The 2.3-A crystal structure of recombinant human dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a binary complex with folate (a poor substrate at neutral pH) and also as a binary complex with an inhibitor, 5-deazafolate. The inhibitor appears to be protonated at N8 on binding, whereas folate is not. Rotation of the peptide plane joining I7 and V8 from its position in the folate complex permits hydrogen bonding of 5-deazafolate's protonated N8 to the backbone carbonyl of I7, thus contributing to the enzyme's greater affinity for 5-deazafolate than for folate. In this respect it is likely that bound 5-deazafolate furnishes a model for 7,8-dihydrofolate binding and, in addition, resembles the transition state for folate reduction. A hypothetical transition-state model for folate reduction, generated by superposition of the DHFR binary complexes human.5-deazafolate and chicken liver.NADPH, reveals a 1-A overlap of the binding sites for folate's pteridine ring and the dihydronicotinamide ring of NADPH. It is proposed that this binding-site overlap accelerates the reduction of both folate and 7,8-dihydrofolate by simultaneously binding substrate and cofactor with a sub van der Waals separation that is optimal for hydride transfer.

A preparation of folate binding protein purified from human placental membranes in the presence of a variety of protease inhibitors followed by deglycosylation with N-glycanase gave a sharp band at Mr approximately 28,000 following SDS-polyacrylamide gel electrophoresis. The deglycosylated protein bound [3H]folic acid as tightly as the native protein. Peptides obtained following digestion of the purified protein with staphylococcal V8 protease and HPLC purification were sequenced. Polyclonal antibodies against the protein preparation were affinity purified and used to screen a placental cDNA expression library. A full-length cDNA for a placental folate binding protein was thus obtained and the corresponding protein sequence deduced. This result, taken together with the peptide sequence data, indicates the expression of at least two homologous folate binding proteins in placenta, one of which appears to be identical with the folate binding protein from human milk and nasopharyngeal epidermoid carcinoma (KB) cells; the cDNA sequence obtained corresponds to the other protein. The deduced protein sequence is characterized by a putative 16-residue amino-terminal signal peptide that is cleaved, resulting in a 239-residue polypeptide. The mature protein exhibits two potential sites for N-linked glycosylation at Asn-99 and Asn-179, eight potential intramolecular disulfide bonds, and a stretch of hydrophobic residues at the carboxyl terminus that could form a transmembrane domain. The protein bears a 68% sequence homology with the KB cell folate binding protein and may represent a fetal folate transport protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-state kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6----Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the kcat for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the Km values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by alpha-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

A series of eight previously undescribed 2,4-diaminothieno[2,3-d]pyrimidine analogues of the potent dihydrofolate reductase (DHFR) inhibitors trimetrexate (TMQ) and piritrexim (PTX) were synthesized as potential drugs against Pneumocystis carinii and Toxoplasma gondii, which are major causes of severe opportunistic infections in AIDS patients. 2,4-Diamino-5-methyl-6-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 3,4,5-trimethoxy or 2,5-dimethoxy substitution in the aryl/aralkyl moiety and 2,4-diamino-5-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 2,5-dimethoxy substitution in the aryl/aralkyl moiety were obtained by reaction of the corresponding 2-amino-3-cyanothiophenes with chloroformamidine hydrochloride. The aryl group in the 5,6-disubstituted analogues was either attached directly to the hetero ring or was separated from it by one or two carbons, whereas the aryl group in the 5-monosubstituted analogues was separated from the hetero ring by two or three carbons. 2-Amino-3-cyano-5-methyl-6-(aryl/alkyl)thiophene intermediates for the preparation of the 5,6-disubstituted analogues were prepared from omega-aryl-2-alkylidene-malononitriles and sulfur in the presence of a secondary amine, and 2-amino-3-cyano-4-(aryl/aralkyl)thiophene intermediates for the preparation of the 5-monosubstituted analogues were obtained from omega-aryl-1-chloro-2-alkylidenemalononitriles and sodium hydrosulfide. Synthetic routes to the heretofore unknown ylidenemalononitriles, and the ketone precursors thereof, were developed. The final products were tested in vitro as inhibitors of DHFR from Pneumocystis carinii, Toxoplasma gondii, rat liver, beef liver, and Lactobacillus casei. A selected number of previously known 2,4-diaminothieno[2,3-d]pyrimidines lacking the 3,4,5-trimethoxyphenyl and 2,5-dimethoxyphenyl substitution pattern of TMQ and PTX, respectively, were also tested for comparison. None of the compounds was as potent as TMQ or PTX, and while some of them showed some selectivity in their binding to Pneumocystis carinii and Toxoplasma gondii versus rat liver DHFR, this effect was not deemed large enough to warrant further preclinical evaluation.