Structure-Activity Relationships of Wollamide Cyclic Hexapeptides with Activity against Drug-Resistant and Intracellular Mycobacterium tuberculosis

Abstract

Wollamides are cyclic hexapeptides, recently isolated from an Australian soil Streptomyces isolate, that exhibit promising in vitro antimycobacterial activity against Mycobacterium bovis Bacille Calmette Guérin without displaying cytotoxicity against a panel of mammalian cells. Here, we report the synthesis and antimycobacterial activity of 36 new synthetic wollamides, collated with all known synthetic and natural wollamides, to reveal structure characteristics responsible for in vitro growth-inhibitory activity against Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, HN878, and HN353). The most potent antimycobacterial wollamides were those where residue VI d-Orn (wollamide B) was replaced by d-Arg (wollamide B1) or d-Lys (wollamide B2), with all activity being lost when residue VI was replaced by Gly, l-Arg, or l-Lys (wollamide B3). Substitution of other amino acid residues mainly reduced or ablated antimycobacterial activity. Significantly, whereas wollamide B2 was the most potent in restricting M. tuberculosisin vitro, wollamide B1 restricted M. tuberculosis intracellular burden in infected macrophages. Wollamide B1 synergized with pretomanid (PA-824) in inhibiting M. tuberculosisin vitro growth but did not antagonize prominent first- and second-line tuberculosis antibiotics. Furthermore, wollamide B1 exerted bactericidal activity against nonreplicating M. tuberculosis and impaired growth of multidrug- and extensively drug-resistant clinical isolates. In vivo pharmacokinetic profiles for wollamide B1 in rats and mice encourage further optimization of the wollamide pharmacophore for in vivo bioavailability. Collectively, these observations highlight the potential of the wollamide antimycobacterial pharmacophore.

Keywords: Mycobacterium tuberculosis; cyclic hexapeptides; multidrug resistance; structure-activity relationships; wollamides.

FIG 1

Chemical structures for selected wollamides.

FIG 2

Summary of an SAR analysis across a library of 82 wollamides compared to the base structure for wollamide B (compound 2).

FIG 3

Wollamide B1 (compound 3) exhibits potent activity against intracellular M. tuberculosis and is bactericidal against replicating and nonreplicating bacteria. (A) Bone marrow-derived macrophages were infected with M. tuberculosis H37Rv (MOI of 0.1 to 0.3) for 4 h and then cultured in the presence of wollamide B (compound 2), wollamide B1 (compound 3), or wollamide B2 (compound 4) (20 µM). DMSO served as a control. Intracellular bacterial burden was analyzed 3 and 6 days postinfection. Means ± standard errors of the means (SEM) from three to five independent experiments are shown. (B) Wollamide B1 (compound 3) dose-dependently reduced the burden of intracellular H37Rv in macrophages treated as described for panel A. Means ± SEM from three independent experiments are shown. (Size of error bars for some data points on day 6 are smaller than the symbol.) Data obtained with 20 µM compound 3 are also part of the five experiments plotted in panel A. The effect of wollamides 2 to 4 compared to that of DMSO was analyzed by multiple t test with Sidak-Holm correction. (C) CFU determination of M. tuberculosis H37Rv cultured in 7H9 liquid medium in the presence of compound 3 at 1, 3, or 10 µM, with DMSO (D) as the solvent control, for 4 and 7 days. Data are means ± SEM from six independent cultures analyzed in two independent experiments. The dotted line indicates detection limit of the assay. (D) M. tuberculosis H37Rv bacteria were starved for 10 days in PBS–0.05% Tween 80 before addition of 1 µM or 10 µM compound 3, or DMSO (D) as a solvent control, with CFU determined at 7 and 21 days. Data are means ± SEM from four independent cultures analyzed in two independent experiments. The dashed line indicates input CFU on day 0; the dotted line indicates detection limit of the assay. Effect of compound 3 treatment compared to that of DMSO (control) treatment was analyzed by two-way analysis of variance with Dunnett correction for multiple comparisons. ***, P < 0.0005; ****, P < 0.0001.

FIG 4

Synergistic effects of wollamide B1 (compound 3) and pretomanid (PA-824) in inhibiting M. tuberculosis growth. (A and B) OD₆₀₀ of M. tuberculosis H37Rv cultured in 7H9 after 7 days of growth in the presence of wollamide B1 (compound 3) (0.01 to 30 μM) alone or across a dose range of 0.0002 to 30 μM, in combination with 1.1 μM pretomanid (PA-824) (A), and pretomanid (PA-824) (0.01 to 30 μM) alone or across a dose range of 0.0002 to 30 μM, in combination with 1.1 μM compound 3 (B). DMSO and isoniazid (INH; 20 μg/ml) served as controls. Data are means ± SEM from six independent cultures examined in 2 independent experiments. (C) MIC (µM) of individual and combination treatments. FICI of <0.5 denotes synergism.

FIG 5

Wollamide B1 (compound 3) impairs growth of clinical drug-resistant and drug-sensitive M. tuberculosis isolates. TTP analyses in standard mycobacterial growth indicator tube (MGIT) analyses for clinical M. tuberculosis isolates, including 555 and 569 (drug sensitive) and 12596 (capreomycin resistant), MDR M. tuberculosis isolates 105 (isoniazid, rifampin, and ethambutol resistant) and 929 (isoniazid, rifampin, ethambutol, and pyrazinamide resistant), and XDR M. tuberculosis isolate 928 (isoniazid, rifampin, ethambutol, pyrazinamide, fluoroquinolones, amikacin, and cycloserine resistant). Wollamide B1 (compound 3) was added to MGIT cultures at the concentrations indicated, and time to positivity was determined by the automated MGIT detection system.

FIG 6

Plasma stability and in vivo pharmacokinetics of wollamide B1 (compound 3). (A) Concentrations of compound 3 in rat and mouse plasma over a 2 h incubation at 37°C. Aliquots (20 µl) were taken at 0, 5, 15, 30, 60, and 120 min, added to MeCN (80 µl), and analyzed by HPLC-MS. Shown are means ± range for two independent experiments. (B to D) C57BL/6 mice were administered a single dose of compound 3, 1 mg/kg intravenously (i.v.) (B) or 5 mg/kg intraperitoneally (i.p.) (C) or subcutaneously (s.c.) (D). Animals were euthanized at 0 h (predose), as well as 0.5, 1, 2, 4, 6, and 8 h after administration of compound 3 (i.v. and s.c., n = 3 to 6 per time point; i.p., n = 1 per time point). Concentrations of compound 3 in plasma as well as samples of liver, lung, kidney, spleen, adipose tissue, and brain were determined by LC-MS (plasma) and HPLC-DAD-MS (organs).