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.