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SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341-Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373-Ala374, which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.

Despite the excellent in vitro potency of a series of benzamide glycoprotein IIb/IIIa antagonists, which have been reported previously, poor in vivo potency in the inhibition of platelet aggregation was observed when the most potent inhibitor XU057 was dosed intravenously to dogs. In this communication, we report that replacement of the benzamide in XU057 with an isoxazolecarboxamide resulted in significant improvement in in vivo potency. More importantly, the analogue XU065 showed an excellent oral antiplatelet effect in dogs.

Objective: To define the antiplatelet efficacy and specificity of the glycoprotein IIb/IIIa complex (GPIIb/IIIa) antagonist prodrug DMP754 and its free acid form, XV459., Methods and Materials: DMP754 has an IC50 > 1 mumol/l, and, upon its conversion with esterases to its free acid form, demonstrated high potency (IC50 20-45 nmol/l) in inhibiting human platelet aggregation induced by 10 mumol/l adenosine diphosphate, 20 micrograms/ml collagen, 1 mmol/l epinephrine, 10 mumol/l platelet activating factor or 0.5 IU/ml thrombin. The in-vitro rate of hydrolysis of DMP754 or XV459 is much faster with human or canine liver esterases (t 1/2 = 2.4-23 min) than with plasma esterases (t 1/2 = 5.5-7.6 h). Platelet GpIIb/IIIa integrin binding affinity and specificity for XV459 were determined using cell binding/adhesion assays., Results: The range of IC50 values of XV459 in inhibiting platelet aggregation in platelet-rich plasma obtained from 12 subjects was 0.035-0.069 mumol/l with a mean IC50 of 0.050 +/- 0.003 mumol/l. Additionally, XV459 inhibited platelets obtained from mongrel dogs, baboons, sheep, guinea pigs, and mice with IC50 in the range 0.024-0.06 mumol/l, and IC50 in the range 0.16-5.8 mumol/l in pigs, rabbits, and rats. XV459 inhibited [125I]-fibrinogen binding to activated human platelets with an IC50 of 0.011 +/- 0.003 mumol/l. XV459 demonstrated a high degree of selectivity in specifically inhibiting fibrinogen binding to the platelet integrin, GPIIb/IIIa (IC50 = 0.00025 +/- 0.00005 mumol/l) compared with inhibiting other integrins (alpha v beta 3, IC50 > 10 mumol/l; or alpha v beta 5, alpha 5 beta 1, or alpha 4 beta 1, for which the IC50 exceeded 100 mumol/l)., Conclusion: DMP754 is a potent antiplatelet agent in inhibiting platelet aggregation, and has a high specificity and affinity for human platelet GPIIb/IIIa receptors.

Analogs of the Saccharomyces cerevisiae alpha-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys7 and Gln10 were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo7,10[Cys7,X9,Cys10,Nle12]alpha-factor , where X=D-Val, D-Ala, L-Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20-30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys7 and Gln10 with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5-10-fold more active than their sulfhydryl-containing homologs, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the alpha-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form beta-turns spanning residues 7-10 than their less active linear counterparts. The results provide strong evidence that this beta-turn is important for optimal signal transduction by alpha-factor.

The a-factor of Saccharomyces cerevisiae (YIIKGVF-WDPAC(Farnesyl)-OCH3) is a peptide pheromone in which post-translational modification with a farnesyl isoprenoid and carboxyl methyl group is required for export and bioactivity. Truncated and carboxyl-terminal modified analogs of the a-factor were synthesized in order to determine the effect of such modifications on bioactivity. Bioactivity studies on carboxyl-terminal analogs in which the chirality, the cysteine thioether, and the carboxyl ester were varied in an attempt to study the influence of topology on a-factor activity indicate that the hydrophobicity conferred by the farnesyl moiety and not its specific spatial orientation is a key determinant of a-factor potency. Analyses on truncated a-factors suggest that sequential removal of NH2-terminal residues leads to a gradient of potency loss, with some amino acids exhibiting a slightly greater contribution to bioactivity than others. Random oligonucleotide-targeted mutagenesis of the gene encoding a-factor was coupled to a biological screen to identify altered a-factor peptides which are secreted yet exhibit a loss of a-factor bioactivity. Transformants exhibiting this phenotype were examined to identify codon changes presumably responsible for the altered phenotype, thus indicating residues that may contribute significantly to a-factor bioactivity.

