Mark E. Zupec

N-myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA: protein N-myristoyltransferase.

Myristoyl-CoA: protein N-myristoyltransferase (Nmt) catalyses the covalent attachment of myristate to the N-terminal glycine of a small subset of cellular proteins produced during vegetative growth of Candida albicans. nmt447D is a mutant NMT allele encoding an enzyme with a Gly447-->ASP substitution and reduced affinity for myristoyl-CoA. Among isogenic NMT/NMT, NMT/ delta nmt and nmt delta/nmt447D strains, only nmt delta/nmt447D cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 degrees C. When switched from YPD/myristate to YPD alone, 60% of the organisms die with 4 h. Antibodies raised against the C-terminal eight residues of Saccharomyces cerevisiae Arf1p were used to probe Western blots of total cellular proteins prepared from these isogenic Candida strains. N-Myristoylation of C. albicans ADP-ribosylation factor (Arf) produced a change in its electrophoretic mobility during SDS-PAGE: the myristoylated species migrated more rapidly than the nonmyristoylated species. In an NMT/nmt delta strain, 100% of the Arf is N-myristoylated based on this mobility shift assay. When exponentially growing nmt delta/nmt447D cells were incubated at 24 degrees C in YPD/myristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, > or = 50% of total cellular Arf was nonmyristoylated. This finding suggests that > or = 50% reduction in Arf N-myristoylation is a biochemical marker of a growth-arrested cell. A similar conclusion was made after assaying isogenic S. cerevisiae strains containing various combinations of NMT1, nmt1-451D, ARF1, arf1 delta, ARF2 and arf2 delta alleles and grown at 24-37 degrees C on YPD of YPD/myristate. Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the N-terminal sequence of an S. cerevisiae Aft. SC-59383 has an IC50 of 1.45 +/- 0.08 microM for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransferase. It had an EC50 of 51 +/- 17 and 67 +/- 6 microM, 24 and 48 h after a single administration of the drug to cultures of C. albicans. The Arf gel mobility shift assay indicated that a single dose of 200 microM produced a < 50% reduction in Arf N-myristoylation after 4 h, which is consistent with the fungistatic, but not fungicidal, activity. The effect on Nmt was specific: an enantiomer, SC-59840, had no inhibitory effect on purified C. albicans Nmt (IC50 > 1,000 microM), and 200 microM of the compound produced no detectable reduction in Arf N-myristoylation in vivo. SC-58272, which is related to SC-59383, was a more potent inhibitor in vitro (IC50 0.056 +/- 0.01 microM), but had no growth inhibitory activity and did not produce any detectable reduction in Arf N-myristoylation. These findings highlight the utility of the Arf protein gel mobility shift assay for demonstrating the mechanism-based antifungal activity of SC-59383, a selective inhibitor of C. albicans Nmt.

In vitro and in vivo effects of a peptide mimetic (SC-47643) of RGD as an antiplatelet and antithrombotic agent

Platelet aggregation requires binding of fibrinogen (fgn) to activated platelets and inhibition of this binding blocks platelet aggregation. Synthetic peptides modeled after the platelet binding sequence on fgn block the platelet glycoprotein IIb/IIIa receptor and effectively inhibit aggregation. SC-47643 (SC) is a mimetic of the RGD-containing peptide sequence that is recognized by the platelet IIb/IIIa receptor. SC inhibited fgn binding to activated platelets (IC50: 1.0 x 10(-5) M) and prevented platelet aggregation in response to a variety of platelet agonists in both washed human platelets and platelet rich plasma (IC50s ranging from 4 x 10(-6) to 1 x 10(-5) M, respectively). SC inhibited collagen induced thrombocytopenia in the rat (ED50 0.07 mg/kg and t1/2 36 min). In dogs ex vivo collagen induced platelet aggregation was inhibited 50% after a bolus injection of 1.7 mg/kg. After a steady state infusion (2 hr), the ED50 was 0.03 mg/kg/min, with no effects on blood pressure, heart rate or platelet count. These data demonstrate that SC, a peptide mimetic of the natural fgn binding sequence, is capable of blocking platelet-fgn interactions and platelet aggregation.

Differential structure-activity relationships of atrial peptides as natriuretics and renal vasodilators in the dog

Natriuretic-diuretic and vasodilator activities of synthetic atriopeptin (AP)-related peptides were examined in the anesthetized dog. We have selected, the naturally occurring, APIII as the reference compound for comparison with various related peptides. APIII is a 24 amino acid peptide with the sequence ser-ser-cys-phe-gly-gly-arg-ile-asp-arg-ile-gly-ala-gln-ser-gly-leu-gly- cys-asn-ser-phe-arg-tyr-OH. APII, another peptide isolated from atrial extracts, lacks the C-terminal arg- of APIII. N-terminal amino acid extensions on APIII or APII, exhibited enhanced natriuretic-diuretic effectiveness. Furthermore, the maximum response obtained by ser-leu-arg-arg-APIII and arg-arg-APIII were significantly higher and the dose-response curve was not parallel to that obtained with APIII. In contrast, there were no significant qualitative or quantitative differences between the renal blood flow responses produced by the N-terminal extended peptides and APII or APIII. These results suggest a heterogeneity of AP receptors in vascular and renal tubular tissues.

