Isoxazoline GPIIb/IIIa antagonists bearing a phosphoramidate

Isoxazolinylacetamides bearing a phosphoramidate group alpha- to the carboxylate moiety (3) were prepared and evaluated for in vitro antiplatelet efficacy. They were found to bind GPIIb/IIIa with high affinity and were potent antagonists of ADP mediated platelet aggregation.     

Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library.

Methods have recently been developed to present vast libraries of random peptides on the surface of filamentous phage. To introduce a degree of conformational constraint into random peptides, a library of hexapeptides flanked by cysteine residues (capable of forming cyclic disulfides) was constructed. This library was screened using the platelet glycoprotein, IIb/IIIa, which mediates the aggregation of platelets through binding of fibrinogen. A variety of peptides containing the sequence Arg-Gly-Asp or Lys-Gly-Asp were discovered and synthesized. The cyclic, disulfide-bonded forms of the peptides bound IIb/IIIa with dissociation constants in the nanomolar range, while reduced forms or an analogue in which Ser replaced the Cys residues bound considerably less tightly. These results demonstrate the feasibility for introducing conformational constraints into random peptide libraries and also demonstrates the potential for using phage peptide libraries to discover pharmacologically active lead compounds.     

1,2,4-triazolo[3,4-a]pyridine as a novel, constrained template for fibrinogen receptor (GPIIb/IIIa) antagonists

Conformationally constrained analogues of the GPIIb/IIIa antagonist elarofiban (RWJ-53308) have been synthesized and biologically evaluated. The 1,2,4-triazolo[3,4-a]pyridine scaffold provided potent antagonists with favorable pharmacodynamic and pharmacokinetic attributes in dogs. Compounds 12a and 13a exhibited enhancements in oral bioavailability, t(1/2), and ex vivo duration of action (inhibition of ADP-induced platelet aggregation) relative to elarofiban.     

Preparation and biological activity of novel tricyclic GPIIb/IIIa antagonists

Antagonists of the glycoprotein GPIIb/IIIa are a promising class of antithrombotic agents offering potential advantages over present antiplatelet agents (i.e., aspirin and ticlopidine). Novel tricyclic nonpeptidal GPIIb/IIIa antagonists have been prepared and evaluated in vitro as antagonists of fibrinogen binding to the purified GPIIb/IIIa receptor and as inhibitors of platelet aggregation. The work presented demonstrates the robustness of the benzodiazepinedione (BZDD) scaffold, which can be functionalized at the N¹---C² amide as well as at C⁷, to provide structural diversity and allow optimization of the physiochemical and pharmacological properties of the BZDD based GPIIb/IIIa antagonists. In addition, the resulting new class of tricyclic GPIIb/IIIa antagonists could be used to probe for additional binding interactions on the GPIIb/IIIa receptor and perhaps lead to BZDD based GPIIb/IIIa antagonists with increased potency. The tricyclic molecules reported herein demonstrate that a heterocyclic ring can be fused to the benzodiazepinedione scaffold with retention of anti-aggregatory potency and in the case of tetrazole 30i, increased potency relative to the bicyclic analogue 1c.     

2-Acylimino-3H-thiazoline derivatives: a novel template for platelet GPIIb/IIIa receptor antagonists

In the course of our research for the low-molecular weight RGD peptide mimics, we have found that a rigid 2-acylimino-3H-thiazoline structure is suitable for the peptide backbone mimics. Introduction of amidinophenyl and beta-alanine moiety as arginine and aspartic acid side-chain surrogates to this backbone mimic resulted in a highly potent fibrinogen receptor antagonist 2-(4-amidinobenzoylimino)-3,4-dimethyl-N-(2-carboxyethyl)-3H-thiazoline-5-carboxamide (7c), namely PS-028 (Ki = 46.5 +/- 5.8 microM).     

Docking based design of diastereoisomeric MTCA as GPIIb/IIIa receptor inhibitor

In GPIIb/IIIa mediated arterial thrombosis platelet activation plays a central role. To discover platelet activation inhibitor the pharmacophores of GPIIb/IIIa receptor inhibitors and anti-thrombotic agents were analyzed. This led to the design of (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids as GPIIb/IIIa inhibitors. Comparing to (1S,3S)-isomer (1R,3S)-isomer had lower cdocker interaction energy. AFM image showed that the minimal effective concentration of (1S,3S)-isomer and (1R,3S)-isomer inhibiting platelet activation were 10^?5?M and 10^?6?M, respectively. In vivo 1?μmol/kg of oral (1S,3S)-isomer effectively inhibited the rats to form arterial thrombus and down regulated GPIIb/IIIa expression, but the activities were significantly lower than those of 1?μmol/kg of oral (1R,3S)-isomer. Both (1S,3S)-isomer and (1R,3S)-isomer can be safely used for structural modifications, but (1R,3S)-isomer should be superior to (1S,3S)-isomer.     