The cyclo7,10[Cys7,Cys10,Nle12], cyclo7,10[Cys7,D-Ala9,Cys10,Nle12], and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] analogues of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of the yeast Saccharomyces cerevisiae were studied in DMSO/water (80:20) and aqueous solution by nmr spectroscopy. In addition, the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor was examined in DMSO/water. Nuclear Overhauser effect (NOE) and NH d delta/dT data indicate that the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor adopts a type II beta-turn in DMSO/water and that the cyclo7,10[Cys7,D-Ala9,Cys10,Nle12]- and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12]alpha-fa ctor analogues adopt type II and type I/III beta-turns, respectively, in both DMSO/water and aqueous solutions. In aqueous solution, residues 8 and 9 of the cyclo7,10[Cys7,Nle12] alpha-factor appear to adopt at least two distinct conformations, one of these being identified as a type I/III beta-turn. In contrast, the cyclo7,10[Cys7,Cys10,Nle12] alpha-factor appears to adopt predominately a type II beta-turn in DMSO/water. Quantitative NOE measurements of the cyclo7,10[Cys7,Cys10,Nle12]-, cyclo7,10[Cys7,D-Val9,Cys10,Nle12]-, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factors in DMSO/water were used to derive three-dimensional structures of the cyclo7,10[Cys7,Pro8,X9,Cys10] portion of these analogues.

Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the reciprocal action of mating pheromones. We recently showed that M-factor, the pheromone released by cells of the cellular mating type Minus, is a nonapeptide in which the C-terminal cysteine residue is carboxyl-methylated and S-alkylated, probably with a farnesyl residue (Davey, 1992): Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(S-farnesyl)- OCH3. Here we describe the chemical synthesis of this modified peptide and show that it exhibits all of the properties of the native pheromone. These results confirm the structure of the M-factor while the production of relatively large amounts of pure pheromone will be invaluable for studying the mating response in this yeast.

We have studied the interaction between phospholipids and a-factor (YIIKGVFWDPAC-[Farn]OMe), S-alkylated forms of a-factor with the farnesyl group substituted by methyl, hexadecanyl, or benzyl groups, and truncated forms of this lipopeptide. Circular dichroism studies suggest that, despite its lack of farnesylation, S-methyl-a-factor is incorporated into vesicles of dimyristoylphosphatidylcholine in a conformation similar to that which a-factor adopts in this membrane. However, studies of the intrinsic fluorescence of the Trp residues of these peptides indicate that this residue is more deeply imbedded into the bilayer in the case of the farnesylated peptide. The a-factor is more effective in raising the bilayer to the hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine than is the S-methyl-a-factor. This bilayer-stabilizing ability is also reflected in a-factor inhibiting leakage from vesicles of N-methyldioleoylphosphatidylethanolamine. Studies on a-factor analogs permit the conclusion that the bilayer-stabilizing effect of a-factor is not solely a consequence of its greater partitioning into the membrane but is also a consequence of the degree of penetration into the bilayer and the specific conformation of the peptide at the membrane interface. These results indicate that the farnesyl group alone, in the absence of cellular factors, bestows a particular physical interaction with membranes.

Small peptides initiate sexual conjugation in the yeast Saccharomyces cerevisiae and this phenomenon is an ideal paradigm for studying the mode of action of mammalian peptide hormones. 1H-nmr spectroscopy was used to examine the conformation of linear and cyclic analogues of the alpha-factor (WHWLQLKPGQPMY) in aqueous solution. In all cases peptides that exhibit nmr parameters expected for a type II beta-turn have higher biological activities than those that do not appear to assume this conformation. Based on a simple model for the interaction of the pheromone with its receptor, we prepared fragments of the alpha-factor. Several of these fragments either antagonize or potentiate the activity of the alpha-factor. The latter represent the first example of peptide fragments that synergize the activity of the parent pheromone.

Biologically inactive, truncated analogues of the Saccharomyces cerevisiae alpha-mating factor (WHWLQLKPGQPMY) either antagonized or synergized the activity of the native pheromone. An amino-terminal truncated pheromone [WLQLKPGQP(Nle)Y] had no activity by itself, but the analogue acted as an antagonist by competing with binding and activity of the mating factor. In contrast, a carboxyl-terminal truncated pheromone [WHWLQLKPGQP] was not active by itself nor did the peptide compete with alpha-factor for binding to the alpha-factor receptor, but it acted as a synergist by causing a marked increase in the activity of alpha-factor. The observation that residues near the amino terminus may be involved in signal transduction whereas those near the carboxyl terminus influence binding allows us to separate binding and signal transduction in the yeast pheromone response pathway. If found for other hormone-receptor systems, synergists may have potential as therapeutic compounds.

Two-Dimensional NMR was used to examine the solution conformation of the lipopeptide a-factor, YIIKGVFWDPAC (S-farnesyl) OCH3, from the yeast Saccharomyces cerevisiae and five analogues containing various S-alkylated cysteines in DMSO-d6. NOESY data, NH temperature coefficients, and 3J alpha NH coupling constants indicate that the a-factor is a predominantly unstructured peptide in DMSO. Similar results were obtained for the other peptides indicating that S-prenylation of Cys12 does not affect the conformation of these peptides.

