Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis.

SUSCEPTIBILITY Developing Synergistic Drug Combinations To Restore Antibiotic Sensitivity in Drug-Resistant Mycobacterium tuberculosis Charles Omollo,a,b,c,d Vinayak Singh,a,b,c,d Elizabeth Kigondu,a,b,c* Antonina Wasuna,a,b,c Pooja Agarwal,c,d Atica Moosa,c,d Thomas R. Ioerger,e Valerie Mizrahi,c,d,f Kelly Chibale,a,b,d Digby F. Warnerc,d,f Department of Chemistry, University of Cape Town, Rondebosch, South Africa a South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch, South Africa b c SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch, South Africa Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, South Africa d Texas A&M University, Department of Computer Science, College Station, Texas, USA e Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, Rondebosch, South Africa f Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis. We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S rRNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis. Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPTmonoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant. These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds. ABSTRACT KEYWORDS Rv1258c, chlorpromazine, efflux, fusidic acid, potentiation, spectinomycin T he increasing prevalence of multidrug-resistant tuberculosis (MDR-TB)—defined as resistance to the frontline anti-TB agents isoniazid (INH) and rifampicin (RIF)— necessitates the urgent development and implementation of new antimycobacterial drugs and therapeutic strategies (1, 2). A number of anti-TB compounds are currently in the drug discovery pipeline, with several others in advanced preclinical development (3). However, with the exception of bedaquiline (BDQ) and delamanid, no TBspecific drugs have been introduced into clinical use within the past 40 years (4). Therefore, new options need to be explored to address the problem of drug resistance. May 2021 Volume 65 Issue 5 e02554-20 Antimicrobial Agents and Chemotherapy Citation Omollo C, Singh V, Kigondu E, Wasuna A, Agarwal P, Moosa A, Ioerger TR, Mizrahi V, Chibale K, Warner DF. 2021. Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 65:e02554-20. https://doi.org/10 .1128/AAC.02554-20. Copyright © 2021 Omollo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Charles Omollo, , or Digby F. Warner, . * Present address: Elizabeth Kigondu, Centre for Traditional Medicine & Drug Research, Kenya Medical Research Institute, Nairobi, Kenya. Received 12 December 2020 Returned for modification 19 January 2021 Accepted 14 February 2021 Accepted manuscript posted online 22 February 2021 Published 19 April 2021 aac.asm.org 1 Omollo et al. Antimicrobial Agents and Chemotherapy Novel combination regimens comprising standard anti-TB agents and repurposed drugs represent a logical approach, especially where the new partner drug has already been approved for other clinical indications (5–7). Recent advances in understanding the physicochemical properties that determine drug distributions within complex tissue and cellular (micro)environments (8, 9), together with improved methods for rapid selection of multiple potentially synergistic drug partners in vitro and in vivo (8, 10–12), suggest the potential for rational development of novel combination approaches. This is important since it might address the long-held belief that developing combinations should be avoided owing to the complexities inherent in ensuring simultaneous and sustained delivery of the optimal partner compounds to the same target site (11). The impact of preexisting drug resistance on the utility of new drug combinations presents an additional challenge and is of particular concern when these combinations comprise current frontline anti-TB agents. To minimize the risks of exposing an individual to effective monotherapy, the likely preexistence of resistance to individual drugs must be recognized and informed combination approaches for drug therapies designed. These should incorporate multiple attributes beyond simply selecting individual molecules based on their biological activities as single agents (13). In this study, we employed spectinomycin (SPT) as an anchor compound in combination with other experimental antibiotics and existing frontline anti-TB agents. SPT is an aminocyclitol antibiotic that inhibits protein synthesis by disrupting mRNA interactions with the 30S ribosome (14). Unlike other aminocyclitol antibiotics (including gentamicin and kanamycin [KAN]), SPT is not ototoxic (15) and has been used extensively in treating Neisseria gonorrhoeae infections in patients who cannot tolerate first-line treatments (16). From the perspective of new regimen design, SPT has been shown in combination screens against M. tuberculosis to synergize with several different classes of antimycobacterial compounds, both in vitro and in a macrophage model (10). Unfortunately, a key liability undermining its utility as a single agent is that SPT is subject to active efflux by M. tuberculosis—an observation that motivated an elegant medicinal chemistry solution in the development of the spectinamides (SPD) as derivative “efflux-resistant” anti-TB antibiotics (17–19). Spectinamides are also known to (...truncated)