High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells.

Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria-derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS-2/B10 and MG-63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARgamma mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG-63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARdelta/NUC-1 but not PPARalpha, indicating that cells that do not express either PPARgamma or PPARalpha are capable of differentiating into adipocyte-like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR-dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35-fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte-like differentiation of both ROS17/2.8 and SaOS-2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte-like cells.

NMR studies of novel inhibitors bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase-bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L-739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse-turn conformations most closely approximating a type III beta-turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 9. The synthesis and biological evaluation of novel simvastatin analogs.

Substitution of hydroxy and hydroxyalkyl functionality at C-7 of the hexahydronaphthalene nucleus of simvastatin has provided novel analogs. The synthetic strategy employed epoxidation or Lewis acid-catalyzed aldol reaction of the 8-keto silyl enol ether as a key reactive intermediate. These analogs were evaluated as potential hypocholesterolemic agents via initial determination of their ability to inhibit HMG-CoA reductase in vitro. Oral activity of these compounds was determined in an acute rat model and a three-week study in cholestyramine-primed dogs. Compounds were identified that possessed in vitro and in vivo activity comparable to that of simvastatin.

Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

Identification of fatty acid methyl ester as naturally occurring transcriptional regulators of the members of the peroxisome proliferator-activated receptor family.

The nuclear hormone receptors NUC-1 (PPAR delta) and PPAR alpha are members of the peroxisome proliferator-activated receptor (PPAR) family. The members of this receptor family are activated by agents that stimulate peroxisome proliferation, free fatty acids, prostaglandin 12 metabolites, and agents considered for the therapy of insulin-independent diabetes mellitus. To identify putative physiological agents that activate NUC-1, we tested the ability of acetone extracts of various rat tissues to activate the transcription of an MMTV-luciferase reporter gene, via a GR/NUC-1 hybrid receptor. GR/NUC-1 contains the ligand binding region of the NUC-1 receptor and the DNA binding domain of the glucocorticoid receptor. Using this assay, we found stimulatory activity in the pancreas, which upon purification and characterization was identified as methyl-palmitate, known to be enriched in pancreatic lipids. In addition, we determined that ethyl esters of palmitic and oleic acids are also potent activators of this receptor. Thus, fatty acid ester formation may control the cellular concentrations of fatty acids, and acyl-ester formation may play a role in the control of metabolic pathways and the activation of the PPAR.

Regioselectivity and stereoselectivity in the metabolism of HMG-CoA reductase inhibitors.

Biotransformation of three analogs of simvastatin, L-672,201, L-157,012 and L-672,220, by rat liver microsomes has been examined. These compounds differ from each other at the 6' position of the hexahydronaphthalene system. When 6'-substituents were in the alpha configuration, rat liver microsomes catalysed biotransformation primarily at the 6' position. Hydroxylation was stereoselective giving 6' beta-hydroxy derivatives as major metabolites. In contrast, when the 6'-substituent had a beta-configuration, metabolism at this site was blocked. Rates of metabolism (nmols/mg protein/min) also indicated that 6' beta-derivatives were poorer substrates than their 6' alpha-counterparts. The results indicate that cytochrome P-450 exhibits a high degree of regio- and stereoselectivity in the metabolism of HMG-CoA reductase inhibitors.

Metabolism of antiparkinson agent dopazinol by rat liver microsomes.

Metabolism of dopazinol (DZ) by liver microsomes from control and phenobarbital- and 3-methylcholanthrene-treated rats has been investigated. Liver microsomes from control and treated rats metabolized DZ to N-despropyl-DZ (39-53% of total metabolites); 8-hydroxy-DZ, a catechol metabolite (32-39%); and 5- or 6-hydroxy-DZ (12-20%). The last metabolite was identified as its dehydration product 5,6-dehydro-DZ. N-Dealkylation was favored only slightly over catechol formation (ratio = 1.2) by liver microsomes from control and phenobarbital-treated rats, whereas with liver microsomes from 3-methylcholanthrene-treated rats, N-dealkylation predominated (ratio = 1.7). Liver microsomes from control rats metabolized DZ at a rate of 0.86 nmol/nmol cytochrome P-450/min. Pretreatment of rats with phenobarbital or 3-methylcholanthrene stimulated rates of metabolism by 2.4- and 3-fold, respectively. Metabolism of DZ was inhibited by SKF 525-A, methimazole, and thiobenzamide. SKF 525-A completely inhibited metabolism of DZ, while methimazole and thiobenzamide, two alternate substrates of the microsomal flavin-containing monooxygenase (MFMO) inhibited N-dealkylation only. These results indicated that while the cytochrome P-450-dependent monooxygenase is the primary enzyme system in DZ oxidation, the MFMO also catalyzes the N-dealkylation reaction. The catechol metabolite was converted to isomeric O-methylated derivatives in approximately 1:1 ratio by purified catechol-O-methyl transferase or 105,000g liver cytosol. The late eluting isomer was 8-methoxy-DZ.

