Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants

Abstract

In drug-discovery, systematic variations of substituents on a common scaffold and bioisosteric replacements are often used to generate diversity and obtain molecules with better biological effects. However, this could saturate the small-molecule diversity pool resulting in drug-resistance. On the other hand, conventional drug-discovery relies on targeting known pockets on protein surfaces leading to drug resistance by mutations of critical pocket residues. Here, we present a two-pronged strategy of designing novel drugs that target unique pockets on a protein’s surface to overcome the above problems. Dihydrofolate reductase, DHFR, is a critical enzyme involved in thymidine and purine nucleotide biosynthesis. Several classes of compounds that are structural-analogues of the substrate dihydrofolate have been explored for their anti-folate activity. Here, we describe 10 novel small-molecule inhibitors of Escherichia coli DHFR, EcDHFR, belonging to the stilbenoid, deoxybenzoin and chalcone family of compounds discovered by a combination of pocket-based virtual ligand screening and systematic scaffold-hopping. These inhibitors show a unique uncompetitive or noncompetitive inhibition mechanism, distinct from those reported for all known inhibitors of DHFR, indicative of binding to a unique pocket distinct from either substrate or cofactor-binding pockets. Furthermore, we demonstrate that rescue mutants of EcDHFR, with reduced affinity to all known classes of DHFR inhibitors, are inhibited at the same concentration as the wild-type. These compounds also exhibit antibacterial activity against E. coli harboring the drug-resistant variant of DHFR. This discovery is the first report on a novel class of inhibitors targeting a unique pocket on EcDHFR.

The crystal structure of a tetrahydrofolate-bound dihydrofolate reductase reveals the origin of slow product release

“…4, on comparing the conformation of AMPQD to FH4 and trimethoprim to FH4, there are subtle differences in the van der Waals envelopes. The trimethoprim eDHFR escape variants of E. coli DHFR possess mutations that also block the inhibitory function of AMPQD 57 . By studying the differences in interactions, one can search for other ligands that minimize these interaction differences with FH2 and FH4.…”

“…The observed Met20 loop’s conformational dynamics in the current eDHFR substrate/product/inhibitor complexes are consistent with ligand-dependent conformational sampling and energy landscape shaping during catalysis. Exploiting the conformational diversity of eDHFR, especially targeting the energetically favorable occluded conformations of the FH4 and AMPQD complexes and DHFR’s allosteric sites, may enable the design of effective next-generation therapeutics to target DHFR with species-specificity 38,57 .…”

Benzothiazolyl Ureas are Low Micromolar and Uncompetitive Inhibitors of 17β-HSD10 with Implications to Alzheimer’s Disease Treatment

“…The inhibitors can bind the target only if the substrate is present and this mechanism of action should be favorable for enzymes with cellular substrate concentration higher than the enzyme K m value [40]. Several drugs act in an uncompetitive manner towards dehydrogenases or reductases with a NAD + /NADH pair as their enzyme cofactor: e.g., mycophenolic acid, reversible inhibitor of inosine 5 -monophosphate dehydrogenase [41,42]; ononetin, an inhibitor of dihydrofolate reductase [43]; or epristeride, an inhibitor of human steroid 5α-reductase [44].…”

Chemical space of Escherichia coli dihydrofolate reductase inhibitors: New approaches for discovering novel drugs for old bugs

“…For instance, the top nine best compounds tested from our studies (along with appropriate positive and negative controls) against a panel of seven organisms belonging to the gram positive, gram negative and yeast cells returned non-significant inhibition at a concentration of 20 μM (unpublished results). However, our pocket based approaches (section 2.6), apart from picking the classical and non-classical inhibitors 60 , 80 , 81 , were also successful in predicting a unique set of inhibitors targeting a novel allosteric pocket. These compounds had MIC values very similar to their IC 50 values for the enzyme strongly suggesting that they can efficiently diffuse across gram negative bacterial cell membranes 80 , 81 .…”