A novel series of orally active antiplatelet agents

A novel series of orally active fibrinogen receptor antagonists has been discovered through structural modification of our lead intravenous (iv) antiplatelet agent, 5-(4-amidinophenyl)pentanoyl-Asp-Phe 1 (SC-52012). The Asp-Phe amide bond was removed through truncation to a 3-substituted beta-amino acid aspartate mimetic which resulted in a tripeptide mimetic inhibitor of lower molecular weight (from 482 to the 330-390 g mol-1). The zwitterionic nature of the inhibitor was masked through the preparation of an ethyl ester prodrug. A lead compound from this series, 5-(4-amidinophenyl)pentanoyl-3-(3-pyridyl)propanoic acid 19a, was found to be a potent inhibitor of canine platelet aggregation in vitro (collagen, platelet rich plasma, PRP, IC50 = 270 nM). In further canine studies, oral administration of different ester pro-drugs of 19a at 10 mg kg-1 resulted in the following oral systemic activities: pivaloyloxymethyl ester derivative 19p (5.1 +/- 1.5% OSA), cyclohexyl ester derivative 19c (9.2 +/- 1.9% OSA), and ethyl ester derivative 19e (9.9 +/- 2.3% OSA).

Isolation of Genomic Sequence Encoding a Biologically Active Bovine TGF- α Protein

AbstractGenomic clones encoding bovine TGF-α were identified by hybridization with probes derived from human TGF-α sequence. Nucleotide sequence of the clones predicts that mature bovine TGF-α is a 50 amino acid polypeptide which shares 96% and 92% homology with human and rat TGF-α, respectively. Bovine TGF-α with the predicted sequence was chemically synthesized and tested for activity. Synthetic bovine TGF-α competes in a radioreceptor assay with labelled mouse EGF with activity parallel to that of human TGF-α and mouse EGF. The mitogenic activity of bovine TGF-α is comparable to that of human EGF in causing proliferation of bovine mammary epithelial cells. An ∼ 5.0 kilobase RNA transcript is observed in polyadenylated RNA from MDBK cells by Northern blot analysis. The polymerase chain reaction detects the presence of a TGF-α transcript in many bovine tissues. These data indicate that bovine TGF-α may be a normal regulator of cell growth in the bovine animal.

Phosphorylation of high- and low-molecular-mass atrial natriuretic peptide analogs by cyclic AMP-dependent protein kinase

Synthetic high‐ and low‐molecular‐mass atrial peptides were phosphorylated in vitro by cyclic AMP‐dependent protein kinase and [32P]ATP. From a series of atrial peptide analogs, it was deduced that the amino acid sequence, Arg101–Ser104 of atriopeptin was required for optimal phosphorylation. Phosphorylated AP(99–126) was less potent than the parent atriopeptin in vasorelaxant activity and receptor‐binding properties. These results indicate that the presence of a phosphate group at the N‐terminus of AP(99–126) decreases the interaction of the peptide with its receptor and, as a consequence, decreases bioactivity. These observations are in contrast to those of Rittenhouse et al. [(1986) J. Biol. Chem. 261, 7607–7610] who reported that phosphorylation of AP(101–126) enhanced the stimulation of Na/K/Cl cotransport in cultured vascular smooth muscle cells.

Chemical synthesis of bovine transforming growth factor-α: Synthesis, characterization and biological activity

Bovine transforming growth factor-alpha (bTGF-alpha) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-alpha was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-alpha competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-alpha. Compared to hEGF, bTGF-alpha elicits a greater response in a bovine mammary cell proliferation.

Design of orally active, non-peptide fibrinogen receptor antagonists. An evolutionary process from the RGD sequence to novel anti-platelet aggregation agents

The evolutionary process from the Arg-Gly-Asp-Phe (RGDF) tetrapeptide to potent orally active anti-platelet agents is presented. The RGD sequence is an important component in the recognition of fibrinogen by its platelet receptor GP IIb-IIIa (integrin alpha IIb beta 3). This work concentrates on the replacement of the Arg-Gly dipeptidyl fragment by an acylated aminobenzamidine. The C-terminal fragment has been replaced by a variety of beta-amino acids, expanding on a previously reported paradigm. The lead compounds showed good potency in an in vitro platelet aggregation assay (dog PRP/ADP). The affinity for the fibrinogen receptor was confirmed in several cases by the ability to inhibit 125I fibrinogen binding to activated human platelets. The ethyl ester prodrug form was tested by oral administration to dogs and monitoring of the anti-platelet effect on ex vivo collagen induced platelet aggregation. From the structural studies reported, the 4-[[(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid (5) was the best surrogate for the Arg-Gly dipeptide. Several conformationally restricted analogues are also reported which are compatible with the hypothesis of RGD binding to the alpha IIb beta 3 in a turn-extended-turn conformation. The structure-activity relationships described also underline the importance of the beta-amino acid substitution for potency. In particular, the absolute configuration at the beta-carbon was crucial for high affinity. The best acid/ester pairs reported in this study had high potency (acid PRP/ADP IC50 approximately 50 nM) and showed good oral activity in dogs at 5 mg/kg per os (ethyl ester).