Biomedical subjects

M Winn

Publications and source records attributed to M Winn.

At least 19 recordsLinked to original sources

PubMed2006

SPINE workshop on automated X-ray analysis: a progress report.

The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.

Algorithms↗

PubMed2001

The crystal and molecular structures of diferric porcine and rabbit serum transferrins at resolutions of 2.15 and 2.60 A, respectively.

The serum transferrins are monomeric proteins with a molecular mass of around 80 kDa and are responsible for the transport of iron in vertebrates. The three-dimensional structures of diferric porcine and rabbit serum transferrin have been refined against X-ray diffraction data extending to 2.15 and 2.60 A, respectively. Data for both proteins were collected using synchrotron radiation at temperatures of 277 K. The porcine protein crystallizes in the space group C2, with unit-cell parameters a = 223.8, b = 44.9, c = 78.9 A, beta = 105.4 degrees with one molecule in the asymmetric unit. The structure was solved by molecular-replacement methods using rabbit serum transferrin as the search model. The structure was refined using REFMAC, with a final residual of 13.8% (R(free) = 18.2% for a 5% data sample) for all data to 2.15 A. The final model comprises 5254 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, one N-acetyl glucosamine moiety and 494 water molecules. The rabbit protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = 127.2, c = 144.9 A and one molecule per asymmetric unit. The structure was solved using the method of multiple isomorphous replacement and refined using REFMAC to give a final residual of 18.6% (R(free) = 22.2% for a 5% data sample) for all data to 2.60 A. The final model comprises 5216 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, a Cl(-) anion and 206 solvent molecules; there is no clear indication of the carbohydrate moiety attached to Asn490 (rabbit serum numbering). Both molecules adopt a bilobal structure typical for members of the transferrin family. Each of the structurally homologous lobes contains two dissimilar domains with a single iron-binding site buried within the interdomain cleft. The porcine serum protein lacks an interdomain disulfide bridge close to the connecting peptide between the lobes, but this seems to have little effect on the overall orientation of the lobes. The N-lobes of both proteins possess lysine residues, one from each of the two domains, that lie in close proximity to one another to form the so-called dilysine trigger. The more acid-labile release of iron from serum transferrins than from lactoferrins is discussed.

Animals↗

PubMed2001

Discovery of novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. 4. Structure-activity relationship of substituents on the benzene ring of the cinnamide.

We have shown that p-arylthio cinnamides can inhibit the interaction of LFA-1 and ICAM-1, which is involved in cell adhesion and the inflammatory process. We now show that 2,3-disubstitution on the aryl portion of the cinnamide results in enhanced activity over mono substitution on the ring. The best 2,3-substituents were chlorine and trifluoromethyl groups. Compounds 39 and 40 which contain two CF3 groups have IC(50) values of 0.5 and 0.1 nM, respectively, in inhibiting JY8 cells expressing LFA-1 on their surface, from adhering to ICAM-1. The structure-activity relationship (SAR) was examined using an NMR based model of the LFA-1 I domain/compound 31 complex. One of our compounds (38) was able to reduce cell migration in two different in vivo experiments.

Amides↗

PubMed2001

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 5. Highly selective, potent, and orally active ET(A) antagonists.

The synthesis and structure-activity relationships (SAR) of a series of pyrrolidine-3-carboxylic acids as endothelin antagonists are described. The data shows an increase in selectivity when the methoxy of Atrasentan (ABT-627) is replaced with methyl, and the benzodioxole is replaced with dihydrobenzofuran. Adding a fluorine further increases the binding activity and provides a metabolically stable and orally bioavailable ET(A)-selective antagonist.

Administration, Oral↗

PubMed1999

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 4. Side chain conformational restriction leads to ET(B) selectivity.

When the dialkylacetamide side chain of the ET(A)-selective antagonist ABT-627 is replaced with a 2,6-dialkylacetanilide, the resultant analogues show a complete reversal of receptor selectivity, preferring ET(B) over ET(A). By optimizing the aniline substitution pattern, as well as the alkoxy group on the 2-aryl substituent, it is possible to prepare antagonists with subnanomolar affinity for ET(B) and with selectivities in excess of 4000-fold. A number of these compounds also show promising pharmacokinetic profiles; a useful balance of properties is found in A-192621 (38). Pharmacology studies with A-192621 serve to reveal the role of the ET(B) receptor in modulating blood pressure; the observed hypertensive response to persistent ET(B) blockade is consistent with previous postulates and indicates that ET(B)-selective antagonists may not be suitable as agents for long-term systemic therapy.

