Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase.

The structure of the complex between E. coli (RT500) form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X-ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. We discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme.

Rapid validation of molecular structures of biological samples by electrospray-mass spectrometry.

A short account is presented of the method of measuring molecular masses (M(r)) of pure biological samples by electrospray ionisation mass spectrometry. It is demonstrated that the technique yields M(r) values with an effective accuracy equal to or better than 0.008% of the calculated M(r), provided that the correct molecular structure is employed in the calculation. It is therefore recommended that this method of measuring M(r)'s should be considered to form an essential part of all studies aimed at elucidating the molecular structure of purified biological macromolecules or for confirming the identity of labelled samples of such molecules.

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme.

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 A resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

Some electrospray mass spectrometric evidence for the existence of covalent O-acyl enzyme intermediates.

Electrospray mass spectrometry has been used to measure the masses of the species present in solutions of three serine proteases (alpha-chymotrypsin, subtilisin Carlsberg and subtilisin BPN') before, during and after completion of the hydrolytic reaction with cinnamoyl imidazole and indole acryloyl imidazole. The masses measured during the reaction demonstrated that covalent O-acyl enzyme intermediates had been formed.

Pseudosymmetry in the structure of luteinizing hormone-releasing hormone. Studies on a series of novel analogs.

Pseudosymmetry in the LH-RH structure is described. Eleven analogs of LH-RH (SMALLER THAN Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) have been synthesized by the fragment condensation method and the repetitive excess mixed anhydride method. Multiple substitutions have been made in the LH-RH sequence, which retain the pseudosymmetry of the LH-RH molecule, while presenting fewer problems of synthesis than the corresponding residues in the natural decapeptide. Thus Trp3, Ser4, Tyr5, Gly6, Leu7, and Arg8 residues were replaced by amino acids having similar properties to the residues that they replace. In all but one of the peptides the Gly10-NH2 residue was replaced by ethylamide, while in the remaining peptide, 1-methyl-5-aminomethyltetrazole (AMT-Me) was substituted at position 10. The compounds were assayed in vitro and in vivo. The following analogs had in vivo and in vitro activities in the range 1-28 percent relative to LH-RH: I, smaller than Glu-His-Phe-Ala-Tyr-Gly-Leu-Arg-Pro-NHEt; II, smaller than Glu-His-Phe-Gly-Tyr-Gly-Leu-Arg-Pro-NHEt; VII, smaller than Glu-His-Phe-Ala-Tyr-Gly-Phe-Arg-Pro-NHEt; IX, smaller than Glu-His-Phe-Ala-Tyr-D-Ala-Leu-Arg-Pro-NHEt; XI, smaller than Glu-His-Phe-Gly-Tyr-Gly-Leu-Arg-Pro-AMT-Me.

Receptor-based design of dihydrofolate reductase inhibitors: comparison of crystallographically determined enzyme binding with enzyme affinity in a series of carboxy-substituted trimethoprim analogues.

By the use of molecular models of Escherichia coli dihydrofolate reductase (DHFR), analogues of trimethoprim (TMP) were designed which incorporated various 3'-carboxyalkoxy moieties in order to acquire ionic interactions with positively charged active-site residues. Certain of these compounds have shown exceptionally high affinity for this enzyme. For example, the 3'-(carboxypentyl)oxy analogue was found to be 55-fold more inhibitory than TMP toward E. coli DHFR (Ki = 0.024 nM vs. 1.32 nM for TMP). X-ray crystallographic studies of E. coli DHFR in binary complexes with TMP and two members of this acid-containing series of compounds defined the binding of these inhibitors and showed the carboxyl group of the latter two inhibitors to be ionically bound to Arg-57. These observations were in agreement with postulated binding modes that were based on receptor modeling.

Species differences in the binding of compounds designed to fit a site of known structure in adult human haemoglobin.

1. Oxygen dissociation curves are reported for human haemoglobins A1, FII, FI, A1c and Raleigh (beta1 valine leads to acetylalanine) and for horse haemoglobin in the absence and presence of 2,3-diphosphoglycerate (DPG), or 4,4'-diformyl-2-bibenzyl oxyacetic acid, or the bisulphite addition compound of the latter. 2. These haemoglobins were selected because their amino acid sequences are different at the DPG receptor site of human adult deoxyhaemoglobin. 3. The size of the shifts of the dissociation curves are in the sequence expected from the postulated numbers of interactions made by each compound with each haemoglobin type, based on the assumption of a common receptor site for the three compounds. 4. Multiple linear regression analysis shows that the free energies of interaction of the compounds with the haemoglobins may be predicted, to a first approximation, by summing the number of ionic and covalent bonds predicted for each effector-receptor combination, a reversible covalent bond contributing about twice as much energy (-6.78 kJmol-1) as an ionic interaction (-3.14 kJmol-1).

Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes.

Substituted benzaldehydes have been designed to bind preferentially to the oxy conformation of human haemoglobin at a site between the amino terminal residues of the alpha-subunits. Such compounds should stabilize the oxygenated form of haemoglobin and thereby increase its oxygen affinity. The compounds produce the expected effect, left-shifting the oxygen saturation curve of dilute haemoglobin solutions and of whole blood, although the binding pattern to haemoglobin is more complex than envisaged by the design hypothesis. The predicted best compound is also a potent inhibitor, at low oxygen pressure, of the sickling of erythrocytes from patients homozygous for sickle cell disease, and may prove to be a clinically useful anti-sickling agent.

Compounds designed to fit a site of known structure in human haemoglobin.

1 The three-dimensional coordinates of the atoms in human haemoglobin are known, and there is a specific site in the deoxygenated form of the protein at which 2,3-diphosphoglycerate (DPG) interacts. 2 Molecular models of this site have been constructed and used to design compounds which should bind to the deoxy conformation and stabilize it. These compounds should therby promote oxygen liberation, as does DPG. 3The compounds so designed were found to promote oxygen liberation. Their relative potencies, as assessed by sigmoidal dose-response curves, are in the predicted sequence.

A conformational analysis for leucine-enkephalin using activity and binding data of synthetic analogues.

1. Leucine-enkephalin and some analogues were assayed for activity in vitro on the mouse vas deferens and for binding to opiate receptors from rat brain. 2. The experimental data were analysed in terms of the stringency for glycine, a D-amino acid or an L-amino acid at each position in the peptide. 3. The observed configurational specificity was compared with the stringency that would be predicted to occur if enkephalin adopted certain hydrogen-bonded conformations at the receptor. 4. A small subset of the conformations examined was found to be compatible with the experimental data.

Quantitative structure-metabolism relationships for substituted benzoic acids in the rat. Computational chemistry, NMR spectroscopy and pattern recognition studies.

An extensive set of computed molecular properties, both steric and electronic, have been calculated using molecular orbital and empirical methods for benzoic acid (1) and a congeneric series of substituted benzoic acids, i.e. 2-, 3- and 4-fluorobenzoic acids (2-4), 2-, 3- and 4-trifluoromethyl benzoic acids (5-7), 2-, 3- and 4-methylbenzoic acids (8-10), 4-amino benzoic acid (11), 2-fluoro-4-trifluoromethyl benzoic acid (12), 4-fluoro-2-trifluoromethyl benzoic acid (13), 3-trifluoromethyl-4-fluorobenzoic acid (14). We have monitored the urinary excretion profiles and determined the metabolic fate of compounds 2-7, 12-14 in the rat using high resolution 1H and 19F NMR spectroscopy. Corresponding data for compounds 1,8-11 are taken from the literature. In all cases phase II glucuronidation or glycine conjugation reactions dominated the metabolism of these compounds. Compounds 5, 7, 12, 13 have ester glucuronides as their major metabolites; the rest primarily form glycine conjugates. Compounds (1-12) have been classified according to their calculated physicochemical properties using pattern recognition methods and principal components maps have been used as a novel type of structure-metabolism diagram. The maps of compounds in the physicochemical property space served to separate the compounds into the two major classes which related to their principal metabolic fate in vivo, namely glucuronidation versus glycine conjugation. Compounds 13 and 14 were used as further probes of the property space, and dominant metabolic fates of glucuronidation and glycine conjugation, respectively, were predicted from the previous "training set map". The metabolic fate of compounds 1-14 can thus be classified according to a simple set of physicochemical rules. Investigation of the physicochemical properties which are important in distinguishing the metabolic fate of the compounds may give insight into key features of the drug-metabolizing enzyme active sites and hence provide information on basic mechanisms of benzoate metabolism.

Raised transaminase activity of blood plasma from rats with experimentally-induced kidney damage detected by spin-echo 1H-NMR spectroscopy.

We report the application of spin-echo 1H-NMR spectroscopy to the detection of raised plasma transaminase activity in rats treated with the nephrotoxic cephalosporin antibiotic cephaloridine (CPH). Spin-echo 1H-NMR analysis of lyophilized plasma, reconstituted in H2O reveals a doublet at delta 1.48 for alanine. However when samples were reconstituted with 2H2O we noted that in samples from CPH-treated rats (but not in control samples) there was a variable degree of appearance of a singlet at delta 1.47 together with a reduction in the doublet at delta 1.48. We suggest that this is due to the release of transaminases from damaged tissue which, via a reversible conversion of alanine to pyruvate, causes selective deuteration of alanine at the alpha-hydrogen (alpha-CH) position. This observation suggests that these 1H-NMR spectral patterns are dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.