An amiloride derivative is active against the F1Fo-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis

Abstract

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo^®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F₁F_o-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa₃ oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.

Fig. 1. Inhibition of the mycobacterial F₁F_o-ATP synthase by amiloride and hexamethylene amiloride.

a Structure of amiloride and b 5-(N,N-hexamethylene)amiloride (HMA). c ATP synthesis activity of NADH-energized M. smegmatis inverted membrane vesicles (IMV), treated with either 64 μM of amiloride, 64 μM of HMA, or 2 μM of BDQ as indicated. Error bars represent standard deviation from triplicate biologically independent measurements. d Time course of DCCD labelling in purified M. phlei c-rings in the presence of the indicated amounts of HMA. Data are representative of duplicate measurements.

Fig. 2. HM2-16F is bactericidal towards mycobacteria in vitro and in macrophages.

a Structure of HM2-16F. b Survival of M. tuberculosis after treatment with: isoniazid (INH 2 μM, 10× MIC), HM2-16F (20 μM, 5× MIC) and bedaquiline (BDQ 2 μM, 10X MIC). c Survival of M. bovis BCG in human THP-1 macrophages after treatment with 5 μM HM2-16F and BDQ. Error bars indicate standard deviation from three biological independent experiments.

Fig. 3. Interactions of HM2-16F with the mycobacterial F₁F_o-ATP synthase.

a Knockdown of the ATP synthase operon (sgRNA targeting atpB – pCi74) was induced with the indicated amounts of ATc and the growth relative to the vehicle control was determined. Error bars represent standard deviation from three biological independent experiments. b Growth after 10 days and IC₅₀ of HM2-16F as a function of F₁F_o-ATP synthase knockdown (sgRNA targeting atpB – pCi74). Error bars represent 95% confidence interval. c Viability of M. tuberculosis after 10 days was determined in an analogous experiment to panel a at 0, 10 and 100 ng mL⁻¹. d Time course of DCCD labelling in purified M. phlei c-rings in the presence of the indicated amounts of compounds. Data are representative of duplicate measurements. e HM2-16F (purple carbons) docked into the BDQ binding site of mycobacterial F₁F_o-ATP synthase c-ring (PDB ID: 4V1F). The high-resolution c-ring-BDQ structure is shown in grey as a reference. The protein is shown as an electrostatic potential surface (red – electronegative, white – neutral, blue – electropositive; generated in PyMOL). Amino acid positions refer to the M. phlei protein. The top three ranked poses are shown in Supplementary Figure 5.

Fig. 4. Bedaquiline-resistant mutants of M. tuberculosis are not cross-resistant to HM2-16F.

The M. tuberculosis strains mc²6206 (WT), AtpE(A63P), and Rv0678(G65fs) were grown in the presence of increasing concentrations of: a BDQ, b HM2-16F or c clofazimine and growth measured after 10 days. Error bars represent standard deviation from four biological independent experiments.

Fig. 5. Metabolomic profiling of HM2-16F-treated cultures of M. tuberculosis indicates HM2-16F inhibits respiration and induces cellular reductive stress.

a Global profile of metabolites with dose-dependent changes, as assessed by log-linear regressions. b Global profile of metabolites with dose-independent changes, as assessed by two sample t-tests with Benjamini & Hochberg False Discovery Rate (FDR) adjustment. c Fold changes of selected metabolites, classified according to whether the metabolite changed in a dose-dependent (increasing/decreasing) or dose-independent manner. d Changes in adenine nucleotide/nucleoside pools. e Relative changes in the indicated metabolites. Raw data are available in Supplementary Data 1.

Fig. 6. HM2-16F synergizes with Q203 by inhibiting cytochrome bd oxidase.

a Cell killing (viability) of M. tuberculosis with increasing amounts of both Q203 and HM2-16F after 14 days challenge with both compounds. Error bars indicate standard deviation from three independent experiments. b Inhibition of the M. tuberculosis cytochrome bd oxidase in IMVs from M. smegmatis mc²155 ΔcydAB with either pYUB28b (empty vector) or pLHcyd- MtbCydABDC⁺. IMVs were pre-inhibited with 500 nM TB47 for 1 min as indicated. Oxygen consumption was initiated with 5 mM malate (final concentration) as the sole electron donor. The indicated treatment: 0.2× MIC aurachin D (AuD) (top panel 6b), 10× MIC HM2-16F (middle panel b) and 500× MIC BDQ (bottom panel b) was added once malate energized oxygen consumption reached steady state (~2 min after malate addition). Error bars represent standard deviation from triplicate measurements. Asterisks indicate significance compared to the corresponding malate energized OCR *** = p < 0.001, ** = p < 0.01, ns = p > 0.05 (One-way ANOVA, Tukey multiple comparisons, 95% CI). c Titration of the indicated compounds in malate-energized IMVs of M. smegmatis mc²155 ΔcydAB pLHcyd- MtbCydABDC⁺ pre-inhibited with 500 nM TB47 (final concentration). Error bars indicate standard error from four technical replicates.

Fig. 7. Summary of the action of HM2-16F at the level of the respiratory chain.

HM2-16F inhibits cytochrome bd and, to a lesser or indirect extent, the F₁F_o-ATP synthase. Disruption of quinol regeneration can affect the primary dehydrogenases of the respiratory chain leading to dysregulated regeneration of reducing equivalents that can lead to a systemic failure of cellular redox reactions. The addition of Q203 (or TB47), direct inhibitors of the cytochrome bcc-aa₃ oxidase branch, removes the only alternative pathway that mycobacteria can use to escape this inhibition of oxygen consumption leading to cell death.