Acetanilides↗

PubMed1999

Design, synthesis, and activity of a series of pyrrolidine-3-carboxylic acid-based, highly specific, orally active ET(B) antagonists containing a diphenylmethylamine acetamide side chain.

The endothelin (ET)-B receptor subtype is expressed on vascular endothelial and smooth muscle cells and mediates both vasodilation and vasoconstriction. On the basis of the pharmacophore of the previously reported ET(A)-specific antagonist 1, (ABT-627), we are reporting the discovery of a novel series of highly specific, orally active ET(B) receptor antagonists. Replacing the dibutylaminoacetamide group of 1 with a diphenylmethylaminoacetamide group resulted in antagonist 2 with a complete reversal of receptor specificity. Structure-activity relationship studies revealed that ortho-alkylation of the phenyl rings could further increase ET(B) affinity and also boost the ET(A)/ET(B) activity ratio of the resulting antagonists. A similar antagonism selectivity profile could also be achieved when one of the phenyl rings of the acetamide side chain was replaced with an alkyl group, preferably a tert-butyl group (10h). Combining these features with modification of the 2-aryl group of the pyrrolidine core, we have identified a potent antagonist (9k, A-308165) with over 27 000-fold selectivity favoring the ET(B) receptor and an acceptable pharmacokinetic profile (F = 24%) in rats.

Acetamides↗

PubMed1999

Discovery of a series of pyrrolidine-based endothelin receptor antagonists with enhanced ET(A) receptor selectivity.

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET(A) and ET(B) receptors. The ET(A) receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET(B) receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET(A)-selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET(A)-selective, ET(B)-selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET(A) selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.

Animals↗

PubMed1998

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 3. Discovery of a potent, 2-nonaryl, highly selective ETA antagonist (A-216546).

Previously we have reported the discovery of ABT-627 (1, A-147627, active enantiomer of A-127722), a 2,4-diaryl substituted pyrrolidine-3-carboxylic acid based endothelin receptor-A antagonist. This compound binds to the ETA receptor with an affinity (Ki) of 0. 034 nM and with a 2000-fold selectivity for the ETA receptor versus the ETB receptor. We have expanded our structure-activity studies in this series, in an attempt to further increase the ETA selectivity. When the p-anisyl group of 1 was replaced by an n-pentyl group, the resultant antagonist 3 exhibited substantially increased ETB/ETA activity ratio, but a decreased ETA affinity. Structure-activity studies revealed that substitution and geometry of this alkyl group, and substitution on the benzodioxolyl ring, are important in optimizing this series of highly ETA selective antagonists. In particular, the combination of a (E)-2,2-dimethyl-3-pentenyl group and a 7-methoxy-1,3-benzodioxol-5-yl group provided hydrophobic compound 10b with subnanomolar affinity for human ETA receptor subtype and with an ETB/ETA activity ratio of over 130000. Meanwhile, synthetic efforts en route to olefinic compounds led to the discovery that 2-pyridylethyl (9o) and 2-(2-oxopyrrolidinyl)ethyl (9u) replacement of the p-anisyl group of 1yielded very hydrophilic ETA antagonists with potency and selectivity equal to those of 10b. On the basis of overall superior affinity, high selectivity for the ETA receptor (Ki, 0.46 nM for ETA and 13000 nM for ETB), and good oral bioavailability (48% in rats), A-216546 (10a) was selected as a potential clinical backup for 1.

Administration, Oral↗

PubMed1997

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 2. Sulfonamide-based ETA/ETB mixed antagonists.

When the N,N-dialkylacetamide side chain of the highly ETA-selective endothelin antagonist ABT-627 (1; [2R,3R,4S]-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-[[N, N-dibutylamino)-carboxyl]methyl]pyrrolidine-3-carboxylic acid; A-147627) is replaced by N,S-dialkylsulfonamidoethyl, the resultant analogs retain ETA affinity, but exhibit substantial ETB affinity as well. Structure-activity studies reveal that modifications in the length of the two alkyl groups, and in the substitution on the anisyl ring, are important in optimizing this "balanced" antagonist profile. In particular the combination of an N-n-propyl group, an S-alkyl chain between four and six carbons in length, and a fluorine atom ortho to the aromatic OCH3 provides compounds with sub-nanomolar affinities for both receptor subtypes, and with ETA/ETB ratios close to 1. A number of these compounds also exhibit oral bioavailabilities (in rats) in the 30-50% range and have substantial plasma half-lives. The balanced receptor-binding profile of these potent and orally bioavailable compounds complements the ETA selectivity observed with 1.

