| Abstract: |
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 μM IC<sub>50</sub>'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. © 2009 American Chemical Society. |
| Keywords: |
unclassified drug; genetics; nonhuman; animal; metabolism; animals; protein binding; high throughput screening; drug effect; drug resistance; drug screening; enzymology; drug discovery; enzyme inhibitor; drug development; reference database; antibiotic resistance; chemistry; drug antagonism; enzyme inhibitors; cell culture techniques; binding site; crystallography, x-ray; binding sites; chemical structure; molecular structure; 4,6 diamino 1,2 dihydro 2,2 dimethyl 1 [3 (2,4,5 trichlorophenoxy)propoxy] 1,3,5 triazine; antimalarial agent; biguanide derivative; dihydrofolate reductase inhibitor; rjf 00670; rjf 00719; rjf 01302; thymidylate synthase inhibitor; dihydrofolate reductase; folic acid antagonist; multienzyme complex; thymidylate synthase; thymidylate synthase dihydrofolate reductase; thymidylate synthase-dihydrofolate reductase; antimalarial activity; computer model; crystallization; drug binding site; ic 50; malaria falciparum; molecular docking; screening test; structure analysis; culture technique; plasmodium falciparum; x ray crystallography; antimalarials; folic acid antagonists; inhibitory concentration 50; malaria, falciparum; multienzyme complexes; tetrahydrofolate dehydrogenase
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