Comparison of ternary complexes of Pneumocystis carinii and wild-type human dihydrofolate reductase with coenzyme NADPH and a novel classical antitumor furo[2,3-d]pyrimidine antifolate

Vivian Cody, Nikolai Galitsky, Joseph R. Luft, Walter Pangborn, Aleem Gangjee, Rajesh Devraj, Sherry Queener, Raymond L. Blakley

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The novel furopyrimidine N-(4-(N-[(2,4-diaminofuro-[2,3-d]pyrimidin-5-yl)methyl]methylamino)benzoyl)-L -glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with wild-type Pneumocystis carinii (pc) and recombinant human wild-type dihydrofolate reductase (hDHFR). These structural data provide the first direct comparison of the binding interactions of the same antifolate inhibitor in the active site for pc and human DHFR. The human ternary DHFR complex crystallizes in the rhombohedral space group R3 and is isomorphous to the ternary complex reported for a γ-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P21 and is isomorphous to that reported for a trimethoprim (TMP) complex. Interpretation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observed for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compared with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a) - C(5) - C(8) - N(9) and C(5) - C(8) - N(9) C(1'), are -156.5/51.9°and -162.6/51.8°, respectively for h and pc, compared with -146.8/57.4°for MTXT. In each case, the p-aminobenzoylglutamate conformation is similar to that observed for MTXT. In the pcDHFR complex, the active-site region is conserved and the additional 20 residues in the sequence compared with the human enzyme are located in external loop regions. There is a significant change in the nicotinamide ribose conformation of the cofactor which places the nicotinamide O atom close to the 4NH2 group of MTXO (2.7 Å), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen bond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 Å contact with the 4NH2 group of MTXO. Although the novel classical antifolate MTXO is not highly active against pcDHFR, there are correlations between its binding interactions consistent with its lower potency as an inhibitor of h and pcDHFR compared with MTX.

Original languageEnglish
Pages (from-to)638-649
Number of pages12
JournalActa Crystallographica Section D: Biological Crystallography
Volume53
Issue number6
DOIs
StatePublished - 1997

Fingerprint

coenzymes
Folic Acid Antagonists
Pneumocystis carinii
Tetrahydrofolate Dehydrogenase
Coenzymes
pyrimidines
nicotinamide
NADP
Niacinamide
Conformations
Methotrexate
inhibitors
Pteridines
rings
Atoms
Trimethoprim
tetrazoles
ribose
glutamates
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics
  • Clinical Biochemistry
  • Structural Biology
  • Condensed Matter Physics

Cite this

Comparison of ternary complexes of Pneumocystis carinii and wild-type human dihydrofolate reductase with coenzyme NADPH and a novel classical antitumor furo[2,3-d]pyrimidine antifolate. / Cody, Vivian; Galitsky, Nikolai; Luft, Joseph R.; Pangborn, Walter; Gangjee, Aleem; Devraj, Rajesh; Queener, Sherry; Blakley, Raymond L.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 53, No. 6, 1997, p. 638-649.

Research output: Contribution to journalArticle

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abstract = "The novel furopyrimidine N-(4-(N-[(2,4-diaminofuro-[2,3-d]pyrimidin-5-yl)methyl]methylamino)benzoyl)-L -glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with wild-type Pneumocystis carinii (pc) and recombinant human wild-type dihydrofolate reductase (hDHFR). These structural data provide the first direct comparison of the binding interactions of the same antifolate inhibitor in the active site for pc and human DHFR. The human ternary DHFR complex crystallizes in the rhombohedral space group R3 and is isomorphous to the ternary complex reported for a γ-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P21 and is isomorphous to that reported for a trimethoprim (TMP) complex. Interpretation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observed for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compared with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a) - C(5) - C(8) - N(9) and C(5) - C(8) - N(9) C(1'), are -156.5/51.9°and -162.6/51.8°, respectively for h and pc, compared with -146.8/57.4°for MTXT. In each case, the p-aminobenzoylglutamate conformation is similar to that observed for MTXT. In the pcDHFR complex, the active-site region is conserved and the additional 20 residues in the sequence compared with the human enzyme are located in external loop regions. There is a significant change in the nicotinamide ribose conformation of the cofactor which places the nicotinamide O atom close to the 4NH2 group of MTXO (2.7 {\AA}), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen bond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 {\AA} contact with the 4NH2 group of MTXO. Although the novel classical antifolate MTXO is not highly active against pcDHFR, there are correlations between its binding interactions consistent with its lower potency as an inhibitor of h and pcDHFR compared with MTX.",
author = "Vivian Cody and Nikolai Galitsky and Luft, {Joseph R.} and Walter Pangborn and Aleem Gangjee and Rajesh Devraj and Sherry Queener and Blakley, {Raymond L.}",
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T1 - Comparison of ternary complexes of Pneumocystis carinii and wild-type human dihydrofolate reductase with coenzyme NADPH and a novel classical antitumor furo[2,3-d]pyrimidine antifolate

