed reduced activity compared to native full-length (FL) ClpC1. Using the native FL-ClpC1, we assessed ecumicin (ECU) and five analogs via ATPase activity and surface plasmon resonance (SPR), using rufomycin (RUF) and cyclomarin A (CYMA) as controls. RUF and CYMA bound tightly (K_D = 0.006–0.023 µM) and inhibited Mtb growth (MIC₉₀ = 0.02–0.094 µM) but modestly stimulated ATPase activity (≤2-fold). In contrast, ECU and its analogs strongly enhanced ATPase activity (4–9-fold) despite slightly weaker binding (K_D = 0.042–0.80 µM) and growth inhibition (MIC₉₀ = 0.19 µM). The partial correlation among AC₅₀, K_D, and MIC values highlights the complementary value of enzymatic, biophysical, and cellular assays. Our assay platform enables mechanistic characterization of ClpC1-targeting compounds and supports rational antitubercular drug development.

Natural products and synthetic small molecules have been extensively studied as potential therapeutics against Mycobacterium tuberculosis (Mtb), particularly drug-resistant strains. The caseinolytic protein C1 (ClpC1) has emerged as a promising drug target for combating Mtb, and accurate measurement of its ATPase activity is essential for elucidating the mechanisms of drug candidates. In this study, we optimized a highly sensitive ATPase assay for ClpC1 and compared the enzymatic activity of various constructs, including N-terminal His6-SUMO-tagged, C-terminal His6-tagged, native full-length (FL) ClpC1, and smaller domains. Our results demonstrated that the N-terminal tag significantly impairs ClpC1 activity, whereas the C-terminal tag has no such effect. Additionally, smaller domains exhibited markedly reduced enzymatic activity compared to FL-native ClpC1. Taking the native ClpC1 forward, we tested three natural products—rufomycin (RUF), ecumicin (ECU), and cyclomarin A (CYMA)—and observed varying degrees of ATPase activity enhancement. ECU and five of its analogs were further evaluated, yielding AC50 values consistent with their binding affinities (KD) measured by surface plasmon resonance (SPR). RUF and CYMA exhibited strong KD values of 0.023 µM and 0.006 µM, respectively, and minimal inhibitory concentrations (MICs) of 0.02 µM and 0.094 µM, respectively. However, their ATPase activity enhancement was modest (<93%). In contrast, ECU and its analogs significantly enhanced ATPase activity (up to 830%) despite weaker MICs and KD values compared to RUF and CYMA. These findings suggest that RUF/CYMA and ECU may operate via distinct mechanisms of action, offering valuable insights for the development of ClpC1-targeting therapeutics against tuberculosis.

The fruits of Amomun tsao-ko (Chinese black cardamom; Zingiberaceae) contain an abundance of essential oils, which have previously demonstrated significant antimicrobial activity. In our preliminary search for natural anti-tuberculosis agents, an acetone extract of A. tsao-ko (AAE) exhibited strong antibacterial activity against Mycobacterium tuberculosis H37Rv. Therefore, the aim of this study was to find the principal compounds in an AAE against M. tuberculosis. Nine aliphatic compounds (1–9) including a new compound (1, tsaokol B) and a new natural unsaturated aliphatic diester (6), together with three acyclic terpenoids (10–12), were isolated from an AAE by repetitive chromatography. The structures of the isolates were determined by spectroscopic data analysis. All isolates were evaluated for activity against M. tuberculosis H37Rv. Isolated compounds 1–6, and 11 had MICs ranging from 0.6–89 µg/mL. In contrast, compounds 7 to 10, and 12 had MICs that were >100 µg/mL. Tsaokol A (3) was the most active compound with MICs of 0.6 µg/mL and 1.4 µg/mL, respectively, against replicating and nonreplicating M. tuberculosis. These results are the first to illustrate the potency of tsaokol A (3) as a natural drug candidate with good selectivity for treating tuberculosis.

Three new lavandulylated flavonoids, (2S,2″S)-6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone (1), (2S,2″S)-6-lavandulyl-5,7,2′,4′-tetrahydroxylflavanone (2), and (2″S)-5′-lavandulyl-2′-methoxy-2,4,4′,6′-tetrahydroxylchalcone (3), along with seven known compounds 4–10 were isolated from culture broth of Streptomyces sp. G248. Their structures were established by spectroscopic data analysis, including 1D and 2D nuclear magnetic resonance (NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The absolute configurations of 1–3 were resolved by comparison of their experimental and calculated electronic circular dichroism spectra. Compounds 1–3 exhibited remarkable antimicrobial activity. Whereas, two known compounds 4 and 5 exhibited inhibitory activity against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 6.0 µg/mL and 11.1 µg/mL, respectively.