Biomedical subjects

V Cody

Publications and source records attributed to V Cody.

At least 37 records · Page 2Linked to original sources

PubMed1997

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.

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 gamma-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P2(1) 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 degrees and -162.6/51.8 degrees, respectively for h and pc, compared with -146.8/57.4 degrees 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 4NH(2) group of MTXO (2.7 A), 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 A contact with the 4NH(2) 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.

Journal Article↗

PubMed1997

The incorporation of butyrophenones and related compounds into a pharmacophore for dopamine D2 antagonists.

This study is an attempt to incorporate the butyrophenones, an important class of nontricyclic antipsychotic drugs, into a previously proposed pharmacophore model of tricyclic dopamine D2 receptor antagonist ligands. Conformational energy calculations were performed using the MM3-92 program on spiperone, as a representative butyrophenone, and milenperone and R48455, as related compounds with more limited conformational freedom. Twenty seven conformers were evaluated for spiperone with MM3-92 calculations and nine of these were within 1.1 kcal/mole of the global minima indicating the flexibility of the compound. A conformational analysis of twenty crystal structures of butyrophenones was also performed and six distinct conformers were represented. All of the energy minimized conformers of spiperone were superimposed in a least squares sense onto loxapine as a relatively rigid, typical D2 antagonist and a pair of mirror image conformers, which are observed in one crystal structure of spiperone, were found to be the best fit. However, it was not possible to discriminate between these two conformers since they fit the pharmacophore model equally well. The para-fluoro and carbonyl group of the butyrophenones were found to correspond best to the oxygen and chlorine atoms of loxapine, respectively. The conformations of milenperone and R48455 were also consistent with the two putative biologically active forms of spiperone and the pharmacophore model. Conformational energy calculations were also performed on molindone, an antipsychotic drug in clinical use, which can be related to the butyrophenones since both have a carbonyl group adjacent to an aromatic ring. A putative biologically active form was proposed for molindone and this was related to the structure of piquindone, a rigid analog of molindone. All of the compounds were found to be entirely consistent with the pharmacophore model. However, as previously found, there is great variability in the distance between the ammonium nitrogen and the center of the relevant aromatic ring with the most extreme case in the present study being R48455 where the distance is 7.2 A. The results of the present study should also be relevant to the structures of novel, atypical antipsychotic drugs such as risperidone which appear to be analogs of the butyrophenones.

Antipsychotic Agents↗

PubMed1996

Structures of human transthyretin complexed with thyroxine at 2.0 A resolution and 3',5'-dinitro-N-acetyl-L-thyronine at 2.2 A resolution.

The molecular structures of two human transthyretin (hTTR, prealbumin) complexes, co-crystallized with thyroxine (3,5,3',5'-tetraiodo-L-thyronine; T(4)), and with 3',5'-dinitro-N-acetyl-LL-thyronine (DNNAT), were determined by X-ray diffraction methods. Crystals of both structures are orthorhombic, space group P2(1)2(1)2, and have two independent monomers in the asymmetric unit of the crystal lattice. These structures have been refined to 17.0% for 8-2.0 A resolution data for the T(4) complex (I), and to R = 18.4% for 8-2.2 A resolution data for the DNNAT structure (II). This report provides a detailed description of T(4) binding to wild-type hTTR at 2.0 A resolution, as well as DNNAT. In both structures, the two independent hormone-binding sites of the TTR tetramer are occupied by ligand. A 50% statistical disorder model was applied to account for the crystallographic twofold symmetry along the binding channel and the lack of such symmetry for the ligands. Results for the co-crystallized T(4) complex show that T(4) binds deep in the hormone-binding channel and displaces the bound water previously reported for T(4) soaked into a native transthyretin crystal [Blake & Oatley (1977). Nature (London), 268, 115-120]. DNNAT also binds deeper in the channel toward the tetramer center than T(4) with the nitro groups occupying the symmetrical innermost halogen pockets. The N-acetyl moiety does not form polar contacts with the protein side chains as it is oriented toward the center of the channel. The weak binding affinity of DNNAT results from the loss of hydrophobic interactions with the halogen binding pockets as observed in T(4) binding. These data suggest that the halogen-binding sites toward the tetramer center are of primary importance as they are occupied by analogues with weak affinity to TTR, and are therefore selected over the other halogen sites which contribute more strongly to the overall binding affinity.

