Effect of Codon 22 Mutations on Substrate and Inhibitor Binding for Human Dihydrofolate Reductase

Ercikan, Emine; Waltham, Mark; Dicker, Adam P.; Schweitzer, Barry; Bertino, Joseph R.

doi:10.1007/978-1-4615-2960-6_104

Cited by 5 publications

(8 citation statements)

References 10 publications

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“…The pteroyl moiety of MTX is also involved in hydrogen bonding with residue Trp24 via a conserved water molecule and with residue Glu30, 8 while the side-chain of residues Leu22 and Phe34 are within van der Waals distance of this portion of the inhibitor ( Figure 1(b)). 9,10 These interactions are formed also with the pteroyl moiety of folate, albeit with the opposite side of the pterin ring. The p-ABA moiety of MTX and DHF interact mainly via van der Waals interactions with residues Phe31 and Phe34 of α-helix 1 (residues [27][28][29][30][31][32][33][34][35][36][37][38][39][40], which also contains Gln35 that is proximal to the γ-glutamate moiety of the bound ligands.…”

mentioning

confidence: 97%

“…[11][12][13][14][15][16][17] Identification of MTX-resistant hDHFR mutants has been performed either ex vivo in cultured cells exposed to MTX [18][19][20] or by performing site-directed mutagenesis at the active site of hDHFR by rational design. 9,[11][12][13][14][15]21 These studies have mostly yielded point mutants that maintain good catalytic efficiency while displaying moderate MTX resistance (e.g. F31S, K i = 0.240 nM; L22R, K i = 4.6 nM).…”

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confidence: 99%

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Increasing Methotrexate Resistance by Combination of Active-site Mutations in Human Dihydrofolate Reductase

Volpato

Fossati

Pelletier

2007

Journal of Molecular Biology

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confidence: 97%

mentioning

confidence: 99%

Increasing Methotrexate Resistance by Combination of Active-site Mutations in Human Dihydrofolate Reductase

Volpato

Fossati

Pelletier

2007

Journal of Molecular Biology

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“…The combined filtrates were freeze-dried; yield 37 mg (total 67 mg, 71%). Ionexchange on a DEAE-cellulose using NH4HC03 (0.2 to 2.4 M) as the eluent, followed by freeze-drying, afforded a white solid indistinguishable (mp, IR, NMR) from the product in the preceding experiment; mp 205-209 °C dec; IR (KBr) v 3420 br, 1640,1600,1570,1500 cm-1; NMR (DMSO-d6) ó 1.93-2.28 (m, 4H, CH2CH2), 4.30 (br s, 1H, a-CH), 4.4 (s, 2H, 9-CH2), 6.25 (s, 2H, 2-NH2), 6.74 (m, 1H, 10-NH), 6.79 (d, 2H, 3'-and 5'-H), 7.07 (m, 1H, 6-H), 7.17 (s, 2H, 4-NH2), 7.21 (m, 1H, 7-H), 7.44 (m, 1H, 8-H), 7.69 (d, 1H, 2'and 6'-H), 8.0 (br s, 1H, CONH). Addition of D20 caused disappearance of the 2-NH2, 4-NH2, and CONH protons.…”

Section: Methodsmentioning

confidence: 87%

“…The precipitated solid was collected, washed with H20, and purified by silica gel column chromatography with solvent D as the eluent. Fractions giving a single TLC spot (,Rf 0.15, silica gel, solvent A) were pooled and evaporated to a white solid, which was recrystallized from boiling EtOH to obtain white needles (135 mg, 11%); mp 168-171 °C dec (softening above 158 °C); IR (KBr) v 3420 br, 2940, 1735, 1630, 1610, 1570, 1560, 1500 cm"1; NMR (DMSO-d6) <5 2.08 m, 2H, 0-CH2), 3.57 (s, 3H, y-COOMe), 3.61 (s, 3H, -COOMe), 4.40 (m, 1H, a-CH), 4.45 (m, 2H, 9-CH2), 5.98 (s, 2H, 2-NH2), 6.78- 6.80 (m, 3H, 3'and 5'-H, 10-NH), 6.99 (s, 2H, 4-NH2), 7.03 (m, 1H, 6-H), 7.18 (m, 1H, 7-H), 7.40 (m, 1H, 8-H), 7.72 (d, 2H, 2'-and 6'-H), 8.37 (d, 1H, amide NH). Anal.…”

Section: Methodsmentioning

confidence: 99%

“…Our interest in 5-substituted 2,4-diaminoquinazolines was sparked by a recent molecular modeling study in which the three-dimensional features of the active site in wild-type human DHFR and a site-directed mutant enzyme with phenylalanine (Phe) in place of leucine (Leu) at position 22 were compared. 4 Interest in this particular mutation derives from the fact that position 22 is believed to be a "hot spot" for mutations in the dhfr gene. Although a natural Leu22 -Phe mutation in human DHFR remains to be found, such a mutation has been observed in two different Chinese hamster ovary cell lines selected for resistance to the classical antifolate methotrexate (MTX, 1; Chart 1) in two independent laboratories.…”

