title: Exploiting structural analysis, in silico screening, and serendipity to identify novel inhibitors of drug-resistant falciparum malaria.
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 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.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA.
authors.forename: Tina
authors.initials: T
authors.lastname: Dasgupta
authors.forename: Penchit
authors.initials: P
authors.lastname: Chitnumsub
authors.forename: Sumalee
authors.initials: S
authors.lastname: Kamchonwongpaisan
authors.forename: Cherdsak
authors.initials: C
authors.lastname: Maneeruttanarungroj
authors.forename: Sara E
authors.initials: SE
authors.lastname: Nichols
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Lyons
authors.forename: Julian
authors.initials: J
authors.lastname: Tirado-Rives
authors.forename: William L
authors.initials: WL
authors.lastname: Jorgensen
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
authors.forename: Karen S
authors.initials: KS
authors.lastname: Anderson
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D001665:Binding Sites; D018929:Cell Culture Techniques; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D020128:Inhibitory Concentration 50; D016778:Malaria, Falciparum / Q000188:drug therapy*; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D011485:Protein Binding; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D000962:Antimalarials; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase