An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary co... more Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.
Acta Crystallographica Section D Biological Crystallography, 2002
Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepine (PT653), an... more Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepine (PT653), an example of structure-based inhibitor design with 21-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR) relative to rat liver dihydrofolate reductase (rlDHFR). These data test the hypothesis that 2,4-diaminopteridines with a bulky N,N-diarylaminomethyl side chain at the 6-position could fit better into the larger active site of pcDHFR than into that of mammalian DHFR. The crystal structure of the ternary complex of NADPH, PT653 and pcDHFR, refined to 2.4 A resolution, reveals that PT653 binds in a different orientation than predicted from modeling studies reported previously [Rosowsky et al. (1999), J. Med. Chem. 42, 4853-4860]. These crystal data show that the pteridine-ring plane is tilted compared with that observed in the crystal structure of the pcDHFR methotrexate (MTX) NADPH ternary complex used as a template to model PT653 binding. Also, as a result of this tilt, the dibenzoazepine ring is bound deeper into the p-aminobenzoyl folate binding pocket of pcDHFR, thereby relieving close intermolecular contacts predicted from the modeling data. By far the most significant structural change, but more subtle in magnitude, is the ligand-induced conformational shift of 1.2 A away from the inhibitor of residues 61-66 in helix C. The other major effect is the unwinding of the short helical segment involving loop 47 which has a different conformation to that observed in other pcDHFR complexes [Cody et al. (1999), Biochemistry, 38, 4303-4312]. The favorable pcDHFR selectivity of PT653 could be a result of ligand-induced fit of the large hydrophobic dibenzazepine ring which occupies regions of the enzyme active site not probed by other antifolates and which take advantage of sequence and conformational differences between the structures of human and pcDHFR. These data suggest that such hydrophobic analogs could be used as lead compounds in the design of more pcDHFR-selective antifolates. Enzyme inhibition data also show that PT653 is 102-fold selective for Toxoplasma gondii (tg) DHFR relative to rlDHFR. Homology-modeling studies of the tgDHFR structure suggest that differences in ligand-binding orientation and enzyme sequence could influence the enhanced selectivity of PT653 for tgDHFR.
Structural data for two independent crystal forms (monoclinic, C2, and orthorhombic, P2(1)2(1)2(1... more Structural data for two independent crystal forms (monoclinic, C2, and orthorhombic, P2(1)2(1)2(1)) of the ternary complex of the potent antitumor agent PT523 [N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine], reduced nicotinamide adenine dinucleotide phosphate (NADPH), and recombinant human dihydrofolate reductase (hDHFR) reveals multiple binding orientations for the hemiphthaloyl group of the inhibitor. Analysis of these data shows that PT523 binds with its pteridine ring in the same orientation observed for methotrexate (MTX) analogues. However, in each structure, the hemiphthaloyl ring occupies three alternate conformations. In the C2 lattice, the phthaloyl moiety binds in two extended conformations, A and C, with each conformer having a 180 degrees flip of the o-carboxylate group, and a third, lower occupancy conformer B, with the phthaloyl group folded within contact of the active-site pocket. In the orthorhombic lattice, PT523 also has three conformers for the phthaloyl group; however, these differ from those observed in the monoclinic lattice. Two major conformers, A and C, are displaced on either side of the extended position observed in the C2 lattice, one near the folded B conformer of the C2 lattice and the other extended. These conformers form tighter intermolecular contacts than those in the C2 lattice. Conformer B is folded back away from the active site in a unique position. There are also significant differences in the conformation of the adenine-ribose moiety of NADPH in both complexes that differ from that observed for other inhibitor-NADPH-hDHFR ternary complexes. These data suggest that the added intermolecular contacts made by the hemiphaloyl group of PT523 contribute to its tighter binding to hDHFR than MTX, which does not extend as far from the active site and cannot make these contacts. These crystallographic observations of multiple conformations for the hemiphthaloyl group are in general agreement with solution NMR data for the binding of PT523 to hDHFR [Johnson et al. (1997) Biochemistry 36, 4399-4411], which show that the hemiphthaloyl group may adopt more than one conformation. However, the crystallographic data reveal more discretely occupied positions than can be interpreted from the solution data. These results suggest that crystal packing interactions may influence their stability.
