Wollamide Cyclic Hexapeptides Synergize with Established and New Tuberculosis Antibiotics in Targeting Mycobacterium tuberculosis

Abstract

Shorter and more effective treatment regimens as well as new drugs are urgent priorities for reducing the immense global burden of tuberculosis (TB). As treatment of TB currently requires multiple antibiotics with diverse mechanisms of action, any new drug lead requires assessment of potential interactions with existing TB antibiotics. We previously described the discovery of wollamides, a new class of Streptomyces-derived cyclic hexapeptides with antimycobacterial activity. To further assess the value of the wollamide pharmacophore as an antimycobacterial lead, we determined wollamide interactions with first- and second-line TB antibiotics by determining fractional inhibitory combination index and zero interaction potency scores. In vitro two-way and multiway interaction analyses revealed that wollamide B1 synergizes with ethambutol, pretomanid, delamanid, and para-aminosalicylic acid in inhibiting the replication and promoting the killing of phylogenetically diverse clinical and reference strains of the Mycobacterium tuberculosis complex (MTBC). Wollamide B1 antimycobacterial activity was not compromised in multi- and extensively drug-resistant MTBC strains. Moreover, growth-inhibitory antimycobacterial activity of the combination of bedaquiline/pretomanid/linezolid was further enhanced by wollamide B1, and wollamide B1 did not compromise the antimycobacterial activity of the isoniazid/rifampicin/ethambutol combination. Collectively, these findings add new dimensions to the desirable characteristics of the wollamide pharmacophore as an antimycobacterial lead compound. IMPORTANCE Tuberculosis (TB) is an infectious disease that affects millions of people globally, with 1.6 million deaths annually. TB treatment requires combinations of multiple different antibiotics for many months, and toxic side effects can occur. Therefore, shorter, safer, more effective TB therapies are required, and these should ideally also be effective against drug-resistant strains of the bacteria that cause TB. This study shows that wollamide B1, a chemically optimized member of a new class of antibacterial compounds, inhibits the growth of drug-sensitive as well as multidrug-resistant Mycobacterium tuberculosis isolated from TB patients. In combination with TB antibiotics, wollamide B1 synergistically enhances the activity of several antibiotics, including complex drug combinations that are currently used for TB treatment. These new insights expand the catalogue of the desirable characteristics of wollamide B1 as an antimycobacterial lead compound that might inspire the development of improved TB treatments.

Keywords: Mycobacterium tuberculosis; antimicrobial combinations; multidrug resistance.

FIG 1

(A to J) Wollamide B1 synergizes with delamanid (A and B), para-aminosalicylic acid (C and D), pretomanid (E and F), and ethambutol (G and H), but not with isoniazid (I and J) in the growth inhibition of M. tuberculosis H37Rv in liquid culture. (A, C, E, G, and I) Checkerboards demonstrate shifts in the percent inhibition of growth of M. tuberculosis H37Rv by combinations of wollamide B1 and the drug of interest. (B, D, F, H, and J) Three-dimensional (3D) interaction profiles show synergistic peaks upon combinations of wollamide B1 and the drug of interest. Zero interaction potency (ZIP) scores represent the percent response beyond the expected response due to drug interactions (21). The data represent mean of 4 to 7 independent cultures analyzed in 3 to 4 independent experiments.

FIG 2

(A to E) Wollamide B1 synergizes with (A) delamanid, (B) para-aminosalicylic acid, (C) pretomanid, (D) ethambutol, and (E) isoniazid in bactericidal activity against M. tuberculosis H37Rv. The addition of wollamide B1 to (A) delamanid, (B) para-aminosalicylic acid, (C) pretomanid, (D) ethambutol, and (E) isoniazid amplified bactericidal activity compared to the drug alone after 10 days of incubation. The data are the mean ± SEM of 3 to 8 independent cultures across three independent experiments. L.O.D., limit of detection at 10² CFU/mL; #, synergistic interaction of wollamide B1 and antibiotic defined as a decrease in CFU/mL greater than 2 × log₁₀ compared to antibiotic alone (22).

FIG 3

Wollamide B1 enhances the inhibition of M. tuberculosis H37Rv growth by the combination of bedaquiline/pretomanid/linezolid. (A, C, and E) Percent inhibition of M. tuberculosis H37Rv growth in liquid culture in the presence of (A) isoniazid (H) and rifampicin (R), (C) isoniazid (H), rifampicin (R), and ethambutol (E), (E) bedaquiline (B), pretomanid (Pa), and linezolid (L), with and without wollamide B1. Data are the mean ± SEM of 10 independent cultures across 5 independent experiments. (B, D, and F) Fractional IC₅₀ of antibiotic combinations with and without wollamide B1 extracted from 10 independent cultures across 5 independent experiments. (A, C, and E) Paired Student’s t test; *, P < 0.05. (G) MIC, MIC₉₀, and IC₅₀ (as fractions of the concentration [μM] of individual agents) of inhibition of M. tuberculosis H37Rv growth in liquid culture by antibiotic combinations with and without wollamide B1 as the means of n = 10 independent cultures across 5 independent experiments (as shown in panels A, C, and E).