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Structural analysis of Pneumocystis carinii dihydrofolate reductase complexed with NADPH and 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine.

ABSTRACT: Structural data are reported for 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine (PY1014) complexed with Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to 1.8 Å resolution. These data reveal that the carboxylate of the ?-carboxyalkynyl side chain of PY1014, the most pcDHFR-selective analog in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR. The reversal of the 2',5'-substitution pattern of this analog compared with the highly selective diaminopyrimidine analog PY1011 (i.e. the 5'-pentynylcarboxy-5'-methoxy pattern of PY1014 versus the 3',4'-dimethoxy-5'-pentynylcarboxy pattern of PY1011) is necessary to achieve optimal interaction with Arg75 as observed in other structures. The larger diaminopyrido[2,3-d]pyrimidine ring of PY1014 places the 5'-methoxy group closer to Leu25 and Ser64 than does the diaminopyrimidine ring of PY1011. The 5'-methoxy O atom forms a hydrogen bond to the amide of Leu25 (O···N, 2.7 Å) and the 5'-methoxy methyl group makes a hydrophobic contact of 3.1 Å with C(?) of Ser64. Although the IC(50) values of PY1014 and PY1011 are similar, inhibition data show that the selectivity of PY1011 for pcDHFR is significantly greater. The greater selectivity for pcDHFR compared with mammalian DHFR of these inhibitors is also influenced by the enhanced hydrophobic interactions of the side-chain methylene atoms with Phe69 of pcDHFR compared with Asn64 of mammalian DHFR.

SUBMITTER: Cody V

PROVIDER: S-EPMC3325810 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Structural analysis of Pneumocystis carinii dihydrofolate reductase complexed with NADPH and 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine.

Cody Vivian V Pace Jim J Stewart Elizabeth E

Acta crystallographica. Section F, Structural biology and crystallization communications 20120328 Pt 4

Structural data are reported for 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine (PY1014) complexed with Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to 1.8 Å resolution. These data reveal that the carboxylate of the ω-carboxyalkynyl side chain of PY1014, the most pcDHFR-selective analog in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR. The reversal of the 2',5'-substitution pa ...[more]

PMID: 22505410

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Project description:To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N(6)-methyl-N(6)-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N(6)-methyl-N(6)-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N(6)-methyl-N(6)-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 24) are reported as ternary complexes with NADPH. This series of bicyclic pyrido[2,3-d]pyrimidines were designed in which there was a transposition of the 5-methyl group of PTX to the N9 position of the pyrido[2,3-d]pyrimidine. It was hypothesized that the N9-methyl group would preferentially interact with Ile123 of pcDHFR (and Ile123 of pjDHFR), but not with the shorter Val115 in hDHFR. Structure-activity data for this series of antifolates revealed that a trifluoro derivative (26) was the most selective against pjDHFR compared with mammalian DHFR (h/pj = 35.7). Structural data for the hDHFR-26 complex revealed that 26 binds in a different conformation from that observed in the pcDHFR-26 complex. In the hDHFR-26 complex the trifluorophenyl ring of 26 occupies a position near the cofactor-binding site, with close intermolecular contacts with Asp21, Ser59 and Ile60, whereas this ring in the pcDHFR-26 complex is positioned away from the cofactor site and near Ile65, with weaker contacts with Ile65, Phe69 and Ile123. Comparison of the intermolecular contacts between the N9-methyl group with Val115/Ile123 validates the hypothesis that the N9-methyl substituent preferentially interacts with Ile123 compared with Val115 of hDHFR, as the weaker contact with Val115 in the hDHFR structure is consistent with its weaker binding affinity compared with pcDHFR. The results for the structures of hDHFR-22 and pcDHFR-24 show that their inhibitor-binding orientation is similar to that observed in pcDHFR-26 and the pcDHFR variant (F69N) reported previously. The naphthyl moiety of 24 makes several intermolecular contacts with the active-site residues in pcDHFR that help to stabilize the binding, resulting in a more potent inhibitor.

Project description:Structural data are reported for five antifolates, namely 2,4-diamino-6-[5'-(5-carboxypentyloxy)-2'-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine, (1), and the 5'-[3-(ethoxycarbonyl)propoxy]-, (2), 5'-[3-(ethoxycarbonyl)butoxy]-, (3), 5'-[3-(ethoxycarbonyl)pentyloxy]-, (4), and 5'-benzyloxy-, (5), derivatives, which are potent and selective for Pneumocystis carinii dihydrofolate reductase (pcDHFR). Crystal structures are reported for their ternary complexes with NADPH and pcDHFR refined to between 1.4 and 2.0 Å resolution and for that of 3 with human DHFR (hDHFR) to 1.8 Å resolution. These data reveal that the carboxylate of the ω-carboxyalkoxy side chain of 1, the most potent inhibitor in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR, whereas the less potent ethyl esters of 2-4 bind with variable side-chain conformations. The benzyloxy side chain of 5 makes no contact with Arg75 and is the least active inhibitor in this series. These structural results suggest that the weaker binding of this series compared with that of their pyrimidine homologs in part arises from the flexibility observed in their side-chain conformations, which do not optimize intermolecular contact to Arg75. Structural data for the binding of 3 to both hDHFR and pcDHFR reveals that the inhibitor binds in two different conformations, one similar to each of the two conformations observed for the parent pyrido[2,3-d]pyrimidine, piritrexim (PTX), bound to hDHFR. The structure of the pcDHFR complex of 4 reveals disorder in the side-chain orientation; one orientation has the ω-carboxyalkoxy side chain positioned in the folate-binding pocket similar to the others in this series, while the second orientation occupies a new site near the nicotinamide ring of NADPH. This alternate binding site has not been observed in other DHFR structures. Structural data for the pcDHFR complex of 5 show that its benzyl side chain forms intermolecular van der Waals interactions with Phe69 in the binding pocket that could account for its enhanced binding selectivity compared with the other analogs in this series.

Dataset Information

Structural analysis of Pneumocystis carinii dihydrofolate reductase complexed with NADPH and 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine.

Publications

Structural analysis of Pneumocystis carinii dihydrofolate reductase complexed with NADPH and 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine.

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