The a-mating factor of Saccharomyces cerevisiae Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(farnesyl)OCH3, and 10 analogs modified at the cysteine side chain and/or the terminal carboxyl were synthesized using a combination of solid phase and solution phase methodologies. The strategy of synthesis involved the condensation of an amine terminal protected decapeptide with a carboxyl terminal S-alkylated dipeptide ester or amide using benzotriazol-l-yloxy-tris(methylamino)-phosphonium hexafluorophosphate as the coupling agent. The protected decapeptide was assembled on a PAM-resin using 9-fluorenylmethoxycarbonyl (Fmoc) for the protection of the Tyr alpha-amine and Lys epsilon-amine and 9-fluorenylmethyl ester (OFm) for the protection of the Asp beta-carboxyl. Premature loss of the OFm group from the HF cleavage was observed at 0-2 degrees, whereas no loss occurred when the cleavage reaction was conducted at -5 degrees. In contrast to these results, the OFm group in Asp(OFm) was partially removed by HF at -5 degrees and was completely stable to HF only at -20 degrees. The S-alkylated dipeptide esters were prepared, in yields from 64% to 88%, via thioalkylation of amine protected or unprotected dipeptide esters using potassium fluoride dihydrate as the base. The use of a tertiary amine as the base of thiohexadecanylation resulted in low reactivity.

The same dialkylphosphite reagent can be used for both N protection and C activation of amino acids. Two N-diisopropyloxyphosphinyl (Dipp) tripeptide esters were prepared, and nine N-Dipp-dipeptide acids were synthesized through the activated amide intermediate. The positive ion FAB-MS of N-Dipp-tripeptide showed novel cleavage patterns in that only the N-phosphoryl fragment ions gave intense peaks while the C-terminal series ions did not appear. This novel character might be useful for peptide sequence analysis. In addition, dialkyloxyphosphinyl group can be examined by 31P-NMR for peptide conformational analysis and inspection for the degree of racemization during the coupling reaction.

The fast atom bombardment mass spectra of peptides containing S-prenylated cysteine display signature fragmentations characteristic of this modified amino acid. The fragmentation is independent of the nature of the cysteine carbonyl substituent, easily differentiates prenyl from nonprenyl alkylation, and readily identifies the oligomer count of the prenyl. This screening method, which requires little time, effort, or material (compared with previous analysis methods based on chemical degradation), greatly facilitates the identification of these prenylated proteins.

The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product. A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs.

The solution phase synthesis of the Saccharomyces cerevisiae a-mating factor and nonfarnesylated and nonmethylated a-factor analogs are reported. The a-factor, a lipopeptide with the sequence Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(S-Farnesyl)OCH3 was synthesized by the condensation of the amine terminal protected decapeptide with the carboxyl terminal farnesylated dipeptide using benzotriazol-l-yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent) as the coupling agent. The synthesis of the decapeptide involved 5 + 5 fragment coupling with the BOP reagent and the successful application of 9-fluorenylmethyl ester(OFm) and 9-fluorenylmethoxycarbonyl(Fmoc) groups for the protection of Asp and Lys side chains and Tyr alpha-amine and of phenacyl esters (OPa) for alpha-carboxyl protection. The OFm and Fmoc groups tolerated repeated couplings and were completely stable to zinc powder in acetic acid, a condition under which the OPa group was removed. The synthesis of the nonfarnesylated alpha-factor was accomplished by the coupling of the decapeptide with tetrapeptide (Ala-CysOCH3)2 followed by the deprotection of the OFm and Fmoc groups with piperidine and the cleavage of the disulfide bond with zinc powder in acetic acid. The nonmethylated a-factor was prepared by 10 + 2 fragment coupling using OFm protection of the dipeptide carboxyl group followed by removal of all protecting groups with piperidine. Attempts to saponify a-factor were not successful. The synthetic nonfarnesylated and nonmethylated a-mating pheromones were 100-1000 times less active than the a-factor, indicating that although the methyl ester and the farnesyl group are not essential for biological activity, they are necessary for high potency.

The binding of the tridecapeptide yeast mating pheromone, alpha-factor, to its receptor represents an excellent model for the investigation of peptide hormone-receptor interactions. In this paper we present a number of strategies to probe the binding site of the alpha-factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the alpha- or epsilon-amine in [Nle12]-alpha-factor was accomplished using 3-[3,5-diiodo-4-hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton-Hunter reagent). At pH 8.0 in a N-N-dimethylformamide/water mixture the ratio of epsilon- to alpha-acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2-dimethoxyethane/water mixture alpha-acylation was favored by more than 3 to 1. The product distribution was found to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially alpha-acylated [Nle12]-alpha-factor and served as a temporary masking group during the synthesis of epsilon-Bolton-Hunter acylated pheromone. Biotin or diiodo Bolton-Hunter reagents were also directly incorporated into [Nle12]-alpha-factor or Lys[Nle12]-alpha-factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylacetamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton-Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays.(ABSTRACT TRUNCATED AT 250 WORDS)

The a-mating factor of Saccharomyces cerevisiae was synthesized using both solution phase and solid phase strategies. Structure of the final peptide was confirmed using amino acid analysis, fast atom bombardment mass spectroscopy and 400 MHz proton NMR. The synthetic farnesylated dodecapeptide, YIIKGVFWDPAC (S-farnesyl) OCH3, exhibited chromatographic and spectroscopic properties identical to the natural pheromone and had significant biological activity at nanomolar concentrations.