Verapamil, and its metabolite norverapamil, inhibit macrophage-induced, bacterial efflux pump-mediated tolerance to multiple anti-tubercular drugs

Abstract

Drug tolerance likely represents an important barrier to tuberculosis treatment shortening. We previously implicated the Mycobacterium tuberculosis efflux pump Rv1258c as mediating macrophage-induced tolerance to rifampicin and intracellular growth. In this study, we infected the human macrophage-like cell line THP-1 with drug-sensitive and drug-resistant M. tuberculosis strains and found that tolerance developed to most antituberculosis drugs, including the newer agents moxifloxacin, PA-824, linezolid, and bedaquiline. Multiple efflux pump inhibitors in clinical use for other indications reversed tolerance to isoniazid and rifampicin and slowed intracellular growth. Moreover, verapamil reduced tolerance to bedaquiline and moxifloxacin. Verapamil's R isomer and its metabolite norverapamil have substantially less calcium channel blocking activity yet were similarly active as verapamil at inhibiting macrophage-induced drug tolerance. Our finding that verapamil inhibits intracellular M. tuberculosis growth and tolerance suggests its potential for treatment shortening. Norverapamil, R-verapamil, and potentially other derivatives present attractive alternatives that may have improved tolerability.

Keywords: R-verapamil; efflux; efflux pump inhibitor; norverapamil; persistence; tolerance; tuberculosis; verapamil.

Figure 1.

Schematic of protocols used to test effect of efflux pump inhibitors on macrophage-induced tolerance as well as intracellular Mycobacterium tuberculosis growth.

Figure 2.

Effects of efflux inhibitors on macrophage-induced tolerance to isoniazid and rifampicin and intracellular Mycobacterium tuberculosis growth. A–F, THP-1 macrophages were infected with CDC 1551 and lysed at 2 hours (gray bars) or 96 hours (black bars) postinfection. The released bacteria were left untreated or were treated for an additional 48 hours with 0.6 µg/mL isoniazid (INH) or 1 µg/mL rifampicin (RIF) in the presence or absence of 80 µg/mL verapamil (VER), 100 µg/mL piperine (PIP), or 2 µg/mL thioridazine (TRZ) prior to enumeration of colony-forming units (CFU). G and H, THP-1 macrophages were infected with CDC 1551 for 48 hours (G) and 96 hours (H) and subsequently left untreated (UNT) or treated for an additional 48 hours with 40 µg/mL VER, 100 µg/mL piperine, or 2 µg/mL TRZ prior to enumeration of CFU. Results (A–H) are representative of at least 3 independent experiments. Error bars represent SEM. Significance testing was performed using 1-way ANOVA with Dunnett's post-test. Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean.

Figure 3.

Tolerance develops to other drugs used for drug-susceptible tuberculosis. THP-1 macrophages were infected with CDC 1551 and lysed at 2 hours (gray bars) or 96 hours (black bars) postinfection. The released bacteria were left untreated or were treated for an additional 48 hours with 0.6 µg/mL INH (A), 10 µg/mL ethambutol (B), 1 µg/mL RIF (C), 1 µg/mL rifabutin (D), 6 µg/mL streptomycin (E), or 0.6 µg/mL INH plus 6 µg/mL streptomycin (F) in the presence or absence of 80 µg/mL VER or 2 µg/mL TRZ (E) prior to enumeration of CFU. Results are representative of at least 3 (A–D) or at least 2 (E and F) independent experiments. Error bars represent SEM. Significance testing was performed using 1-way ANOVA with Dunnett's post-test. Abbreviations: ANOVA, analysis of variance; CFU, colony-forming units; INH, isoniazid; RIF, rifampicin; SEM, standard error of the mean; TRZ, thioridazine; VER, verapamil.

Figure 4.

Verapamil promotes killing of intracellular drug-resistant Mycobacterium tuberculosis. A and B, THP-1 macrophages were infected with H37Rv (WT) or an isogenic rpoB H526Y mutant. Starting 2 hours (gray bars) or 96 hours (black bars) postinfection, the cells were either left untreated or were treated with 1 µg/mL rifampicin for an additional 48 hours prior to lysis and enumeration of CFU. At the 96-hour time point, they were additionally treated with no efflux inhibitor, 40 µg/mL VER, or 2 µg/mL TRZ for 48 hours. C and D, THP-1 macrophages were infected with CDC1551 (WT) or an isogenic katG mutant. Starting 2 hours (gray bars) or 96 hours (black bars) postinfection, the cells were either left untreated or were treated with 0.6 µg/mL INH for an additional 48 hours prior to lysis and enumeration of CFU. At the 96-hour time point, they were additionally treated with no efflux inhibitor, 40 µg/mL VER, or 2 µg/mL TRZ for 48 hours. Results are representative of 3 independent experiments for panels A and B. For panels C and D, the results of the only experiment performed are shown. Error bars represent SEM. Significance testing was performed using 1-way ANOVA with Dunnett's post-test. Abbreviations: ANOVA, analysis of variance; CFU, colony-forming units; INH, isoniazid; SEM, standard error of the mean; TRZ, thioridazine; VER, verapamil.