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confidence: 99%

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2,4-Diamino-5-substituted-quinazolines as Inhibitors of a Human Dihydrofolate Reductase with a Site-Directed Mutation at Position 22 and of the Dihydrofolate Reductases from Pneumocystis carinii and Toxoplasma gondii

Rosowsky¹,

Mota²,

Queener³

et al. 1995

J. Med. Chem.

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2,4-Diaminoquinazoline antifolates with a lipophilic side chain at the 5-position, and in one case with a classical (p-aminobenzoyl)-L-glutamate side chain, were synthesized as potentially selective inhibitors of a site-directed mutant of human dihydrofolate reductase (DHFR) containing phenylalanine instead of leucine at position 22. This mutant enzyme is approximately 100-fold more resistant than native enzyme to the classical antifolate methotrexate (MTX), yet shows minimal cross resistance to the nonclassical antifolates piritrexim (PTX) and trimetrexate (TMQ). Although they were much less potent than trimetrexate and piritrexim, the lipophilic 5-substituted analogues were all found to bind approximately 10 times better to the mutant DHFR than to the wild-type enzyme. The potency of the analogue with a classical (p-aminobenzoyl)-L-glutamate side chain was similarly diminished in comparison with MTX, but the difference in its binding affinity to the two DHFR species was only 5-fold. Thus, by making subtle structural changes in the antifolate molecule, it may be possible to attack resistance due to mutational alterations in the active site of the target enzyme. Also, to test the hypothesis that DHFR from Pneumocystis carinii and Toxoplasma gondii may have a less sterically restrictive active site than the enzyme from mammalian cells, inhibition assays using several of the lipophilic analogues in the series were carried out against the P. carinii and T. gondii reductases in comparison with the enzyme from rat liver. In contrast to their preferential binding to mutant versus wild-type human DHFR, binding of these analogues to the P. carinii and T. gondii enzymes was weaker than binding to rat enzyme. It thus appears that, if the active site of the DHFR from these parasites is less sterically restrictive than the active site of the mammalian enzyme, this difference cannot be successfully exploited by moving the side chain from the 6-position to the 5-position.

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Dihydrofolate Reductase from Kaposi's Sarcoma-Associated Herpesvirus

et al. 2000

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Kaposi's sarcoma-associated herpesvirus (KSHV) is the first human virus known to encode dihydrofolate reductase (DHFR), an enzyme required for nucleotide and methionine biosynthesis. We have studied the purified KSHV-DHFR enzyme in vitro and analyzed its expression in cultured B-cell lines derived from primary effusion lymphoma (PEL), an AIDS-associated malignancy. The amino acid sequence of KSHV-DHFR is most similar to human DHFR (hDHFR), but the viral enzyme contains an additional 23 amino acids at the carboxyl-terminus. The viral DHFR, overexpressed and purified from E. coli, was catalytically active in vitro. The K(m) of KSHV-DHFR for dihydrofolate (FH(2)) was 2.4 microM, which is significantly higher than the K(m) of recombinant hDHFR (rhDHFR) for FH(2) (390 nM). K(m) values for NADPH were similar for the two enzymes, about 1 microM. KSHV-DHFR was inhibited by folate antagonists such as methotrexate (K(i): 200 pM), aminopterin (K(i): 610 pM), pyrimethamine (K(i): 29 nM), trimethoprim (K(i): 2.3 microM), and piritrexim (K(i): 3.9 nM). In all cases, K(i) values for these folate antagonists were higher for KSHV-DHFR than for rhDHFR. The viral enzyme was expressed at levels two- to tenfold higher than hDHFR in PEL cell lines as an early lytic cycle gene. KSHV-DHFR mRNA and protein appeared from 6 to 24 h after chemical induction of the KSHV lytic cycle. Epitope-tagged KSHV-DHFR and rhDHFR both localized to the nucleus of transfected cells, while other KSHV nucleotide metabolism genes localized to the cytoplasm. DHFR activity was not essential for viral replication in cultured PEL cells. Since hDHFR was not detectable in peripheral blood mononuclear cells (PBMCs), KSHV-DHFR may function to provide increased DHFR activity in vivo in infected cells that have little or none of their own enzyme.

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Effect of Codon 22 Mutations on Substrate and Inhibitor Binding for Human Dihydrofolate Reductase

Cited by 5 publications

References 10 publications

Increasing Methotrexate Resistance by Combination of Active-site Mutations in Human Dihydrofolate Reductase

Increasing Methotrexate Resistance by Combination of Active-site Mutations in Human Dihydrofolate Reductase

2,4-Diamino-5-substituted-quinazolines as Inhibitors of a Human Dihydrofolate Reductase with a Site-Directed Mutation at Position 22 and of the Dihydrofolate Reductases from Pneumocystis carinii and Toxoplasma gondii

Dihydrofolate Reductase from Kaposi's Sarcoma-Associated Herpesvirus

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