Administration, Oral↗

PubMed1997

Potent and selective non-benzodioxole-containing endothelin-A receptor antagonists.

The benzodioxole ((methylenedioxy)benzene) group is present in a number of endothelin (ET) receptor antagonists thus far reported. As part of our own endothelin antagonist program we have developed (2R*,3R*,4S*)-1-(N,N-dibutylacetamido)-4-(1,3-benzodioxol-5- yl)-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (A-127722). This is a potent antagonist, binding to the ETA and ETB receptor subtypes with affinities (IC50) of 0.4 and 520 nM, respectively, and also contains the aforementioned benzodioxole. While this compound was seemingly optimized at its N-terminus, no effort had been directed toward understanding the contributions to binding affinity or receptor subtype selectivity conferred by the benzodioxole. Substitution by 1- or 2-naphthyl yielded weak antagonists. Oxygenated benzenes, such as p-anisyl, were potent compounds with IC50s in the low-nanomolar range. Simple deletion of either of the two oxygen atoms (dihydrobenzofurans) yielded extremely potent agents, possessing subnanomolar affinity for the ETA receptor. Additionally, the compounds showed enhanced selectivity, binding to the ETB receptor subtype in the micromolar range. This paper describes the development of this novel class of compounds.

Acetamides↗

PubMed1996

2,4-Diarylpyrrolidine-3-carboxylic acids--potent ETA selective endothelin receptor antagonists. 1. Discovery of A-127722.

We have discovered a novel class of endothelin (ET) receptor antagonists through pharmacophore analysis of the existing non-peptide ET antagonists. On the basis of this analysis, we determined that a pyrrolidine ring might replace the indian ring in SB 209670. The resultant compounds were readily prepared and amenable to extensive SAR studies. Thus a series of N-substituted trans,trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrroli din e-3- carboxylic acids (8) have been synthesized and evaluated for binding at ET(A) and ET(B) receptors. Compounds with N-acyl and simple N-alkyl substituents had weak activity. Compounds with N-alkyl substituents containing ethers, sulfoxides, or sulfones showed increased activity. Much improved activity resulted from compounds where the N-substituents were acetamides. Compound 17u (A-127722) with the N,N-dibutylacetamide substituent is the best of the series. It has an IC(50)=0.36 nM for inhibition of ET-1 radioligand binding at the ET(A) receptor, with a 1000-fold selectivity for the ET(A) vs the ET(B) receptor. It is also a potent inhibitor (IC(50)=0.16 nM) of phosphoinositol hydrolysis stimulated by ET-1, and it antagonized the ET-1-induced contraction of the rabbit aorta with a pA(2)=9.20. The compound has 70% oral bioavailability in rats.

Animals↗

PubMed1996

Endothelins and endothelin receptor antagonists: binding to plasma proteins.

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. PD-156707, L-749329, Ro-470203, and A-127722 are potent non-peptide ET receptor antagonists developed recently. When tested in human and rat plasma, both ET-1 and -3 and the four aforementioned antagonists exhibited a high degree (> 98%) of plasma protein binding. When ET-1 binding to the receptors was examined, 5% (v/v) of human plasma inhibited ET-1 binding to both ETA and ETB receptors by 80 - 90%. Similarly, 5% (w/v) of human serum albumin inhibited ET-1 binding by 82%, suggesting that the major protein component in plasma which interfered with ET-1 binding to the receptors was serum albumin. Competition studies show that, in the absence of human serum albumin, the IC50 values of PD-156707, L-749329, Ro-47-0203, and A-127722 were 0.37, 0.29, 5.7, and 0.22 nM, respectively. Addition of increasing doses of human serum albumin incrementally decreased the potency of the antagonists; in the presence of 5% of human serum albumin, the IC50 values increased to 62.8, 50.2, 122.7, and 6.72 nM for PD-156707, L-749329, Ro-47-0203, and A-127722, respectively. In conclusion, ET and ET receptor antagonists exhibit a high degree of binding to plasma proteins, especially serum albumin. Consequently, serum albumin inhibits ET binding to its receptors, and also decreases the potency of ET receptor antagonists. Our findings may explain the discrepancy observed for ET receptor antagonists between in vitro and in vivo potencies.

3T3 Cells↗

PubMed1996

Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist.