Journal Article↗

PubMed1995

Structure-activity relationships of milrinone analogues determined in vitro in a rabbit heart membrane Ca(2+)-ATPase model.

The cardiac activity of a series of analogues of the positive inotropic bipyridines amrinone (5-amino-[3,4'-bipyridin]-6(1H)-one) and milrinone (2-methyl-5-cyano-[3,4'-bipyridin]-6(1H)-one) was evaluated in vitro in a rabbit myocardial membrane Mg(2+)-dependent, Ca(2+)-stimulable adenosine triphosphatase (Ca(2+)-ATPase) model and structure-activity relationships were compared for nine closely related derivatives. In the present studies, a 5-bromo analogue of milrinone stimulated myocardial membrane Ca(2+)-ATPase significantly (10(-7) M; P < 0.001 vs control, with 67% of the activity of milrinone), whereas a 2'-methyl-2H-milrinone derivative was inactive. Although amrinone was inactive in this assay, its 2-methyl analogue was stimulatory. However, analogues lacking a 2-substituent (with or without a 5-cyano group) or with the 3-N position blocked by a methyl group did not stimulate myocardial membrane Ca(2+)-ATPase activity. Structural data for these bipyridines show that those with either a 2- or 2'-methyl substituent have a twist conformation, whereas those without are nearly planar. Activity data reveal that those bipyridines with a nonplanar conformation are more active in the Ca(2+)-ATPase assay. Further study of milrinone analogues with a 2'-methyl substituent shows that even though the effect on the twist angle is equivalent to that of 2-methyl substitution, these analogues are less potent. Data for this series reveal that the prerequisites for Ca(2+)-ATPase stimulation include not only a 2-methyl to maintain a twist conformation but also a free 3-N position and a 5-substituent. This model for optimal activity in the myocardial membrane Ca(2+)-ATPase system differs from those proposed for phosphodiesterase enzyme receptor recognition only in the requirement for a nonplanar molecule. We have previously shown that milrinone, but not amrinone, shares structural homology with thyroxine and was able to stimulate myocardial membrane Ca(2+)-ATPase activity in a manner similar to the thyroid hormone. Additionally, milrinone, but not amrinone, was an effective competitor for thyroxine binding to the serum transport protein transthyretin. Analysis of the milrinone-transthyretin crystal complex confirms the structural homology between milrinone and thyroid hormone which is not shared by amrinone. Modeling studies of the binding interactions of milrinone analogues indicate that the 2-desmethylmilrinone analogue, the most inhibitory analogue, lacks the hydrophobic contacts present in milrinone in its transthyretin-bound complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Amrinone↗

PubMed1995

Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

Amino Acid Sequence↗

PubMed1995

Absolute configurations and conformations of the opioid agonist and antagonist enantiomers of picenadol.

The absolute configurations of the enantiomers of the opioid picenadol [cis-1,3-dimethyl-4-propyl-4-(3-hydroxyphenyl)piperidine; cis-3-methyl, 4-propyl] have been determined by an X-ray crystallographic study of the chloride salt of the (+)-enantiomer. The agonist (+)-enantiomer and the antagonist (-)-enantiomer were found to have the 3R,4R and 3S,4S absolute configurations, respectively. The conformational properties of the enantiomers were also examined with MM2-87 calculations. There was good agreement between the computed global minimum and the crystallographic structure with the phenyl ring approximately bisecting the piperidine ring by both methods. This orientation of the phenyl ring differs from that of related opioids such as the phenylmorphans, prodines, meperidine, and ketobemidone in which the phenyl ring tends to eclipse one edge of the piperidine ring. Because the phenyl ring bisects the piperidine ring in picenadol, there is little difference in the three-dimensional orientations of the phenyl rings of the two enantiomers when one superimposes the piperidine rings. The agonist (+)-enantiomer is ambiguous with respect to an opioid ligand model, which suggests that agonist activity requires a specific range of dihedral angles for the phenyl ring. While the global minimum of the agonist is not consistent with the model, a second conformer that is only 1.2 kcal/mol above the global minimum is consistent. An alternative explanation is that agonist or antagonist activity is solely due to the presence of the 3-methyl group on the different edges of the piperidine ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗

PubMed1995

Conformational analysis of methylphenidate and its structural relationship to other dopamine reuptake blockers such as CFT.

PURPOSE: This work was performed 1) to determine the conformational preferences of the threo and erythro isomers of the dopamine reuptake blocker methylphenidate, 2) to determine the crystal conformation of the threo isomer, 3) to confirm the absolute configuration of the more active threo enantiomer, and 4) to incorporate the compound into a previously determined pharmacophore for dopamine reuptake blockers. METHODS: A conformational analysis was performed with the MM2-87 program, a crystal of the (-)-threo HCl salt was analyzed by x-ray crystallography, and the global minima of the (+)-threo isomer and the potent dopamine reuptake blocker CFT were superimposed. RESULTS: In the global minimum of the threo isomer, the carbonyl oxygen of the ester group is oriented toward the ammonium group as was also found in the crystal state. In the erythro isomer, the ester group prefers an extended conformation relative to the piperidine group. The absolute configuration of the biologically active (+)-threo enantiomer was confirmed to be R,R. The atomic sequence from the amine group through the ester group is identical in the active enantiomers of methylphenidate and CFT. CONCLUSIONS: The dopamine reuptake protein requires a precise orientation of the ammonium and ester groups but allows considerable leeway in the position of the phenyl ring. The pKa of the threo isomer is predicted to be higher than that of the erythro isomer.

Cocaine↗

PubMed1995

Dopaminergic (4aR,10bS)-cis- and (4aS,10bS)-trans-octahydrobenzo[F]quinolines have similar pharmacophores.

The structures and absolute configurations of two N-phenethyl substituted cis- and trans-octahydrobenzo[f]quinolines were determined by X-ray crystallography. The absolute configurations of the enantiomers that have high affinity for dopaminergic receptors were found to be (4aR,10bS) and (4aS,10bS) for the (-)-cis- and (-)-trans-8,9-dihydroxy substituted compounds. This is consistent with previous results for a dopamine agonist pharmacophore. MM2-87 calculations for a cis isomer, which has two alternative chair conformations of the piperidine ring, indicated that the preferred conformer is the same as that observed in the crystal structure. Superposition of the more active cis and trans enantiomers showed that the three dimensional orientations of the phenyl ring and the ammonium group are similar in the two geometrical isomers. The cis isomer, however, has steric bulk out of the plane of the molecule and this appears to result in a loss of agonist efficacy. The addition of the N-phenethyl group to the 7-OH and 7,8-diOH cis compounds, however, appears to be sufficient to restore high affinity for dopaminergic receptors unlike previously synthesized cis compounds. These cis compounds, however, appear to be mixed agonist/antagonists or antagonists on functional assays of dopaminergic activity.

Benzoquinones↗

PubMed1994

Methotrexate-resistant variants of human dihydrofolate reductase. Effects of Phe31 substitutions.

Substitution of glycine or alanine for phenylalanine 31 in human dihydrofolate reductase produces variants that are inhibited less by methotrexate (MTX) than the previously reported serine variant. The 100 times decrease in MTX affinity for the glycine variant is due to slower binding, and to inability of the initial complex to isomerize to a nondissociating conformer. A polar group at position 31 is unnecessary for resistance, but residues larger than serine confer no resistance. The glycine variant best fulfills criteria for gene therapy: low Km for H2folate, high kcat, and good stability. Although kcat is unaltered by these mutations, the rate of hydride transfer is greatly decreased. Presteady-state measurements have enabled a complete catalytic scheme to be constructed for the glycine variant that predicts observed steady-state behavior. The crystal structures of inhibitor complexes of the serine, alanine, and glycine mutants and of the wild-type enzyme show that the mutations cause little perturbation of the protein backbone, of side chains of residues at the active site, or of the bound inhibitor. A molecule of bound water occupies the space vacated by the phenyl group.

Amino Acid Sequence↗

PubMed1994

Threonine for alanine substitution at position 109 of transthyretin differentially alters human transthyretin's affinity for iodothyronines.

