title: Targeted mutations of Bacillus anthracis dihydrofolate reductase condense complex structure−activity relationships.
abstract: Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Road, Storrs, Connecticut 06269, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Beierlein
authors.forename: Nanda G
authors.initials: NG
authors.lastname: Karri
authors.forename: Amy C
authors.initials: AC
authors.lastname: Anderson
mesh_terms: D000480:Alkynes / Q000138:chemical synthesis / Q000737:chemistry; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000737:chemistry; D001408:Bacillus anthracis / Q000201:enzymology*; D001713:Biphenyl Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011743:Pyrimidines / Q000138:chemical synthesis* / Q000737:chemistry; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*
chemical_list: D000480:Alkynes; D000900:Anti-Bacterial Agents; D001713:Biphenyl Compounds; D005493:Folic Acid Antagonists; D011743:Pyrimidines; D013762:Tetrahydrofolate Dehydrogenase