Foe Siong Tjoeng

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.

Specific receptors for atriopeptin III in rabbit lung.

Binding studies revealed the presence of a single class of high affinity binding sites for atriopeptin III on rabbit lung membranes. An apparent dissociation constant (Kd) of 0.32 nM and a binding capacity of 166 fmol/mg protein was determined. Binding was time-dependent and saturable. The relative binding affinities of atrial peptide analogs correlated well with their potencies in eliciting relaxation of norepinephrine-contracted rabbit aorta strips. Unrelated peptide hormones did not compete for the atriopeptin binding site on rabbit lung membranes. The atrial peptide binding data are similar to those obtained for other tissues and indicate the presence of a physiologically relevant atrial peptide receptor in lung.

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).

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.

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).