The heterozygous substitution of threonine for alanine at amino acid 109 of human transthyretin (TTR) increases its affinity for T4. We compared the affinity of recombinant wild-type (WT) and Thr109-TTRs for various iodothyronines in an attempt to elucidate how this mutation alters the T4-binding site. Homozygous WT and Thr109-TTRs were expressed recombinantly in Escherichia coli, and heterozygous Thr109-TTR was purified from plasma. The affinities of the iodothyronines for TTR were determined by measuring [125I]T4 bound by TTR in the presence of increasing concentrations of unlabeled iodothyronines. Homozygous Thr109-TTR bound T4 with an affinity slightly, but not significantly, greater than that of heterozygous Thr109-TTR. The affinity of Thr109-TTR for all iodothyronines was higher than that of WT TTR. However, the Thr109 mutation increased TTR's affinity for T4, Triac (triiodothyroacetic acid), and T3 to a greater extent than it did for Tetrac (tetraiodothyroacetic acid), EMD21388 (3',5'-dibromo-4',6'-dihydroxy-3-methylflavone), and dextro-T4. These data demonstrate that a subtle change in the structure of the T4-binding channel in TTR differentially alters the affinity of binding of various iodothyronines and suggests that site-directed mutagenesis of residues within the binding channel might clarify the relative importance of specific domains of this binding channel.

Alanine↗

PubMed1994

Computer-assisted molecular modeling of benzodiazepine and thyromimetic inhibitors of the HepG2 iodothyronine membrane transporter.

T3 cellular uptake is inhibited in the presence of benzodiazepines (BZs). The structure-activity relationship of BZ inhibition correlates strongly with halogen substitution of the nonfused phenyl ring and indicates that this ring is required for activity. A structure-activity series of thyromimetic (TH) inhibitors of the HepG2 iodothyronine transporter further point out the critical importance of the amino group of the alanine side chain, its L-stereo configuration, and the size of the substituents of the inner and outer phenyl rings. A third series of compounds, reported to interact at related sites, were inactive as HepG2 iodothyronine transport inhibitors, and therefore the potent inhibitors were restricted to the BZ and TH compounds. Using both of these BZ and TH structure-activity series along with computer-assisted molecular modeling techniques, we determined which chemical structural components were important at the transporter interaction site. By superimposing structures from active chemicals, excluding residues from poor inhibitors, and incorporating molecular electropotential data, we developed a five-point model of BZ conformational similarity to the endogenous transporter ligand, L-T3: the alkyl substitution at the N1 of the BZ ring seems to simulate the alanine side chain of T3, and the electro-negative halogen and oxygen atoms of substituents at R3/R7/R2'/R4' of BZ form a pyramidal pharmacophore that seems to correspond with the 3-l/5-l/3'-l/4'-OH substituents of T3, respectively. These points, suggesting a tilted cross-bow formation, may be sites for ligand interaction with the iodothyronine transporter.

Benzodiazepines↗

PubMed1993

Rabbit skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase activity: stimulation in vitro by thyroid hormone analogues and bipyridines.

Sarcoplasmic reticulum-enriched membranes from rabbit skeletal muscle contained Ca(2+)-ATPase activity which was significantly enhanced (26% increase, P < 0.001) in vitro by physiological concentrations (10(-10) M) of L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3). In contrast, the biologically inactive iodothyronine analogues D-T4 and 3,3',5,5'-tetraiodothyroacetic acid (Tetrac) (10(-10) M) were without effect on enzyme activity. 3,5-Dimethyl-3'-isopropyl-L-thyronine (Dimit), a bioactive analogue, was highly effective as a Ca(2+)-ATPase stimulator, increasing enzyme activity by 43% (P < 0.02 vs. T4 effect). A bipyridine cardiac inotropic agent, milrinone, has been reported to be thyromimetic in a myocardial membrane Ca(2+)-ATPase system, and in concentrations from 10(-10) to 10(-5) M enhanced skeletal muscle SR membrane Ca(2+)-ATPase activity in vitro (P < 0.001). Milrinone analogues which have been previously shown to enhance rabbit myocardial membrane Ca(2+)-ATPase activity, and which have a twist relationship of the pyridine rings, were also striated muscle Ca(2+)-ATPase stimulators. We conclude that (1) striated muscle is a mammalian tissue in which physiological levels of biologically relevant thyroid hormone analogues, particularly Dimit, stimulate Ca(2+)-ATPase activity in vitro by a non-genomic mechanism; (2) cardiac bipyridine analogues which are thyromimetic in vitro in rabbit heart, and which have structural homologies with thyroid hormone, are stimulators of rabbit striated muscle sarcoplasmic reticulum Ca(2+)-ATPase activity.