Physiological disposition and metabolism of L-365,260, a potent antagonist of brain cholecystokinin receptor, in laboratory animals.

L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl)-N'-(3-methylphenylurea)], a potent nonpeptide antagonist of the CCKB receptor, is currently under investigation to treat anxiety and panic disorders. This study describes absorption and disposition of the drug in rats, dogs, and monkeys. Following iv administration (5 mg/kg), L-365,260 was cleared very rapidly in rats, dogs, and monkeys. In all species, the concentrations of the drug in plasma declined in a polyphasic manner. There was no difference in total blood clearance among species, whereas considerable species differences were observed in volume of distribution and terminal half-lives. Binding of 14C-L-365,260 to plasma protein was extensive for all test species (greater than 96%). Interspecies differences in absorption were also observed. The bioavailability for rats, dogs, and monkeys was approximately 14%, 9%, and 2%, respectively. HPLC radiohistograms of urine and bile revealed that only trace amounts of intact drug were present; the drug was mainly eliminated by biotransformation. NMR and mass spectral analyses indicate that hydroxylation and glucuronide conjugation are the major biotransformation pathways.

Proton NMR assignments and secondary structure of the snake venom protein echistatin.

The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C alpha H, and side-chain resonances, except for the eta-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from 3JC alpha H-NH coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I beta-turn, a short beta-hairpin, and a short, irregular, antiparallel beta-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel beta-sheet.

Characterization of a solid state reaction product from a lyophilized formulation of a cyclic heptapeptide. A novel example of an excipient-induced oxidation.

PURPOSE: To elucidate the structure of a degradation product arising from a lyophilized formulation of a cyclic heptapeptide, and to provide a mechanism to account for its formation. METHODS: Preparative HPLC was used to isolate the degradate in quantities sufficient for structural studies. A structure assignment was made on the basis of the compounds spectroscopic properties (UV, MS, NMR) and the results of amino acid analysis. RESULTS: The degradate was identified as a benzaldehyde derivative arising from the oxidative deamination of an aminomethyl phenylalanine moiety. The extent of formation of this product is influenced by the amount of mannitol used as an excipient in the formulation. A mechanism is proposed whereby reducing sugar impurities in mannitol act as an oxidizing agent via the intermediacy of Schiff base adducts which subsequently undergo tautomerization and hydrolysis. CONCLUSIONS: Reducing sugar impurities in mannitol are responsible for the oxidative degradation of the peptide via a mechanism that involves Schiff base intermediates. This mechanism may be a potential route of degradation of other arylmethyl amines in mannitol-based formulations.

Hepatic metabolism of spironolactone. Production of 3-hydroxy-thiomethyl metabolites.

Spironolactone (SP) is used clinically as a renal aldosterone antagonist and as an antiandrogen. It is known that the drug is extensively metabolized and that metabolites mediate its therapeutic actions, but hepatic metabolism of SP has not been comprehensively investigated. Hepatic disposition may also be important in the toxicity of SP, because the parent compound prevents the hepatocarcinogenic effects of its metabolite, canrenone (CAN). Using a combination of in vivo and in vitro approaches, we studied the metabolism of SP by guinea pig livers. The major compounds detected in livers in vivo following SP treatment were the known metabolites, 7 alpha-thiomethyl-spirolactone (TM) and CAN, and a previously uncharacterized compound whose mass spectral and UV absorption characteristics suggested that it was an A-ring-reduced derivative of TM. In vitro incubation of liver homogenates with SP also resulted in the formation of the unknown metabolite. A combination of MS and NMR spectroscopy was used to identify unequivocally the unknown metabolites as 3 alpha-hydroxy-TM. Another metabolite produced in vitro was identified as 3 beta-hydroxy-TM. It is possible that these two new metabolites of SP contribute to the pharmacological actions of the drug. In addition, production of 3 alpha-hydroxy-TM suggests a mechanism to account for the prevention of CAN-induced carcinogenicity by SP. TM may block the conversion of CAN to mutagenic 3-hydroxy-CAN metabolites by serving as a competitive substrate for hepatic 3-keto reductases.

Disposition and metabolism of sodium 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl]-gamma -oxobenzenebutanoate in rats and dogs.