The synthesis and biological activities of 14 6-substituted 2, 4-diaminoquinazolines are reported... more The synthesis and biological activities of 14 6-substituted 2, 4-diaminoquinazolines are reported. These compounds were designed to improve the cell penetration of a previously reported series of 2, 4-diamino-6-substituted-pyrido [2, 3-d] pyrimidines which had shown ...
International journal of peptide and protein research, 1993
The crystal structures of two solvated forms of ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NM... more The crystal structures of two solvated forms of ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala-] are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P2(1)2(1)2(1), a = 11.563(1), b = 21.863(2), c = 36.330(4) A. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P2(1)2(1)2(1), a = 14.067(2), b = 16.695(1), c = 36.824(6) A. N-Methylation of four of the seven residues of ternatin appears to reduce the number of low-energy conformations the molecule can assume. The same H-bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino-acid side chains, with the exception of D-Ile2, have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala3 and (NMe)Leu4, (ii) a type II beta-bend, involving residues Leu5-(NMe)Ala6-D...
The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is ... more The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the delta-endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an R(free) of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64-294); II, a three-sheet domain (residues 295-502); and III, a beta-sandwich domain (residues 503-652). The monomers in the orthorhombic C222(1) crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers...
The novel furopyrimidine, N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)-methyl]-methylamino] -benz... more The novel furopyrimidine, N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)-methyl]-methylamino] -benzoyl]-L-glutamate (MTXO), a classical antifolate with weak antitumor activity compared with methotrexate (MTX), has been studied as inhibitorcofactor ternary crystal complexes with recombinant Phe-31 to Ser (F31S) and Phe-31 to Gly (F31G) variant human dihydrofolate reductase (hDHFR). Kinetic data show that the binding affinity of MTXO is significantly weaker for the variant hDHFR enzyme than for the wild type enzyme. Structural data for the Phe-31 variants, along with wild type hDHFR, provide the first direct comparison of the binding interactions of a single antifolate in a family of variant hDHFR. These ternary hDHFR complexes crystallize in the rhombohedral space group R3, isomorphous to that reported for wild type hDHFR MTXO-NADPH ternary complex. MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu-30 in hDHFR. The greatest change on modification of the side chain...
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary co... more Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.
Acta Crystallographica Section D Biological Crystallography, 2002
Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepine (PT653), an... more Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepine (PT653), an example of structure-based inhibitor design with 21-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR) relative to rat liver dihydrofolate reductase (rlDHFR). These data test the hypothesis that 2,4-diaminopteridines with a bulky N,N-diarylaminomethyl side chain at the 6-position could fit better into the larger active site of pcDHFR than into that of mammalian DHFR. The crystal structure of the ternary complex of NADPH, PT653 and pcDHFR, refined to 2.4 A resolution, reveals that PT653 binds in a different orientation than predicted from modeling studies reported previously [Rosowsky et al. (1999), J. Med. Chem. 42, 4853-4860]. These crystal data show that the pteridine-ring plane is tilted compared with that observed in the crystal structure of the pcDHFR methotrexate (MTX) NADPH ternary complex used as a template to model PT653 binding. Also, as a result of this tilt, the dibenzoazepine ring is bound deeper into the p-aminobenzoyl folate binding pocket of pcDHFR, thereby relieving close intermolecular contacts predicted from the modeling data. By far the most significant structural change, but more subtle in magnitude, is the ligand-induced conformational shift of 1.2 A away from the inhibitor of residues 61-66 in helix C. The other major effect is the unwinding of the short helical segment involving loop 47 which has a different conformation to that observed in other pcDHFR complexes [Cody et al. (1999), Biochemistry, 38, 4303-4312]. The favorable pcDHFR selectivity of PT653 could be a result of ligand-induced fit of the large hydrophobic dibenzazepine ring which occupies regions of the enzyme active site not probed by other antifolates and which take advantage of sequence and conformational differences between the structures of human and pcDHFR. These data suggest that such hydrophobic analogs could be used as lead compounds in the design of more pcDHFR-selective antifolates. Enzyme inhibition data also show that PT653 is 102-fold selective for Toxoplasma gondii (tg) DHFR relative to rlDHFR. Homology-modeling studies of the tgDHFR structure suggest that differences in ligand-binding orientation and enzyme sequence could influence the enhanced selectivity of PT653 for tgDHFR.