Animals↗

PubMed1993

Biologically active conformers of phenothiazines and thioxanthenes. Further evidence for a ligand model of dopamine D2 receptor antagonists.

Conformational analyses have been performed on several phenothiazine and thioxanthene dopamine antagonists using the MM2-87 program and parameter set. The compounds that were examined are thioridazine (2), methotrimeprazine (3), cis- and trans-chlorprothixene, and a piperidylidene derivative of chlorprothixene. In addition, (+)-2 and (-)-3 were determined by X-ray crystallography to have the R absolute configuration. The above compounds were superimposed onto loxapine, which was used as a template for the previously proposed dopamine D2 receptor ligand model. The conformational properties and receptor affinities of these compounds were found to be entirely consistent with the ligand model. For example, a conformer of (+)-R-2 that is consistent with the ligand model is lower in energy than a consistent conformer for (-)-S-2, which agrees with the higher D2 receptor affinity of the former. Similarly, in agreement with the much higher affinity of (-)-R-3 relative to (+)-S-3, only the former contains a low energy conformer consistent with the ligand model. The ligand model is also consistent with the greater potency of cis-thioxanthenes over the trans isomers. These results emphasize the importance of the correct orientation of the ammonium hydrogen for high affinity at the D2 receptor. The pharmacophore for D2 receptor ligands is compared with a recently proposed pharmacophore for D1 ligands.

Antipsychotic Agents↗

PubMed1993

Structural aspects of inotropic bipyridine binding. Crystal structure determination to 1.9 A of the human serum transthyretin-milrinone complex.

The crystal structure of human transthyretin (TTR) complexed with milrinone (2-methyl-5-cyano-3,4'-bipyridin-6(1H)-one), a positive inotropic cardiac agent, has been refined to R = 17.4% for 8-1.9-A resolution data. This report provides the first detailed description of protein interactions for an inotropic bipyridine agent which is an effective thyroid hormone binding competitor to transthyretin. Milrinone is bound along the 2-fold axis in the binding site with its substituted pyridone ring located deep within the channel of the two identical binding domains of the TTR tetramer. In this orientation the 5-cyano group occupies the same site as the 3'-iodine in the TTR complex with 3,3'-diiodothyronine (Wojtczak, A., Luft, J., and Cody, V. (1992) J. Biol. Chem. 267, 353-357), which is 3.5 A deeper in the channel than thyroxine (Blake, C. C. F., and Oately, S. J., (1977) Nature 268, 115-120). These structural results confirm computer modeling studies of milrinone structural homology with thyroxine and its TTR binding interactions and explain the effectiveness of milrinone competition for thyroxine binding to TTR. To understand the weaker binding affinity of the parent inotropic drug, amrinone (5-amino-3,4'-bipyridin-6(1H)-one), modeling studies of its TTR binding were carried out which indicate that the 5-amino group cannot participate in strong interactions with TTR and the lack of the 2-methyl further weakens amrinone binding.

Binding Sites↗

PubMed1993

Enantiomeric conformers of the opioid agonist ketobemidone HCl in the crystal state.

A crystal of the potent opioid agonist ketobemidone [1-methyl-4-(3-hydroxyphenyl)-4-propionylipiperidine] HCl was analyzed by X-ray crystallography. The crystal was monoclinic, space group P2(1)/n with four molecules in the unit cell. In agreement with MM2 calculations (J. Med. Chem. 25:1127-1133, 1982), the crystal contains mirror image conformers in which the phenyl ring is equatorial to the piperidine ring. The conformers are enantiomers since they are not superimposable. One conformer is predicted to be responsible for the typical morphine-like activity of the compound since it closely matches the preferred conformer of the morphine-like (+)-phenylmorphan whereas the other conformer resembles the preferred conformers of (+)-beta-prodine and (-)-phenylmorphan which have atypical opioid properties and/or structure-activity relationships. The importance of considering the conformational enantiomers of a nonchiral receptor ligand in centrosymmetric crystal structures is emphasized.

Analgesics, Opioid↗

PubMed1993

Conformational analysis of human dihydrofolate reductase inhibitor complexes: crystal structure determination of wild type and F31 mutant binary and ternary inhibitor complexes.

