Analysis of high-affinity binding determinants in the receptor binding epitope of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.

Glu-96 of basic fibroblast growth factor is essential for high affinity receptor binding. Identification by structure-based site-directed mutagenesis.

The importance of basic fibroblast growth factor (bFGF) in several pathophysiological processes has stimulated interest in the design of receptor antagonists to mitigate such effects. Of key importance in this connection is the characterization of the functional binding epitopes of the growth factor for its receptor. Based on peptide mapping and molecular dynamics calculations of the three-dimensional structure of basic fibroblast growth factor, we employed site-directed mutagenesis to investigate the effect of altering residues at positions 107, 109-114, and 96 on bFGF on receptor binding affinity. All muteins were cloned and expressed in Escherichia coli, purified to homogeneity employing heparin-Sepharose columns, and evaluated for receptor binding affinity. We found that replacement of residues at positions 107 and 109-114 by alanine or phenylalanine had little effect on receptor binding affinities compared with wild type bFGF, in agreement with previous evidence that bFGF residues 109-114 comprise a low affinity binding site. By contrast, substitution of Glu-96 with alanine yielded a molecule having about 0.1% of the affinity of the wild type bFGF. The affinity of the corresponding lysine and glutamine muteins was 0.3 and 10%, respectively, emphasizing the importance of a negative charge at this position. Our findings are consistent with the view that residues 106-115 on bFGF represent a low affinity binding site on bFGF. In addition, we identify Glu-96 as a crucial residue for binding to fibroblast growth factor receptor-1.

Identification of a new class of ETA selective endothelin antagonists by pharmacophore directed screening.

Novel endothelin antagonists were identified through a "pharmacophore directed screening" strategy. The sulfanilamide antibacterial agent sulfisoxazole was found to be a good endothelin receptor antagonist (IC50's of 0.60 microM and 22 microM for the ETA and ETB receptors, respectively). The structurally similar sulfamethoxazole was found to be a weaker antagonist (IC50 for ETA 16 microM and for ETB 230 microM). These compounds represent a new class of low molecular weight and ETA-selective non-peptide endothelin antagonists.

Endoluminal sealing of vascular wall disruptions with radiofrequency-heated balloon angioplasty.

Suboptimal luminal widening or acute closure secondary to arterial dissection remain significant risks of percutaneous transluminal balloon angioplasty. Non surgical techniques are often employed in an attempt to repair dissections either as temporary or definitive treatment. The aim of this study was to test the hypothesis that radiofrequency thermal balloon angioplasty at an operating temperature of 70 degrees C and low inflation pressure could seal dissections and perforations in a model of severe arterial wall damage. Dissections and perforations were created in renal (n = 4) and carotid (n = 4) arteries in four mongrel dogs. Endoluminal sealing was then attempted with conventional balloon angioplasty or radiofrequency thermal balloon angioplasty (2 atm at 70 degrees C). Contrast dye extravasation persisted in all cases following conventional balloon angioplasty but completely resolved with radiofrequency balloon angioplasty in all but one artery. Histologic examination of the arteries treated with radiofrequency balloon angioplasty showed extensive thermal injury, including transmural coagulation necrosis, flattening of the internal elastic lamina, and medial thinning. On the basis of these results, the utility of thermal balloon angioplasty for endoluminal sealing of dissections and perforations complicating angioplasty deserves further evaluation.

Antibody-directed drug discovery.

By harnessing the remarkable power of the immune system to access the three-dimensional chemical information sequestered in target sites for drug ligands, antibody-directed drug discovery speeds the design of new therapeutic entities.

Skin and hair reflectance in women with red hair.

Skin and hair reflectance were measured with the Photovolt Spectrophotometer in 33 young women with red hair. Four skin sites were measured: forehead, check, inner arm and shoulder. A subsample of 15 red-headed women were tested before and at the end of the summer to test for tanning effects. The study was designed to test for relationships between hair and skin colour by reflectance spectrophotometry. As expected, subjects generally had very pale skin colour, pronounced freckling and very little tanning ability. Eumelanin (brown-black) pigment, as estimated by reflectance at 650 nm, was minimal at all skin and hair sites. Hair and inner arm reflectance values were highly correlated at short wavelengths, suggesting phaeomelanin as the source of the relationship. Estimates of tanning ability by use of the R/G ratio were very low in this sample of redheads.

Conformational analysis of synthetic androgens. IV. 1,2-seco-a-bisnor-5 alpha-androstan-17 beta-ol acetate.

The crystal and molecular structure of 1,2-seco-A-bisnor-5 alpha-androstan-17beta-ol acetate has been determined to evaluated the conformational importance of the intact steroid nucleus. The resulting tricyclic compound retains nearly the same steric profile for the remainder of the molecule when compared to the structures of dihydrotestosterone derivatives with intact A-rings. This may help to explain why these types of molecules retain a significant level of androgenic activity.

Syntheses and biological activities of 7 beta-methyl steroids.

