title: Stoichiometric selection of tight-binding inhibitors by wild-type and mutant forms of malarial (Plasmodium falciparum) dihydrofolate reductase.
abstract: A simple method for screening combinatorial and other libraries of inhibitors of malarial (Plasmodium falciparum) dihydrofolate reductase (PfDHFR) has been developed, based on the affinities of the inhibitors with the enzyme. In the presence of limiting amounts of the enzyme, a number of inhibitors in the library were bound to extents reflecting the relative binding affinities. Following ultrafiltration and guanidine hydrochloride treatment to release bound inhibitors, the amounts of free and bound inhibitors could be determined by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The differences in the patterns reflected the binding of high-affinity components compared with the other members in the library. A good correlation was found between the inhibition constants (Ki values) and the extent of binding of inhibitors to wild-type, double (C59R+S108N) and quadruple mutant (N51I+C59R+S108N+I164L) of PfDHFR, as well as human DHFR. In addition to identifying lead components of the libraries with high affinities (low Ki values) and stabilities (low k(off) rates), this simple method also provides an alternative way for quickly and accurately calculating enzyme binding affinities of inhibitors in combinatorial chemical libraries.
authors.affiliation: National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand.
authors.forename: Sumalee
authors.initials: S
authors.lastname: Kamchonwongpaisan
authors.forename: Jarunee
authors.initials: J
authors.lastname: Vanichtanankul
authors.forename: Bongkoch
authors.initials: B
authors.lastname: Tarnchompoo
authors.forename: Jirundon
authors.initials: J
authors.lastname: Yuvaniyama
authors.forename: Supannee
authors.initials: S
authors.lastname: Taweechai
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
mesh_terms: D000465:Algorithms; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D020650:Combinatorial Chemistry Techniques; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D002727:Proguanil / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D011739:Pyrimethamine / Q000031:analogs & derivatives / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014227:Triazines / Q000737:chemistry; D014295:Trimethoprim / Q000737:chemistry
chemical_list: D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D014227:Triazines; C026009:cycloguanil; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D002727:Proguanil; D011739:Pyrimethamine