Exploiting structural analysis, in silico screening, and serendipity to identify novel inhibitors of drug-resistant falciparum malaria.

Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in folate biosynthesis and a major malarial drug target. This bifunctional enzyme thus presents different design approaches for developing novel inhibitors against drug-resistant mutants. We performed a high-throughput in silico screen of a database of diverse, drug-like molecules against a non-active-site pocket of TS-DHFR. The top compounds from this virtual screen were evaluated by in vitro enzymatic and cellular culture studies. Three compounds active to 20 microM IC(50)'s in both wildtype and antifolate-resistant P. falciparum parasites were identified; moreover, no inhibition of human DHFR enzyme was observed, indicating that the inhibitory effects appeared to be parasite-specific. Notably, all three compounds had a biguanide scaffold. However, relative free energy of binding calculations suggested that the compounds might preferentially interact with the active site over the screened non-active-site region. To resolve the two possible modes of binding, co-crystallization studies of the compounds complexed with TS-DHFR enzyme were performed. Surprisingly, the structural analysis revealed that these novel, biguanide compounds do indeed bind at the active site of DHFR and additionally revealed the molecular basis by which they overcome drug resistance. To our knowledge, these are the first co-crystal structures of novel, biguanide, non-WR99210 compounds that are active against folate-resistant malaria parasites in cell culture.

Search for non-nucleoside inhibitors of HIV-1 reverse transcriptase using chemical similarity, molecular docking, and MM-GB/SA scoring.

A virtual screening protocol has been applied to seek non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) and its K103N mutant. First, a chemical similarity search on the Maybridge library was performed using known NNRTIs as reference structures. The top-ranked molecules obtained from this procedure plus 26 known NNRTIs were then docked into the binding sites of the wild-type reverse transcriptase (HIV-RT) and its K103N variant (K103N-RT) using Glide 3.5. The top-ranked 100 compounds from the docking for both proteins were post-scored with a procedure using molecular mechanics and continuum solvation (MM-GB/SA). The validity of the virtual screening protocol was supported by (i) testing of the MM-GB/SA procedure, (ii) agreement between predicted and crystallographic binding poses, (iii) recovery of known potent NNRTIs at the top of both rankings, and (iv) identification of top-scoring library compounds that are close in structure to recently reported NNRTI HTS hits. However, purchase and assaying of selected top-scoring compounds from the library failed to yield active anti-HIV agents. Nevertheless, the highest-ranked database compound, S10087, was pursued as containing a potentially viable core. Subsequent synthesis and assaying of S10087 analogues proposed by further computational analysis yielded anti-HIV agents with EC50 values as low as 310 nM. Thus, with the aid of computational tools, it was possible to evolve a false positive into a true active.