The preparation of 7 beta-methyl-5 alpha-dihydrotestosterone acetate and its 2-thia-A-nor analogue is described. Biological evaluation shows that a 7 beta-Me largely decreases myotrophic-androgenic activity in both 5 alpha-dihydrotestosterone and the 2-thia-A nor analogue. Testing for antitumor activity shows that the reduction in breast tumor weight was not significant for either compound, but the final tumor size in the animals treated with 7 beta-methyl-2-thia-A nor steroid at 10 (mg/kg)/day was significantly reduced. The effects of 7 beta-Me steroids on the various organ weights are also described. The influence of 7 beta-Me substituent on the biological activities of androgens may be mediated through direct interaction of the substituent with the receptor surface in contact with the third dimension of the steroid molecule.

Selective catalytic reduction of 7-methyl-6-dehydrotestosterone acetate to 7 beta-methyltestosterone acetate by benzyl alcohol.

Using benzyl alcohol as a hydrogen donor in the presence of Pd on charcoal, 7-methyl-6-dehydrotestosterone acetate was selectively reduced to 7 beta-methyltestosterone acetate in 90% yield. The addition of hydrogen atoms to the 6, 7 double bond proceeded from the less hindered alpha-face of the steroid molecule, giving rise to the 7 beta-methyl product. Gas chromatograph analysis indicated small amounts of the 7 alpha-methyl epimer, 7 beta-methyl-5 alpha-dihydrotestosterone acetate and the 5 beta-epimer. The 6,7 double bond was hydrogenated in preference to 4,5 double bond, although both are trisubstituted.

Structure-activity relationships in glucocorticoids.

Meaningful answers to the question of the relationship between glucocorticoid structure and activity have emerged. Structural change has predictable effects on susceptibility to the action of metabolizing enzymes, on receptor affinity, and on intrinsic activity. These effects are, in principle, amenable to mathematical modeling techniques. The fascinating possibility of being able to calculate receptor affinity directly from chemical structure has already been realized through the development of an equation [19] that allows the calculation of receptor binding of any glucocorticoid from structural parameters. Utilizing knowledge of the free energy contributions of the substituents and the hydrophobicity and A-ring conformation of the steroids, receptor affinity for a large number of compounds could be described in terms of four parameters. A general relationship was derived relating the equilibrium dissociation constant to a surface area term, a polar interaction term, and A-ring tilt term, and a size limitation function for the 9 alpha-substituent. The excellent correlation obtained suggests that these four factors are the major determinants of glucocorticoid receptor interactions. It is clear that the use of a mathematical relationship that defines the strength of steroid-receptor interaction is a valuable tool for investigating structure-activity relationships. This would be especially true in the design of steroid drugs. The use of a linear free-energy equation is superior to the assumption of substituent additivity in predicting binding affinities. This type of relationship will be useful in the preparation of steroids for use in affinity labeling studies and should be adaptable to other binding systems in which it is desirable to obtain synthetic analogs for more potent activity or specificity.

Apparent bioisosteric replacement of--S--by NCN: synthesis of N--cyano-2-aza-A-nor-5alpha-androstan-17beta-ol acetate, an aza steroid androgen.

The synthesis of N-cyano-2-aza-A-nor-5alpha-androstan-17beta-ol acetate is described. Cyclization of 1,4-dibromo-1,4-seco-2,3bisnor-5alpha-androstan-17beta-ol acetate with benzylamine in the presence of potassium iodide gives the N-benzyl-2-aza-A-nor steroid. Debenzylation with cyanogen bromide (Von Braun reaction) affords the N-cyano-2-aza-A-nor steroid, which has androgenic activity slightly weaker than that of the corresponding thia compound. The results indicate that NCN may be substituted for--S--as well as for =S. This compound is the first hormonally active steroid containing nitrogen as a heteroatom in the perhydrocyclopentanophenanthrene nucleus.

Quantitative relationships between steroid structure and binding to putative progesterone receptors.

Relationships between chemical structure of androst-4-en-3-one derivatives and their affinity for putative progesterone receptors are described. The binding affinity for 55 derivatives can be expressed by the equation log relative binding affinity (rabbit receptor) = 1.79 + 0.18 (+/-0.11) pia + 1.45 (+/-0.21) pib + 0.010 (+/-0.002) (surface area in hydrophobic pockets) - 0.012 (+/-0.003) (surface area out of hydrophobic pockets) - 0.99 (+/-0.21) MK - 0.33 (+/-0.08) (conformational changes). For this equation, r=0.88. The equation successfully predicts the affinities of other compounds in the literature. The importance of the surface area terms is discussed.

Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan.

[Differentiation in vitro of the nasolacrymal canal of Discoglossus pictus Otth (anuran amphibian)]. / Différenciation in vitro du canal nasolacrymal de Discoglossus pictus Otth (Amphibien Anoure)

The nasolacrymal duct of Discoglossus pictus is able to differentiate in vitro from prometamorphosis, without thyroxine added to the culture medium if it is taken off late or with thyroxine added to the culture medium if it is taken off earlier (presumptive anlage).