Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases

Abstract

Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.

Figure 1

The homotetrameric DfrBs are evolutionarily distinct from the chromosomal Dfrs. (A) The homotetrameric DfrB1 (PDB: 2RK1) is intrinsically TMP-resistant: its large, symmetrical active-site tunnel does not bind TMP. Each protomer is colored differently. The 20 N-terminal residues of each protomer are unstructured and are not represented. For clarity, only I68 of the V66-Q67-I68-Y69 (VQIY) region and K32 are represented as sticks on each protomer. (B) The chromosomal Dfrs are evolutionarily and structurally unrelated. The E. coli Dfr (PDB: 1DDR), shown at scale, is strongly inhibited by TMP. (C) Sequence alignment of the DfrB family; there is no DfrB8. The weakly conserved N-termini and the highly conserved β-barrel core including the VQIY residues that line the active site tunnel 4-fold are identified.

Figure 2

Microbial pathway for biosynthesis of tetrahydrofolate (H₄folate). Enzymes, shown in italics, are GTPCH, guanosine triphosphate (GTP) cyclohydrolase; PPase, phosphatase; DHNA, dihydroneopterin aldolase; HPPK, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; DHPS, dihydropteroate synthase; FPGS, folylpolyglutamate synthase; DHFR, dihydrofolate reductase. The target enzymes in this study, HPPK and DHFR, are highlighted in red.

Scheme 1. Bisubstrate Molecules Include the Pterin Moiety of DHF (Green) and the Adenosyl Moiety of NAD(P)H (Purple)

Atoms involved in hydride transfer catalyzed by DfrB1 are in red. The linker is variable.

Scheme 2. Bisbenzimidazole Inhibitors 6 and 7(30)

Figure 3

Docking of 1 (yellow) into the DfrB1 tunnel with NADPH (cyan) (PDB: 2RK1). A pose with the adenosine moiety of 1 overlaying best with that of NADPH is shown (Figure S3). In the top 25 poses, inhibitor 1 forms contacts most frequently with K32, V66, and I68 (Figure 1; Figure S4). Contacts are rarely or not established with G35 and A36 that participate in binding the 2′-phosphate of NADPH, and with the YTT cluster. (A) Front view. (B) Side view; two subunits are represented.