Endothelins (ET) are potent vasoactive peptides implicated in the pathogenesis of a number of vascular diseases. The effects of ET on mammalian organs and cells are initiated by binding to ETA or ETB receptors. In this report, we document the pharmacology of A-127722, a novel ETA-selective receptor antagonist. A-127722 inhibits [125I]ET-1 binding to cloned human ETA and ETB receptors competitively with Ki values of 69 pM and 139 nM, respectively. A-127722 exhibits a dose-dependent inhibition of ET-1-induced arachidonic acid release in human pericardium smooth muscle cells with a pA2 value of 10.5 and inhibits ET-1-induced vasoconstriction in isolated rat aorta with a pA2 value of 9.2. In vivo, A-127722 dose-dependently blocks the pressor response to ET-1 (0.3 nmol/kg i.v.) in conscious rats. Statistically significant (P < .05) antagonism is seen at doses greater than 0.1 mg/kg p.o. Maximal inhibition, at 10 mg/kg, remains constant for at least 8 hr after dosing. No effect is seen on the ETB-mediated transient vasodepressor effect of exogenous ET-1. In conclusion, A-127722 is ETA-selective, orally bioavailable and efficacious for inhibiting the effects of ET in the rat, and A-127722 is the most potent ET receptor antagonist yet reported.

Administration, Oral↗

PubMed1994

Cardiovascular effects of orally administered ABBOTT-81988, an angiotensin II antagonist, in conscious spontaneously hypertensive rats.

ABBOTT-81988 (A-81988), 2-(N-propyl-N[(2'-[1H-tetrazol-5-yl]biphenyl- 4yl)methyl] amino) pyridine-3-carboxylic acid, a nonpeptide angiotensin II (AII) antagonist was studied in the conscious spontaneously hypertensive rate (SHR) (male, 18 to 21 weeks) for cardiovascular effects of oral administration. Oral A-81988 at 0.3 to 3 mg/kg produced a dose-related 10 to 29% decrease in mean arterial pressure (MAP) in SHR (control, 161 to 177 mm Hg; n = 19) for 12 to 24 h without changing heart rate. Oral A-81988 at 3 mg/kg daily maintained MAP in SHR at normotensive levels (97 to 120 mm Hg) during a 5-day protocol with no rebound hypertension at termination of treatment. There was an increase in plasma renin activity in nanograms AI/milliliter/hour in SHR treated with A-81988 (32 +/- 3, n = 6 v 5 +/- 2, n = 6 for vehicle) during its antihypertensive action. The oral potency of A-81988 was enhanced about 10-fold in furosemide-treated SHR. The pressor response to AII was inhibited selectively in SHR even after an 8-day treatment with A-81988 (approximately 3 mg/kg/day orally). Total peripheral resistance was lowered and cardiac output unchanged in SHR administered A-81988 (3 mg/kg/day orally for 2 days). A-81988 (3 mg/kg orally) did not cause orthostatic hypotension in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1994

Receptor interactions of Abbott-81988, a highly potent, non-peptide angiotensin-II antagonist selective for type-1 receptors.

Abbott-81988 (A-81988) was selected from a series of related compounds as a highly potent and selective antagonist of angiotensin receptors. In the rabbit aorta, A-81988 exhibited a pA2 of 10.12 (+/- 0.08) vs. angiotensin-II, for type 1 receptors (AT1), and the antagonism appeared competitive. These results agreed with radioligand assays in which A-81988 inhibited the binding of [125I]-Sar1-Ile8-Angiotensin-II to rat liver membranes with a pKI of 9.12 (+/- 0.63). A-81988 was selective for AT1 receptors based on its lack of activity at other sites, such as aortic alpha 1 receptors. Moreover, A-81988 lacked affinity for AT2 receptors of bovine cerebellar membranes or for alpha or beta adrenergic receptors in binding assays. A-81988 lowered blood pressure significantly in vivo in renal artery-ligated rats at doses of 0.3 mg/kg administered either i.v. or p.o. The compound was rapidly and almost completely absorbed from the duodenum of anesthetized rats and demonstrated very low first-pass metabolism in the rat liver. These properties of selectivity toward and potency for antagonizing AT1 receptors, activity in lowering blood pressure in experimental animals, and favorable pharmacokinetic properties indicate that A-81988 should be a useful antihypertensive agent in man.

Angiotensin II↗

PubMed1994

Characterization of antihypertensive activity of ABBOTT-81988, a nonpeptide angiotensin II antagonist in the renal hypertensive rat.