Figure 5.

Macrophage-induced tolerance develops to second and third-line anti-tubercular drugs. THP-1 macrophages were infected with CDC 1551 and lysed at 2 hours (gray bars) or 96 hours (black bars) postinfection. The released bacteria were left untreated or were treated for an additional 48 hours with 1.5 µg/mL moxifloxacin (A), 6 µg/mL kanamycin (B), 6 µg/mL capreomycin (C), 75 µg/mL cycloserine (D), 1 µg/mL ethionamide (E), 25 µg/mL PAS (F), 1 µg/mL PA-824 (G), 0.5 µg/mL clofazimine (H), 10 µg/mL linezolid (I), or 0.3 µg/mL bedaquiline (J) in the presence or absence of 80 µg/mL VER prior to enumeration of CFU. Results are representative of at least 3 independent experiments. Error bars represent SEM. Significance testing was performed using 1-way ANOVA with Dunnett's post-test. Abbreviations: ANOVA, analysis of variance; CFU, colony-forming units; PAS, para-aminosalicylic acid; SEM, standard error of the mean; VER, verapamil.

Figure 6.

Macrophage-induced bedaquiline tolerance develops rapidly upon macrophage infection and is inhibited by verapamil. A–C, THP-1 macrophages were infected with CDC 1551 and lysed at 2 hours (gray bars) or 96 hours (black bars) postinfection. The released bacteria were left untreated or were treated for an additional 48 hours with 0.3 µg/mL bedaquiline (A), 0.6 µg/mL INH (B), or 1 µg/mL RIF (C) in the presence or absence of VER (80 µg/mL for bedaquiline and INH and 40 µg/mL for RIF) prior to enumeration of CFU. Results are representative of at least 3 (A and B) or 2 (C) independent experiments. D, Logarithmic-phase CDC 1551 was left untreated or was treated for 48 or 168 hours with 0.3 µg/mL bedaquiline (TMC), 80 µg/mL VER, 20 µg/mL VER, or bedaquiline combined with VER at each dose prior to enumeration of CFU. E and F, THP-1 macrophages were infected and treated as described in panel A, with treatment of macrophage lysates extending to 168 hours for both 2-hour (E) and 96-hour (F) time points. Error bars represent SEM. Significance testing was performed using 1-way ANOVA with Dunnett's post-test. Abbreviations: ANOVA, analysis of variance; CFU, colony-forming units; INH, isoniazid; RIF, rifampicin; SEM, standard error of the mean; VER, verapamil.

Figure 7.

The major metabolite of verapamil, norverapamil, is an antimicrobial agent and reduces macrophage-induced drug tolerance. A, Molecular structures of verapamil and norverapamil. B, THP-1 macrophages were infected with CDC 1551 and lysed at 2 hours (gray bars) or 96 hours (black bars) postinfection. The released bacteria were left untreated or were treated for an additional 48 hours with 1 µg/mL RIF or 0.6 µg/mL INH in the presence or absence of 80 µg/mL VER or 80 µg/mL NOR prior to enumeration of CFU. C, THP-1 macrophages were infected with CDC 1551 for 48 hours and subsequently left untreated or treated for an additional 48 hours with 40 µg/mL VER or 40 µg/mL NOR prior to lysis and enumeration of CFU. D, THP-1 macrophages were infected with CDC 1551 and lysed at 2 hours (gray bars) or 96 hours (black bars) postinfection. The released bacteria were left untreated or were treated for an additional 48 hours with 0.6 µg/mL INH in the presence or absence of 80 µg/mL VER, 80 µg/mL R-verapamil (VER-R), or 80 µg/mL S-verapamil (VER-S) prior to enumeration of CFU. Results are representative of at least 3 independent experiments (B and C). For panel D the results of the only experiment performed are shown. Error bars represent SEM. Significance testing was performed using 1-way ANOVA with Dunnett's post-test. Abbreviations: ANOVA, analysis of variance; CFU, colony-forming units; INH, isoniazid; NOR, norverapamil; RIF, rifampicin; SEM, standard error of the mean; VER, verapamil.