Verapamil, and Its Metabolite Norverapamil, Inhibit Macrophage-induced, Bacterial Efflux Pump-mediated Tolerance to Multiple Anti-tubercular Drugs

MAJOR ARTICLE Verapamil, and Its Metabolite Norverapamil, Inhibit Macrophage-induced, Bacterial Efflux Pump-mediated Tolerance to Multiple Anti-tubercular Drugs 1 Department of Microbiology, 2Department of Medicine, Division of Infectious Diseases, and 3Department of Immunology, University of Washington, Seattle, Washington 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. tuberculosis; tolerance; persistence; efflux; verapamil; norverapamil; R-verapamil; efflux pump Keywords. inhibitor. The long duration of therapy required to reliably cure tuberculosis [1] presents a fundamental hurdle to its eradication. However, attempts to shorten the firstline tuberculosis drug regimen for smear-positive cases have met with unacceptably high relapse rates [2, 3]. It has been long recognized that when relapses occur, they typically involve genetically drug-susceptible organisms [1]. Consequently, relapse and the need for long treatment have been attributed to phenotypic Received 10 December 2013; accepted 10 February 2014; electronically published 14 February 2014. This work was presented in part at the July 2013 Gordon Conference on Tuberculosis Drug Development (Lucca, Italy) and the May 2012 Keystone Conference on Drug Resistance and Persistence in Tuberculosis (Kampala, Uganda). a These authors contributed equally. Correspondence: Dr Lalita Ramakrishnan, University of Washington, 1959 NE Pacific St, UW Box 357735, Seattle, WA 98195-6423 (). The Journal of Infectious Diseases 2014;210:456–66 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jiu095 456 • JID 2014:210 (1 August) • Adams et al drug resistance, also known as tolerance [3, 4]. Drugtolerant organisms are killed poorly, yet their minimal inhibitory concentration (MIC) is unchanged. Existing tolerance models use indirect evidence to invoke a metabolically dormant bacterial population that is not easily killed by existing drugs [5]. Consequently, there has been considerable excitement about the recently developed compounds PA824 and bedaquiline (TMC-207) [6, 7]. Bedaquiline has been shown to be active against both replicating and nonreplicating bacteria in culture [8]. It has recently been approved for multidrug resistant (MDR) tuberculosis based on increased culture conversion rates [9]. However, in murine models with drug-sensitive tuberculosis, bedaquiline shortens treatment only moderately [10]. One explanation for its failure to shorten treatment even further is that additional or alternative tolerance mechanism(s) may be present in vivo. We recently uncovered a completely different mechanism for drug tolerance [11]. We found that Mycobacterium tuberculosis develops bacterial efflux Kristin N. Adams,1,a John D. Szumowski,2,a and Lalita Ramakrishnan1,2,3 METHODS Bacterial Strains, Methods, and Chemicals The M. tuberculosis strain CDC1551 was a gift from W. R. Bishai (Johns Hopkins University). H37Rv and an isogenic rpoB mutant (H526Y) were from D. R. Sherman (Seattle BioMed). Mycobacterium marinum strain M (BAA-535) was obtained from ATCC. M. tuberculosis were grown to mid log phase in Middlebrook 7H9 medium (Becton Dickinson) with 0.05% Tween-80 and albumin, dextrose, catalase (Middlebrook ADC Enrichment, BBL Microbiology) prior to infection. Rifampicin, isoniazid, streptomycin, rifabutin, ethambutol, ethionamide, kanamycin, cycloserine, capreomycin, clofazimine, para-aminosalicylic acid (PAS), linezolid, verapamil, thioridazine, piperine, and R- and S-verapamil were purchased from Sigma. Norverapamil and moxifloxacin was purchased from Santa Cruz Biotechnology. PA-824 was provided by David Sherman (Seattle BioMed) and bedaquiline was provided by Clifton Barry (NIAID). Macrophage Growth and Infection THP-1 macrophages were grown in RPMI, supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. THP-1 cells were differentiated with 100 nM phorbol 12-myristate 13-acetate for 48 hours and allowed to recover for 24 hours prior to infection. Subsequently, 5 × 105 THP-1 macrophages were infected at a multiplicity of infection of 1 for 3 hours at 37°C. Cells were washed with media, and 6 µg/mL streptomycin was added to the media for the duration of the intracellular growth (Figure 1). Media was changed daily. To lyse macrophages and release bacteria, each well was washed once with 1× phosphate-buffered saline (PBS) and then with diH2O, with the latter being removed immediately. Then, 100 µL of diH20 was added, and the cells were incubated at 37°C for 15 minutes. Finally, 900 µL of 7H9 medium with 0.05% Tween80 was added and the wells scraped with a pipette tip. Figure 1. Schematic of protocols used to test effect of efflux pump inhibitors on macrophage-induced tolerance as well as intracellular Mycobacterium tuberculosis growth. Verapamil Inhibits M. tuberculosis Drug Tolerance • JID 2014:210 (1 August) • 457 pump-mediated tolerance to isoniazid and rifampicin following macrophage residence [11]. Moreover, we observed that tolerant bacteria are enriched in the actively-dividing M. tuberculosis population. This macrophage-induced rifampicin tolerance was inhibited by verapamil, a calcium channel antagonist recognized to inhibit bacterial efflux pumps in vitro [12]. Subsequent work in murine tuberculosis models has validated these findings. Verapamil has been shown to accelerate bacterial killing in mice infected with drug-resistant [13] or drug-sensitive tuberculosis [14] and decrease relapse rates with shortened treatment courses [14]. These data suggest the promise of strategies combining efflux inhibitors with existing tuberculosis drugs. In this work, we have extended our prior findings by studying macrophage-induced tole (...truncated)