Para-aminosalicylic acid acts as an alternative substrate of folate metabolism in Mycobacterium tuberculosis

Abstract

Folate biosynthesis is an established anti-infective target, and the antifolate para-aminosalicylic acid (PAS) was one of the first anti-infectives introduced into clinical practice on the basis of target-based drug discovery. Fifty years later, PAS continues to be used to treat tuberculosis. PAS is assumed to inhibit dihydropteroate synthase (DHPS) in Mycobacterium tuberculosis by mimicking the substrate p-aminobenzoate (PABA). However, we found that sulfonamide inhibitors of DHPS inhibited growth of M. tuberculosis only weakly because of their intracellular metabolism. In contrast, PAS served as a replacement substrate for DHPS. Products of PAS metabolism at this and subsequent steps in folate metabolism inhibited those enzymes, competing with their substrates. PAS is thus a prodrug that blocks growth of M. tuberculosis when its active forms are generated by enzymes in the pathway they poison.

Fig. 1

[A. Diagram of canonical folate biosynthetic pathway and chemical structures of DHPS inhibitors; MOVED 1A TO SOM] A. Concentration-dependent accumulation of drug within M. tuberculosis following incubation of filter-laden M. tuberculosis cultures atop drug-impregnated Middlebrook 7H9-based agar media for 18 h. Values on the x-axis denote the relative drug concentration expressed in multiples of the drug’s minimum inhibitory concentration (MIC) in plate agar [or, for aminophenylsulfone (APS) and sulfanilamide (SNL)], molar equivalent of sulfamethoxazole (SMX) doses used) determined at day 14. Y-axis values denote relative molar abundance per unit cell biomass as reported by residual protein content of each sample; B. Concentration-dependent accumulation of PABA in M. tuberculosis following incubation as in 1B; All values are the average of experimental triplicates ± SEM. APS, aminophenylsulfone; MIC, minimal inhibitory concentration; PAS, para-aminosalicylic acid; SNL, sulfanilamide; SMX, sulfamethoxazole.

Fig. 2

A. Time-dependent accumulation of PAS, PABA, N-methyl-PAS (N-Me-PAS) and N, N-dimethyl PAS (N-(Me)₂-PAS) in M. tuberculosis following incubation with PAS as in Fig. 1C; B. Mass spectral profiles displaying concentration-dependent formation of PAS-derived pteroate and folate analogs following exposure of M. tuberculosis to PAS for 18 hours; C. Chemical and kinetic competence of PABA and PAS as in vitro substrates for purified recombinant M. tuberculosis DHPS (top) and FolC (bottom); [D. Chemical scheme of PAS-derived biotransformation products. All values are the average of experimental triplicates ± SEM. RT, retention time; MOVED 2D TO SOM].

Fig. 3

A. Heatmap representation of 6 key folate-dependent metabolites in M. tuberculosis following treatment with bioequivalent doses of PAS, SMX, APS, or SNL as in 1A.Numbers denote drug concentration expressed in relation to MIC or, for APS and SNL, molar equivalent of SMX doses used. Color intensities correspond to relative levels of metabolites, and are expressed as the log₂-transformed ratio of the normalized signal intensity in drug-treated cells at each concentration to the normalized signal intensity in the drug-free sample, and visually scaled as indicated in upper right inset. Signal intensities were normalized to cell protein biomass at each concentration; B. Time-dependent effects of PAS on folate-dependent metabolite levels shown in 3A. Values on y-axes denote relative molar abundance per unit cell biomass as reported by residual protein content of each sample; [C. Metabolic pathway diagram of folate-dependent reactions; MOVED 3C TO SOM] C. Heatmap representation demonstrating a time-dependent metabolic rescue of the PAS-induced effects with a 10-fold molar excess of exogenous PABA. All values are the average of experimental triplicates ± SEM. Labels and abbreviations as in 1B and 3A.