Synergistic drug combinations for tuberculosis therapy identified by a novel high-throughput screen

Abstract

Therapeutic options for tuberculosis (TB) are limited and notoriously ineffective despite the wide variety of potent antibiotics available for treating other bacterial infections. We investigated an approach that enables an expansion of TB therapeutic strategies by using synergistic combinations of drugs. To achieve this, we devised a high-throughput synergy screen (HTSS) of chemical libraries having known pharmaceutical properties, including thousands that are clinically approved. Spectinomycin was used to test the concept that clinically available antibiotics with limited efficacy against Mycobacterium tuberculosis might be used for TB treatment when coadministered with a synergistic partner compound used as a sensitizer. Screens using Mycobacterium smegmatis revealed many compounds in our libraries that acted synergistically with spectinomycin. Among them, several families of antimicrobial compounds, including macrolides and azoles, were also synergistic against M. tuberculosis in vitro and in a macrophage model of M. tuberculosis infection. Strikingly, each sensitizer identified for synergy with spectinomycin uniquely enhanced the activities of other clinically used antibiotics, revealing a remarkable number of unexplored synergistic drug combinations. HTSS also revealed a novel activity for bromperidol, a butyrophenone used as an antipsychotic drug, which was discovered to be bactericidal and greatly enhanced the activities of several antibiotics and drug combinations against M. tuberculosis. Our results suggest that many compounds in the currently available pharmacopoeia could be readily mobilized for TB treatment, including disease caused by multi- and extensively drug-resistant strains for which there are no effective therapies.

Fig. 1.

Combinatorial drug discovery program.

Fig. 2.

High-throughput synergy screening (HTSS). Compounds spotted against M. smegmatis in an agar-based HTSS plate in the absence or in the presence of subinhibitory concentrations of spectinomycin (1/4×MIC). Compounds whose zones of inhibition were larger in the presence of spectinomycin than in plates without spectinomycin (i.e., example A or B) were selected for further studies. Up to 1,536 compounds (4 × 4 × 96) could be tested per plate; however, groups of active compounds on such high-density plates often needed to be tested by spotting at a lower density (deconvolution; 96 compounds per OmniTray), as illustrated in the figure.

Fig. 3.

Chemical structure clustering of the compounds identified by HTSS as enhancers of spectinomycin activity against M. smegmatis. A total of 48 chemical structures were clustered using the Chemical Structure Clustering tool available at PubChem (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?p=clustering). Cutoff values for similarity are between 0.85 and 0.70. A value of 0.5 to 0.6 or lower indicates lack of similarity.

Fig. 4.

Spectinomycin sensitizers have their own characteristic pattern of synergy with other antibiotics. Seven compounds that were synergistically active with spectinomycin in M. smegmatis (sensitizers, horizontal axis) were assayed for their abilities to induce sensitivity to an array of 11 representative antibiotics (vertical axis). A sensitivity “barcode” was generated for each compound. Sensitivity was determined in the presence or absence of a 1/4×MIC concentration of the sensitizers. Solid lines indicate a ≥4-fold increase in sensitivity to one of the 11 antibiotics in the presence of the sensitizer.