Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

Abstract

The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus. Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis, exerts whole cell activity against M. abscessus. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus. As observed in M. tuberculosis, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus. PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus.

Keywords: DHFR; DHPS; NTM; ThyA; folate pathway; non-tuberculous mycobacteria; synergy.

Fig 1

Structures of tested DHFR inhibitors. See Table 1 for references.

Fig 2

Activity of selected DHFR inhibitors against recombinant M. abscessus ATCC 19977 DHFR enzyme. Enzyme inhibition dose-response curves are shown for (A) PQD-1, (B) trimetrexate (TMX), (C) WR99210, and (D) trimethoprim (TMP). The data were fitted to a non-linear regression curve using the variable slope model, and the IC₅₀ values were calculated using GraphPad Prism 9. Percent activity values are the means of three independently performed experiments, and error bars indicate standard deviations.

Fig 3

In silico model of PQD-1 and trimethoprim binding to M. abscessus ATCC 19977 DHFR (PDB code: 7K6C). (A, C) Optimized poses of PQD-1 (yellow sticks) and trimethoprim (green sticks) with interactions with DHFR active site residues. DHFR is colored in cyan, with the residues in the binding pockets shown as cyan sticks. (B, D) Two-dimensional presentation of the key interactions of PQD-1 (B) and trimethoprim (D) with their binding pockets shown in (A, C). The hydrogen bonds, salt bridges, and hydrophobic and π-π stacking interactions are depicted as green, orange, pink, and warm pink dashed lines, respectively.

Fig 4

Mechanism of action of PQD-1. (A) Effect of DHFR over-expression on PQD-1 susceptibility of M. abscessus ATCC 19977. Cultures were treated for 3 days with PQD-1 (top) or TMX (bottom), the second most potent M. abscessus inhibitor identified in the initial whole cell screen (Table 1) and confirmed as a biochemical inhibitor of M. abscessus DHFR (Fig. 2). Percent growth values are the means of three independently carried out experiments, and error bars indicate standard deviations. (B) Schematic of mycobacterial folate pathway and role of thymidylate synthase (TYMS) ThyA. DHPS, inhibited by SMX, converts p-aminobenzoic acid (pABA) into dihydropteroate (DHP), which in turn is converted to dihydrofolate (DHF). DHFR, inhibited by PQD-1, reduces DHF to tetrahydrofolate (THF), which is converted into methyl-tetrahydrofolate (mTHF). The TYMS ThyA catalyzes the reductive methylation of deoxyuridine-monophosphate (dUMP) to deoxythymidine-monophosphate (dTMP) utilizing mTHF as the methyl donor and reductant in the reaction, yielding DHF, the substrate of DHFR, as a by-product. ThyX is a second thymidylate synthase catalyzing the reductive methylation of dUMP to dTMP utilizing mTHF only as the methyl donor (hence generating THF instead of DHF) and NADPH as the reductant. The pathway is according to Hajian et al. (31). (C) Plot of the fractional inhibitory concentrations (FIC) of PQD-1 (top) and TMX (bottom) versus DHPS inhibitor SMX. The FIC index (FICI), calculated as (MIC_A ^combi/MIC_A ^alone) + (MIC_B ^combi/MIC_B ^alone), indicates synergy if <0.5. The FICs were calculated based on the MIC₅₀ values. The experiments were carried out three times independently in duplicate, and the presented data are one representative example.