The disposition of sodium 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl]-gamma-o xo benzenebutanoate (L-648,051) was determined in rats and dogs. L-648,051 is a potent receptor antagonist for leukotriene D4 and is potentially useful in the treatment of asthma and other allergic disorders. After a dosage of 10 mg/kg iv, L-648,051 declined rapidly with a half-life of approximately 2 min in rat and dog plasma. Although the compound was well absorbed, it exhibited poor bioavailability due to efficient first-pass metabolism. In rats receiving 25, 50, and 150 mg/kg po, bioavailabilities were 0.5, 4.8, and 38.7%, respectively. In dogs, bioavailability of 10 and 50 mg/kg po was 0 and 23%, respectively. Two metabolites were identified, 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl-gamma- hydroxybenzenebutanoic acid (metabolite I), formed by ketoreduction, and 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl] benzeneacetic acid (metabolite II) formed by catabolic oxidation of the butanoic acid moiety of L-648,051. Ketoreduction resulted in the production of a chiral center and two enantiomers of metabolite I. In vitro studies suggest that rat erythrocytes formed the (+)-enantiomer exclusively. When L-648,051 was administered orally or iv to rats, both the (+)- and (-)-enantiomers were observed in the plasma. The data suggest that either two L-648,051 ketoreductases were present or that inversion of the hydroxyl stereocenter of metabolite I occurred.

Metabolism of L-689,502 by rat liver slices to potent HIV-1 protease inhibitors.

L-689,502, N-[2(R)-hydroxy-1(S)-indanyl]-5(S)-(1,1-dimethylethoxy- carbonyl-amino)-4(S)-hydroxy-6-phenyl-2(R)-(4-[2(R)-(4-morpholinyl) ethoxy]phenyl)methylhexamide, is a potent and specific inhibitor of human immunodeficiency virus-type 1 (HIV-1) protease in vitro. Metabolism of this compound in rat liver slices produced four major and several minor metabolites. The major metabolites were identified as morpholin-2-one, 3'(S)-hydroxyindan and 4'-hydroxyindan analogs, and a 4-O-glucuronic acid conjugate of the parent compound. The metabolites were characterized by Heteronuclear Multiple Quantum Coherence and Nuclear Overhauser Effect techniques in NMR spectroscopy, by MS, and/or comparison with authentic standards. Two of the minor metabolites were similarly characterized as a 2(R)-[4-(2-carboxymethoxy)phenyl]methyl analog and a product with a degraded morpholino ring. The hydroxyindan metabolites were lower in activity than L-689,502, whereas the morpholin-2-one and carboxymethoxyphenyl analogs were approximately 6- and 11-fold more potent as inhibitors of HIV-1 protease, respectively.

Solution structure of the cytoplasmic domain of phopholamban: phosphorylation leads to a local perturbation in secondary structure.

Peptides representing the N-terminal domain (Ia) of the cardiac sarcoplasmic reticulum protein phospholamban (residues 1-25 [PLB(1-25)] and a phosphorylated form [pPLB(1-25)]) were synthesized and their conformations examined using circular dichroism and nuclear magnetic resonance spectroscopy. In aqueous solution, both PLB(1-25) and pPLB(1-25) adopt a primarily disordered conformation. In 30% trifluoroethanol/10 mM phosphate, PLB(1-25) exhibits a CD spectrum consistent with 60% helical structure. This value decreases to 27% for the phosphorylated peptide. CD spectra in 2% SDS indicate 40% alpha-helix for PLB(1-25) and 20% for pPLB(1-25). Full chemical shift assignments were obtained by conventional homonuclear NMR methodologies for both PLB(1-25) and pPLB(1-25) in 30% trifluoroethanol/water and 300 mM SDS. The solution structure of PLB(1-25) in 30% TFE/water was determined from distance geometry calculations using 54 NOE distance constraints and 17 torsion angle constraints. In the family of 20 calculated conformers, the root mean square deviation from the mean structure is 0.79 A for backbone heavy atoms of residues 1-17. The structure comprises a regular alpha-helix extending from M1 to S16 with the remaining C-terminal residues disordered. The calculated structure is supported by analysis of C alpha H secondary shifts which are significantly negative for residues 1-16. Chemical shift degeneracy is substantially more extensive in the phospho form and precludes a direct comparison of calculated structures. However, the magnitudes of upfield secondary shifts are decreased by 20% in residues 1-11 and are not significantly helical for residues 12-16 according to the criteria of Wishart et al. [(1992) Biochemistry 31, 1647-1651]. 3JHN alpha coupling constants measured for I12, R13, A15, and S16 also suggest that residues 12-16 undergo a local unwinding of the helix upon phosphorylation. Similar results are obtained for PLB(1-25) and pPLB(1-25) in 300 mM perdeuterated sodium dodecyl sulfate except that differences in backbone dynamics for the helical and nonhelical regions of the peptide are evident in the DQF-COSY line shapes for fingerprint cross-peaks. This disruption of structure at the C-terminus of the helix suggests a model for phosphorylation-induced dissociation of the PLB/Ca(2+)-ATPase complex.