AU - Cody, Vivian

AU - Galitsky, Nikolai

AU - Luft, Joseph R.

AU - Pangborn, Walter

AU - Gangjee, Aleem

AU - Devraj, Rajesh

AU - Queener, Sherry

AU - Blakley, Raymond L.

PY - 1997

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N2 - The novel furopyrimidine N-(4-(N-[(2,4-diaminofuro-[2,3-d]pyrimidin-5-yl)methyl]methylamino)benzoyl)-L -glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with wild-type Pneumocystis carinii (pc) and recombinant human wild-type dihydrofolate reductase (hDHFR). These structural data provide the first direct comparison of the binding interactions of the same antifolate inhibitor in the active site for pc and human DHFR. The human ternary DHFR complex crystallizes in the rhombohedral space group R3 and is isomorphous to the ternary complex reported for a γ-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P21 and is isomorphous to that reported for a trimethoprim (TMP) complex. Interpretation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observed for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compared with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a) - C(5) - C(8) - N(9) and C(5) - C(8) - N(9) C(1'), are -156.5/51.9°and -162.6/51.8°, respectively for h and pc, compared with -146.8/57.4°for MTXT. In each case, the p-aminobenzoylglutamate conformation is similar to that observed for MTXT. In the pcDHFR complex, the active-site region is conserved and the additional 20 residues in the sequence compared with the human enzyme are located in external loop regions. There is a significant change in the nicotinamide ribose conformation of the cofactor which places the nicotinamide O atom close to the 4NH2 group of MTXO (2.7 Å), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen bond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 Å contact with the 4NH2 group of MTXO. Although the novel classical antifolate MTXO is not highly active against pcDHFR, there are correlations between its binding interactions consistent with its lower potency as an inhibitor of h and pcDHFR compared with MTX.

AB - The novel furopyrimidine N-(4-(N-[(2,4-diaminofuro-[2,3-d]pyrimidin-5-yl)methyl]methylamino)benzoyl)-L -glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with wild-type Pneumocystis carinii (pc) and recombinant human wild-type dihydrofolate reductase (hDHFR). These structural data provide the first direct comparison of the binding interactions of the same antifolate inhibitor in the active site for pc and human DHFR. The human ternary DHFR complex crystallizes in the rhombohedral space group R3 and is isomorphous to the ternary complex reported for a γ-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P21 and is isomorphous to that reported for a trimethoprim (TMP) complex. Interpretation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observed for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compared with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a) - C(5) - C(8) - N(9) and C(5) - C(8) - N(9) C(1'), are -156.5/51.9°and -162.6/51.8°, respectively for h and pc, compared with -146.8/57.4°for MTXT. In each case, the p-aminobenzoylglutamate conformation is similar to that observed for MTXT. In the pcDHFR complex, the active-site region is conserved and the additional 20 residues in the sequence compared with the human enzyme are located in external loop regions. There is a significant change in the nicotinamide ribose conformation of the cofactor which places the nicotinamide O atom close to the 4NH2 group of MTXO (2.7 Å), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen bond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 Å contact with the 4NH2 group of MTXO. Although the novel classical antifolate MTXO is not highly active against pcDHFR, there are correlations between its binding interactions consistent with its lower potency as an inhibitor of h and pcDHFR compared with MTX.

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