Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus

Abstract

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 μM) and Mycobacterium abscessus (MIC, 0.4 μM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

Keywords: ClpC1; Mycobacterium abscessus; Mycobacterium tuberculosis; cyclic peptide; rufomycin.

FIG 1

Structures of cyclomarin A (CYMA) and ecumicin (ECU).

FIG 2

Rufomycin (RUF) analogues. ^aMIC against M. tuberculosis.

FIG 3

MIC distribution of RUFI against pan-susceptible (blue), MDR (orange), and XDR (gray) M. tuberculosis strains.

FIG 4

Rufomycin I (RUFI) has time- and concentration-dependent bactericidal activity against M. tuberculosis. The inoculum concentration was 6.0 × 10⁴ CFU/ml. Error bars represent the SDs of three measurements.

FIG 5

Activity of RUFI against M. tuberculosis in murine macrophages. Bars represent CFU prior to treatment (T₀), no treatment (T₆), and treatment with rifampin (RIF) or RUFI at the indicated concentrations. Values are means ± SDs from six measurements. According to the two-tailed t test, significant differences (P < 0.02 [**]) were observed between the untreated group (T₀) and the groups treated with RUFI at 0.48 and 0.098 μM.

FIG 6

Activity of RUFI against M. abscessus in BMDMs. Bars represent CFU on the day of infection (D0), the second day with untreated cells (D2), and the second day for cells treated with clarithromycin (CLR) or RUFI at the indicated concentrations. Values are means ± SDs from six measurements with duplication. According to the two-tailed t test, significant differences (P < 0.001 [**]) were observed between D2 and all treatment groups.

FIG 7

ClpC1 ATPase activity (A) and proteolytic activity of the ClpC1/P1/P2 complex (B) in response to ECU and RUFI treatment. This experiment was carried out in triplicate.

FIG 8

Binding of RUFI (A), ECU (B), and CYMA (C) to wild-type full-length ClpC1 from M. abscessus.

FIG 9

Mean plasma concentrations of RUFI in mice (n = 3) after a single i.v. dose of RUFI at 5 mg/kg.