Species differences in the metabolism of a potent HIV-1 reverse transcriptase inhibitor L-738,372. In vivo and in vitro studies in rats, dogs, monkeys, and human.

In vivo and in vitro metabolism of 6-chloro-4(S)-cyclopropyl-3,4-dihydro-4-((2-pyridyl) ethynyl)quinazolin-2(1H)-one (L-738,372), a potent human immunodeficiency virus-type 1 reverse transcriptase inhibitor, has been investigated in rats, dogs, and monkeys. Following 0.9 mg/kg iv and 9 mg/kg po doses, systemic blood clearance (CLB) and bioavailability (F) of L-738,372 were species-dependent and inversely related (CLB = 48, 15, and 3 ml/min/kg; F = 6, 62 and 94%, in dogs, rats, and monkeys, respectively). Incubation of L-738,372 with rat liver slices and liver microsomes from all species studied led to the formation of two hydroxylated metabolites, M1 and M2. Kinetic studies of the microsomal metabolism of L-738,372 indicated that M1 was formed by a much higher affinity, but lower capacity enzyme(s) than that which catalyzed M2 formation in rats, dogs, and monkeys. The total intrinsic clearance of metabolite formation (CL(int) total = CL(int) M1 + CL(int) M2) was highest in dogs, followed by rats and monkeys. In dogs, CL(int) total was caused almost exclusively by CL(int) M1. Extrapolation of the CL(int) total values to the hepatic clearances (19, 8.4, and 0.9ml/min/kg in dogs, rats, and monkeys, respectively) showed a similar rank order to the CLB observed in vivo. Good agreement between these in vivo and in vitro results suggests that the species differences in hepatic first-pass metabolism, and not the intrinsic absorption, contributed significantly to the observed differences in F.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional structure of echistatin and dynamics of the active site.

The snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser effect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment from Gly5 to Cys20 and from Asp30 to Asn42 has a well-defined conformation and the Arg-Gly-Asp sequence, which adopts a turn-like structure, is located at the apex of a nine-residue loop connecting the two strands of a distorted beta-sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by 15N relaxation measurements. The overall correlation time of echistatin was determined from fluorescence measurements, and was used in a model-free analysis to determine internal motional parameters. The active site has order parameters of 0.3-0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution structures was made by calculating generalized order parameters from the ensemble of three-dimensional structures. The motion of the RGD site detected experimentally is more extensive than a simple RGD loop 'wagging' motional model, suggested by an examination of superposed solution structures.

Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation.

The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.

Chemical synthesis of echistatin, a potent inhibitor of platelet aggregation from Echis carinatus: synthesis and biological activity of selected analogs.

Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogen-dependent platelet aggregation stimulated by ADP with IC50 = 3.3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg24-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor--a heterodimeric complex composed of glycoproteins IIb and IIIa. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50 = 1.05 x 10(-7) M. Substitution with Ala-24 gave IC50 = 6.1 x 10(-7) M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50 = 6 x 10(-6) M), Orn-Gly-Asp-Phe (IC50 = 1.3 x 10(-4) M), and Ala-Gly-Asp-Phe (IC50 = 5.0 x 10(-4) M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin.

The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist.

The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist, was characterized in DMSO-d6 by the combination of nmr and molecular modeling. The conformational space available to the peptide was explored using a distance geometry algorithm with distance constraints derived from 1H-nmr spectra. The dynamics of the peptide were examined by relaxation time measurements and low temperature studies. The results from the low temperature studies suggest that the peptide backbone does not exist in a single, well-defined conformation but undergoes exchange between multiple conformers. This result is consistent with the inability to find a single structure that satisfies all the nmr-derived constraints. The constraints could only be satisfied by considering pairs of conformers to represent the experimental data. The low energy conformers comprise type II' or type V beta-turns with distinct side-chain directionality. The Arg-Gly-Asp portion of the ring is flexible and can be described by amide-plane rotations of the Arg-Gly and Gly-Asp peptide bonds. Although some backbone flexibility is evident, the incorporation of beta,beta-dimethyl cysteine imparted greater conformational rigidity as compared to the previously studied cyclic pentapeptide, Ac-Cys-Arg-Gly-Asp-Cys-OH.