Structural data for two independent crystal forms (monoclinic, C2, and orthorhombic, P2(1)2(1)2(1... more Structural data for two independent crystal forms (monoclinic, C2, and orthorhombic, P2(1)2(1)2(1)) of the ternary complex of the potent antitumor agent PT523 [N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine], reduced nicotinamide adenine dinucleotide phosphate (NADPH), and recombinant human dihydrofolate reductase (hDHFR) reveals multiple binding orientations for the hemiphthaloyl group of the inhibitor. Analysis of these data shows that PT523 binds with its pteridine ring in the same orientation observed for methotrexate (MTX) analogues. However, in each structure, the hemiphthaloyl ring occupies three alternate conformations. In the C2 lattice, the phthaloyl moiety binds in two extended conformations, A and C, with each conformer having a 180 degrees flip of the o-carboxylate group, and a third, lower occupancy conformer B, with the phthaloyl group folded within contact of the active-site pocket. In the orthorhombic lattice, PT523 also has three conformers for the phthaloyl group; however, these differ from those observed in the monoclinic lattice. Two major conformers, A and C, are displaced on either side of the extended position observed in the C2 lattice, one near the folded B conformer of the C2 lattice and the other extended. These conformers form tighter intermolecular contacts than those in the C2 lattice. Conformer B is folded back away from the active site in a unique position. There are also significant differences in the conformation of the adenine-ribose moiety of NADPH in both complexes that differ from that observed for other inhibitor-NADPH-hDHFR ternary complexes. These data suggest that the added intermolecular contacts made by the hemiphaloyl group of PT523 contribute to its tighter binding to hDHFR than MTX, which does not extend as far from the active site and cannot make these contacts. These crystallographic observations of multiple conformations for the hemiphthaloyl group are in general agreement with solution NMR data for the binding of PT523 to hDHFR [Johnson et al. (1997) Biochemistry 36, 4399-4411], which show that the hemiphthaloyl group may adopt more than one conformation. However, the crystallographic data reveal more discretely occupied positions than can be interpreted from the solution data. These results suggest that crystal packing interactions may influence their stability.
The synthesis and biological activities of 14 6-substituted 2, 4-diaminoquinazolines are reported... more The synthesis and biological activities of 14 6-substituted 2, 4-diaminoquinazolines are reported. These compounds were designed to improve the cell penetration of a previously reported series of 2, 4-diamino-6-substituted-pyrido [2, 3-d] pyrimidines which had shown ...
International journal of peptide and protein research, 1993
The crystal structures of two solvated forms of ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NM... more The crystal structures of two solvated forms of ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala-] are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P2(1)2(1)2(1), a = 11.563(1), b = 21.863(2), c = 36.330(4) A. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P2(1)2(1)2(1), a = 14.067(2), b = 16.695(1), c = 36.824(6) A. N-Methylation of four of the seven residues of ternatin appears to reduce the number of low-energy conformations the molecule can assume. The same H-bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino-acid side chains, with the exception of D-Ile2, have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala3 and (NMe)Leu4, (ii) a type II beta-bend, involving residues Leu5-(NMe)Ala6-D...
The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is ... more The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the delta-endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an R(free) of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64-294); II, a three-sheet domain (residues 295-502); and III, a beta-sandwich domain (residues 503-652). The monomers in the orthorhombic C222(1) crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers...
The novel furopyrimidine, N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)-methyl]-methylamino] -benz... more The novel furopyrimidine, N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)-methyl]-methylamino] -benzoyl]-L-glutamate (MTXO), a classical antifolate with weak antitumor activity compared with methotrexate (MTX), has been studied as inhibitorcofactor ternary crystal complexes with recombinant Phe-31 to Ser (F31S) and Phe-31 to Gly (F31G) variant human dihydrofolate reductase (hDHFR). Kinetic data show that the binding affinity of MTXO is significantly weaker for the variant hDHFR enzyme than for the wild type enzyme. Structural data for the Phe-31 variants, along with wild type hDHFR, provide the first direct comparison of the binding interactions of a single antifolate in a family of variant hDHFR. These ternary hDHFR complexes crystallize in the rhombohedral space group R3, isomorphous to that reported for wild type hDHFR MTXO-NADPH ternary complex. MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu-30 in hDHFR. The greatest change on modification of the side chain...
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