The structure of mouse L1210 dihydrofolate reductase‐drug complexes and the construction of a model of human enzyme

Stammers, D.K.; Champness, J.N.; Beddell, C.R.; Dann, J. G.; Eliopoulos, Elias; Geddes, A. J.; Ogg, D.; North, A. C. T.

doi:10.1016/0014-5793(87)81042-6

Cited by 92 publications

(54 citation statements)

References 19 publications

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“…The conformations of the chemically very similar ligands folate and methotrexate, one a substrate the other a potent inhibitor, differ substantially in that their pteridine rings are in inverse orientations relative to their p-aminobenzoyl-L-glutamate moieties. Methotrexate binding is similar to that previously observed in two bacterial enzymes but is quite different from that observed in the enzyme from a mouse lymphoma cell line [Stammers et al (1987) FEBS Lett. 218, 178-1841.…”

supporting

confidence: 76%

Crystal structure of human dihydrofolate reductase complexed with folate

Oefner

D’Arcy

Winkler

1988

European Journal of Biochemistry

223

171

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The crystal structure of recombinant human dihydrofolate reductase with folate bound in the active site has been determined and the structural model refined at 0.2-nm resolution. Preliminary studies of the binding of the inhibitors methotrexate and trimethoprim to the human apoenzyme have been performed at 0.35-nm resolution. The conformations of the chemically very similar ligands folate and methotrexate, one a substrate the other a potent inhibitor, differ substantially in that their pteridine rings are in inverse orientations relative to their p-aminobenzoyl-L-glutamate moieties. Methotrexate binding is similar to that previously observed in two bacterial enzymes but is quite different from that observed in the enzyme from a mouse lymphoma cell line [Stammers et al. (1987) FEBS Lett. 218, 178-1841. The geometry of the polypeptide chain around the folate binding site in the human enzyme is not consistent with conclusions previously drawn with regard to the species selectivity of the inhibitor trimethoprim J. Biol. Chem. 260, Dihydrofolate reductase (DHFR; tetrahydrofolate : NADP oxidoreductase) catalyzes the reduction of dihydrofolate or folate to tetrahydrofolate in an NADPH-dependent manner. The enzyme is present in all dividing cells and maintains the level of fully reduced folate coenzymes, which are essential for the synthesis of thymidylate and other metabolites [I].DHFR is the target enzyme for a number of clinically important drugs of which the antineoplastic agent methotrexate and the antibacterial agent trimethoprim are perhaps best known (see Fig. 1). For the purpose of rational drug design of novel antibacterial agents, knowledge of the structure of one or more representative bacterial enzymes as well as that of a mammalian DHFR, ideally from human cell line, is needed. The refined structures of a bacterial and an avian DHFR, from Escherichia coli and chicken liver respectively, have provided the basis for a detailed analysis of inhibitor selectivity, in particular of the remarkably higher affinity of trimethoprim for bacterial as compared to vertebrate DHFRs [2]. While this analysis has revealed important differences between the two classes of enzymes, the chicken and the human sequence are sufficiently different (overall homology 75%) that experimental determination of the structure of the human enzyme was considered necessary to provide a safer basis for drug design purposes.Very recently the refined structures of two ternary complexes of mouse L1210 DHFR, containing the cofxtor NADPH together with trimethoprim and methotrexate respectively, have been reported [3].Although binding of methotrexate and trimethoprim to the human enzyme, as reported in this paper, has thus far only been studied by difference Fourier methods at moderate resolution (0.35 nm), there are, somewhat surprisingly, distinct differences between these two mammalian enzymes, most notably in the side-chain conformation of the active-site residue Phe-31 and in the binding of methotrexate. Despite a wealth of stuctural infor...

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supporting

confidence: 76%

Crystal structure of human dihydrofolate reductase complexed with folate

Oefner

D’Arcy

Winkler

1988

European Journal of Biochemistry

223

171

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“…Gly-15 is located in a loop region that connects elements of protein secondary structure, a (-sheet near the N terminus (P3A, residues 4-10) and an a-helical region (aB, residues 27-40) (30). All except one residue of this loop (residue 11 of region [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] is conserved identically in all vertebrate species of DHFR (2). The loop has high sequence homology with, and is analogous to, the flexible Met-20 loop described for Escherichia coli DHFR (31).…”

Section: Discussionmentioning

confidence: 99%

Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.

Dicker

Waltham

Volkenandt

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DEHFR) 5,6,7,8-tetrahydrofolate, an essential carrier of one-carbon units in the biosynthesis of thymidylate, purine nucleotides, and methyl compounds. DHFR has been the subject of intense study for >40 years as it is the target enzyme for several important drugs, including methotrexate (MTX; 4-amino-4-deoxy-10-methylfolic acid), trimethoprim [TMP; 2,4-diamino-5-(3,4,5-trimethoxybenzyl) repeated fixed maximally tolerated doses of MTX. To this end, a series of L1210 leukemic cells resistant to MTX were developed in vivo through drug dosage schedules analogous to clinical schedules (7). Twenty independently derived sublines were selected for maximum resistance to MTX and all displayed an elevated DHFR activity. Ten of these sublines had only this phenotype, while seven sublines also demonstrated a reduced influx for MTX (7). The remaining three sublines (designated L1210/MTX-1, -2, and -3) were found to have a DHFR with reduced affinity for MTX on the basis of preliminary DHFR activity titration experiments with MTX using crude tumor lysates (8). Here we describe the basis for this decreased antifolate binding. MATERIALS AND METHODSMaterials. General molecular biology reagents (including items for mRNA isolation and cDNA synthesis) and enzyme purification materials were from sources as indicated (9, 10

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“…The binding of TPM to vertebrate dihydrofolate reducatase in a conformation similar to that found for the E. coli enzyme would not be energetically favorable. Fewer interactions occur between the diaminopyrimidine moiety and the vertebrate enzyme, consistent with the drug's lower affinity for mouse enzyme (28). TPM has a 3,000-fold greater affinity for the bacterial enzyme than for vertebrate enzymes (25) and is used as an antibacterial drug.…”

Section: B Antifolatesmentioning

confidence: 85%