Structure-Based Design of Dimeric Bisbenzimidazole Inhibitors to an Emergent Trimethoprim-Resistant Type II Dihydrofolate Reductase Guides the Design of Monomeric Analogues

Abstract

The worldwide use of the broad-spectrum antimicrobial trimethoprim (TMP) has induced the rise of TMP-resistant microorganisms. In addition to resistance-causing mutations of the microbial chromosomal dihydrofolate reductase (Dfr), the evolutionarily and structurally unrelated type II Dfrs (DfrBs) have been identified in TMP-resistant microorganisms. DfrBs are intrinsically TMP-resistant and allow bacterial proliferation when the microbial chromosomal Dfr is TMP-inhibited, making these enzymes important targets for inhibitor development. Furthermore, DfrBs occur in multiresistance plasmids, potentially accelerating their dissemination. We previously reported symmetrical bisbenzimidazoles that are the first selective inhibitors of the only well-characterized DfrB, DfrB1. Here, their diversification provides a new series of inhibitors ( K i = 1.7–12.0 μM). Our results reveal two prominent features: terminal carboxylates and inhibitor length allow the establishment of essential interactions with DfrB1. Two crystal structures demonstrate the simultaneous binding of two inhibitor molecules in the symmetrical active site. Observations of those dimeric inhibitors inspired the design of monomeric analogues, binding in a single copy yet offering similar inhibition potency ( K i = 1.1 and 7.4 μM). Inhibition of a second member of the DfrB family, DfrB4, suggests the generality of these inhibitors. These results provide key insights into inhibition of the highly TMP-resistant DfrBs, opening avenues to downstream development of antibiotics for combatting this emergent source of resistance.

Computational Development of Inhibitors of Plasmid-Borne Bacterial Dihydrofolate Reductase

“…Due to the remarkable differences in selectivity of the different classes of DHFR, the development of drugs targeting the trimethoprim and methotrexate-resistant, plasmid-borne, bacterial R67 dihydrofolate reductases may enable potent antibiotic activity accompanied by low toxicity to human cells. In spite of this, limited effort has been expended so far in this endeavor which has resulted in the discovery of a single class of symmetrical competitive inhibitors based on 1 H -benzimidazole-5-carboxylic acid [ 13 , 14 ] with low toxicity towards mammalian cells, but limited potential for use as drugs due to relatively low affinity towards the target DHFR (K i = 2–4 µM, instead of the nanomolar range usually required for a successful drug). The present paper describes a computational search for dihydrofolate analogues, which may competitively bind to DHFR.…”

Design, Synthesis, and Antimicrobial Evaluation of a New Series of N-Sulfonamide 2-Pyridones as Dual Inhibitors of DHPS and DHFR Enzymes

“…23,24 Moreover, N-sulfonamide 2-pyridone derivatives have exhibited good antimicrobial activity against a variety of antimicrobial strains. 25,26 Development of drug resistance by different bacterial strains against both sulfonamide drugs and TMP 27 has encouraged us to design new compounds containing the sulfonamide group as DHPS inhibitors that are bonded to the six-membered ring such as pyridine substituted with the amino/hydroxyl group at the 6position to inhibit DHFR, as shown in Figure 1. Based on our experience in the synthesis of novel compounds containing benzothiazoles, pyridines, and sulfonamide moieties, 28−33 new compounds of N-sulfonamide 2-pyridones incorporating the benzothiazole moiety were envisaged and synthesized via the reaction of novel benzothiazol sulfonylhydrazide with ketene dithioacetal derivatives.…”

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

“…Therefore, it is possible to form nonproductive complexes between two substrates or two cofactors as well as the productive complex with one substrate and one cofactor 4 . Despite several decades of research on R67 DHFR, there are no drugs that target the enzyme, and only recently have inhibitors been designed that specifically target the enzyme 5,6 …”

“…4 Despite several decades of research on R67 DHFR, there are no drugs that target the enzyme, and only recently have inhibitors been designed that specifically target the enzyme. 5,6 Binding of the cofactor analog, NADP + , can be monitored by HSQC NMR; however, substrate, dihydrofolate (DHF), exhibits complex NMR spectra due to tighter binding of the ligand slowing the exchange kinetics and breaking up the protein symmetry, which increased the number of peaks. 8 Therefore, a different NMR method is needed to study ligand and inhibitor binding to R67 DHFR.…”