2-(N-Propyl-N[(2'-[1H-tetrazol-5-yl]biphenyl-4yl)methyl]amin o) pyridine-3-carboxylic acid (ABBOTT-81988), a novel nonpeptide angiotensin II (AII) antagonist, was evaluated to characterize its antihypertensive activity in the conscious renal hypertensive rat. Oral or i.v. administration of ABBOTT-81988 at 0.03 to 0.3 mg/kg produced a dose-dependent, sustained decrease in mean arterial pressure (MAP; control 162-173 mm Hg, n = 27) of approximately 20 to 70 mm Hg. At a dose of 0.3 mg/kg p.o., ABBOTT-81988 lowered MAP to a normotensive level for more than 24 hr and did not change heart rate. During its antihypertensive effect (delta MAP, -28% approximately -35%), ABBOTT-81988 (0.1-03 mg/kg i.v.) decreased total peripheral resistance (delta resistance, -31% approximately -43%), and cardiac output remained either unchanged or slightly elevated. ABBOTT-81988 (0.3 mg/kg i.v.) produced an additional antihypertensive effect (delta MAP, -12 +/- 2%, n = 5) in captopril-pretreated (10 mg/kg i.v.) hypertensive rats, but captopril (10 mg/kg i.v.) had no effect in ABBOTT-81988-pretreated (0.3 mg/kg i.v.) rats. In the normotensive rat, ABBOTT-81988 (0.3 mg/kg p.o.) had no effect on basal MAP, but it inhibited the AII-induced (0.1 microgram/kg i.v.) pressor response by 51% to 91% for 24 hr, whereas the responses to norepinephrine (0.3 microgram/kg i.v.), vasopressin (0.03 IU/kg i.v.) and bradykinin (3 micrograms/kg i.v.) were not affected. It is concluded that ABBOTT-81988 is a safe and efficacious AII antagonist that may have use in the treatment of human hypertension.

Angiotensin II↗

PubMed1993

2-(Alkylamino)nicotinic acid and analogs. Potent angiotensin II antagonists.

A series of pyridines and other six-membered ring heterocycles connected to a biphenyltetrazole with a -CH2-NR'-link (1) were discovered to be potent angiotensin II antagonists. In the pyrimidine carboxylic acid series (W = CR, X = N, Y = CH, Z = COOH), compounds with an alkyl group (R') on the exocyclic nitrogen were much more potent than compounds with an alkyl group (R) on the heterocyclic ring. The corresponding pyridine, pyridazine, pyrazine, and 1,2,4-triazine carboxylic acids also showed potent in vitro angiotensin II antagonism. The pyridine (W, X, Y = CH, Z = COOH, R' = n-C3H7) demonstrated potent in vitro activity (pA2 = 10.10, rabbit aorta, and Ki = 0.61 nM, receptor binding in rat liver) as well as exceptional oral antihypertensive activity and bioavailability. Any nonacidic replacement for the carboxylic acid was detrimental for activity.

Angiotensin II↗

PubMed1993

Pharmacological characterization of Abbott-81282, a novel, non-peptide angiotensin-II antagonist selective for type-1 receptors.

Abbott-81282 (A-81282) has been identified among a series of related compounds as being a highly potent and selective antagonist of angiotensin receptors. At AT1 receptors of the rabbit aorta, A-81282 exhibited a pA2 of 9.64 (+/- 0.33) vs. angiotensin-II, and demonstrated characteristics consistent with competitive antagonism of this receptor. These results were supported in radioligand binding assays in which A-81282 inhibited the binding of [125I]-Sar-Il8-Angiotensin-II to rat liver membranes with a pKI of 8.505 (+/- 0.102). Selectivity of this agent for AT1 receptors was validated by its lack of activity at other receptor sites, such as alpha 1 receptors of isolated rabbit aorta. Moreover, A-81282 lacked affinity for AT2 receptors of bovine cerebellar membranes or for alpha or beta adrenergic receptor sites in radioligand binding assays. A-81282 lowered blood pressure significantly in vivo in renal artery-ligated rats at doses of 1 mg/kg i.v. or 5 mg/kg p.o. The compound was slowly and moderately absorbed from the duodenum of anesthetized rats and demonstrated low first-pass metabolism in the rat liver. Because of its selectivity and potency for antagonizing AT1 receptors, and its activity in lowering blood pressure in experimental animals, A-81282 has the potential to be a useful antihypertensive agent in man.

Angiotensin Receptor Antagonists↗