Open AccessArticle
Characterization of Cyclic Peptides for Antituberculosis Drug Development Targeting ClpC1
by
Robel Demissie, Tasneem M. Vaid, Youngjin Kwon, Gauri Shetye, Thao Tran, Fatema Nomani, Shengnan Jin, Joo-Won Suh, Hanki Lee, Yern-Hyerk Shin, Jinsheng Cui, Dong-Chan Oh, Scott G. Franzblau, Sanghyun Cho and Hyun Lee
Appl. Sci. 2025, 15(21), 11425; https://doi.org/10.3390/app152111425 (registering DOI) - 25 Oct 2025
Abstract
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
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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 (
KD = 0.006–0.023 µM) and inhibited
Mtb growth (MIC
90 = 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 (
KD = 0.042–0.80 µM) and growth inhibition (MIC
90 = 0.19 µM). The partial correlation among AC
50,
KD, 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.
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