These structural studies reveal unusual intermolecular interactions for the binding of inhibitors and cofactor in ternary complexes with both wild type and F31 mutant recombinant human DHFR and show that these inhibitors have flexibility in occupying the active site. These studies also possibly indicate the first structural data for a ternary complex with a folate inhibitor and a polyglutamate side chain. However, further refinement of this data is necessary before this can be confirmed. In contrast to the ternary complexes of folate and MTX, the lipophilic antifolate PTX binds with its methoxybenzoyl ring oriented toward the cofactor nicotinamide ring, while that of TMQ it is bound closer to the Phe-31 position. Furthermore, the nicotinamide ring makes a close contact to the N10 amine of TMQ, significantly different from its binding site interactions in MTX complexes. These data also reveal that the conserved contacts between the cofactor carboxyamide with the enzyme backbone residues Ala-9 and Ile-16 are dictated by the enzyme and that changes in the orientation of the structural elements requires only subtle changes in the secondary structural units in which they are contained. Therefore, only by careful analysis of a series of enzyme complexes can the mechanisms of binding action be delineated.

Binding Sites↗

PubMed1993

Structural and conformational studies on bio-active flavonoids. Crystal and molecular structure of a complex formed between 2',6'-dimethoxyflavone and orthophosphoric acid: a model for flavone-nucleotide interactions.

In order to investigate mechanisms of action of flavones at the molecular level, we have prepared a complex between a flavone and orthophosphoric acid which can be considered as a simplest model of interaction between a flavone and a more complex biological phosphate such as nucleotide, coenzyme or DNA. With orthophosphoric acid, the title flavone forms crystals which have 1:2 stoichiometry (C17H14O4.2H3PO4). This compound was found to be the 1:1 salt co-crystallized with the unionized molecular acid C17H15O4+.H2PO4-.H3PO4. The symmetry is monoclinic, space group P2(1)/n and unit cell dimensions a = 15.571(2), b = 7.369(1), c = 17.837(2) A, beta = 100.84(1) degrees. One molecule of phosphoric acid is present as a solvate molecule of crystallization, but the other is ionized and protonates the carbonyl oxygen, introducing conformational and bond distance changes in the flavone. The dihedral angle between the benzopyrone and phenyl rings is 47 degrees. Complexes with phosphate groups involve strong hydrogen bonds and are expected to play important roles in biomolecular structures.

Crystallography↗

PubMed1992

Crystal structure determination at 2.3-A resolution of human transthyretin-3',5'-dibromo-2',4,4',6-tetrahydroxyaurone complex.

The crystal structure of the complex of 3',5'-dibromo-2',4,4',6-tetrahydroxyaurone, a flavone derivative, with human transthyretin (TTR), a serum thyroid hormone transport protein, has been determined and refined to R = 17.9% for data to 2.3-A resolution and provides a detailed description of a protein-bound flavonoid structure. This bromoaurone is a potent competitor for thyroid hormone binding to TTR, a 54,980-dalton alpha 4 tetrameric protein of 222 molecular symmetry, as well as an inhibitor of iodothyronine deiodinase. Crystals of the TTR-bromoaurone complex are isomorphous to those of native TTR. Interpretation of difference Fourier electron density maps revealed two binding modes for the bromoaurone in each of the two independent binding sites of the TTR tetramer: deep in the channel near Ser-117 (mode I) and near the channel entrance (mode II). None of the binding modes can be fully occupied because of overlap between binding positions. A statistical disorder for bromoaurone binding was also applied, as it binds along the twofold crystallographic axis and does not possess such symmetry. The binding of mode I and that of mode II were refined at half occupancy, resulting in two molecules per tetramer. The bromoaurone binds in a nonplanar antiskewed conformation. The molecular pattern for TTR binding consists of halogen groups able to anchor between beta-sheets to form both hydrophobic and hydrophilic contacts. Comparison of structural data for bromoaurone- and thyroxine-TTR complexes indicates that bromoaurone binding mode I is 3 A deeper in the channel and binding mode II is 4 A further from the channel center than thyroxine. The bromoaurone binding observed in this TTR complex differs significantly from that based upon computer modeling studies.

Benzofurans↗