Rational design of novel allosteric dihydrofolate reductase inhibitors showing antibacterial-effects on drug-resistant E. coli escape-variants

摘要

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.