Cation-dependent changes in the binding specificity of the platelet receptor GPIIb/IIIa

The presence of manganese (Mn2+) significantly increases the binding of the platelet surface receptor GPIIb/IIIa to two synthetic peptides Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) and Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (L10) that contain the recognition sequences RGD and KQAGDV, respectively. This results in an increase in the amount of GPIIb/IIIa adsorbed by GRGDSPK- and L10-Sepharose by 12-20-fold. Additionally, Mn2+ eliminates contaminating platelet vitronectin receptor, alpha v beta 3, which copurifies with GPIIb/IIIa on the peptide affinity columns in the absence of Mn2+. In contrast to this increased peptide binding of GPIIb/IIIa, Mn2+ reduces the binding of GPIIb/IIIa to its macromolecular RGD-containing ligands fibrinogen, fibronectin, and vitronectin. These results could mean that Mn2+ changes the structure of the binding site on GPIIb/IIIa such that it is now better suited to accommodate conformations available to the RGD sequence within short, linear synthetic peptides but not available to the RGD sequences within the natural ligands. To support this hypothesis we tested a conformationally restricted cyclic peptide, cyclic 2,10-GPenGHRGDLRCA, which in competition assays, preferentially inhibits the binding of GPIIb/IIIa to fibrinogen but does not inhibit well the binding of other RGD-dependent integrins, alpha v beta 3 and alpha 5 beta 1 to their respective ligands. In such assays, the presence of Mn2+ dramatically changed the binding specificity of GPIIb/IIIa by shifting the preference of the receptor away from the selective peptide, cyclic 2,10-GPen-GHRGDLRCA toward the nonselective GRGDSP peptide. This shift parallels the Mn2(+)-dependent change of the binding of GPIIb/IIIa to its natural protein ligands.     

Platelet glycoprotein IIb/IIIa receptor antagonists derived from isoxazolidines

A series of isoxazolidines has been synthesized as mimetics of the RGD sequence and was evaluated as antagonists of the platelet glycoprotein IIb/IIIa receptor. These compounds were shown to be highly potent GPIIb/IIIa antagonists, exhibiting submicromolar potencies.     

Design, synthesis, and structure-activity relationships of potent GPIIb/IIIa antagonists: discovery of FK419

The discovery of the non-peptide antiplatelet injectable agent FK419 is reported. Based on the beta-turn structure of RGD peptide sequences in the alpha chain of fibrinogen, which binds the glycoprotein IIb/IIIa (GPIIb/IIIa) on the surface of platelets to induce platelet aggregation, the prototype 2 was designed. After further substituent effects were investigated at the alpha-position of the carboxylic acid in 2, we enhanced platelet aggregation inhibition, and discovered the useful feature of reduced prolongation of bleeding time. Finally, the potent platelet aggregation inhibitor FK419 (3) could be discovered. FK419 shows a safe feature of reduced prolongation of bleeding time, as well as potent inhibition of platelet aggregation.     

Evaluation of an antibody-based genotype classification of the platelet fibrinogen receptor (GPIIb/IIIa)

Platelet membrane glycoprotein (GP) IIb/IIIa is the central molecule in platelet adhesion and aggregation by high-affinity binding of fibrinogen. Polymorphism of the beta chain of the receptor, especially the GPIIIa-proline33 allele [HPA-1b, Zwb, PI(A2)], has been suggested to be associated with a variety of vascular diseases, such as coronary stenosis, myocardial infarction, cerebral ischemia, or venous thrombosis. Using clinical chemistry standards, we evaluate a flow cytometric whole-blood, antibody-based method to determine the genotype [PI(A1A1), PI(A1A2), PI(A2A)] versus polymerase chain reaction (PCR)-based DNA restriction fragment length analysis in 220 individuals. Both homozygous and heterozygous genotypes differ in the expression of binding sites for the monoclonal antibody, SZ21. Agreement between the two methods was achieved in 187 cases, which reflects a test validity of 85%, a sensitivity of 83.6%, and a specificity of 85.4%. We conclude that flow cytometry is reliable for classifying the PI(AX) genotype. The performance characteristics are easy, fast, and cheap (genomics by proteomics). These features make it suitable for screening patients and broad populations for the future risk of cardiovascular ischemic events.     

Secondary signals mediated by GPIIb/IIIa in thrombin-activated platelets

We have previously found that stimulation of aequorin-loaded platelets by thrombin produced a two-peaked increase in intracellular free calcium concentration ([Ca2+]i), and the development of the second peak of [Ca2+]i was closely related with the aggregation. In this report, we studied the interrelationship between the GPIIb/IIIa complex, aggregation, cytoskeletons and [Ca2+]i of platelets. The pretreatment of the platelets with dihydrocytochalasin B (4 microM), an actin polymerization inhibitor, did not inhibit aggregation and TXB2 production, but did inhibit both actin polymerization and the second peak of [Ca2+]i increase induced by thrombin, suggesting that actin polymerization and the second peak of [Ca2+]i are interrelated. GRGDSP (100 microM), a synthetic anti-adhesive peptide, has already been reported to inhibit platelet aggregation and the second peak of [Ca2+]i induced by thrombin. It also inhibited actin polymerization and TXB2 production, suggesting that aggregation was important for not only the generation of the second peak of [Ca2+]i but also for actin polymerization and TXB2 production. PGI2 (5 nM) did not abolish but only delayed aggregation, TXB2 production, actin polymerization and the second peak of [Ca2+]i increase. These findings suggest that the secondary signals are caused by aggregation (fibrinogen-binding to the GPIIb/IIIa) in thrombin-aggregated platelets, which results in the TXA2 production and the secondary peak of [Ca2+]i increase, and the latter was dependent on actin polymerization.     

Ring constrained analogues of the orvinols: the furanomorphides

A series of furanomorphides were synthesised as ring-constrained analogues of buprenorphine and related orvinols. Evaluation in binding and functional assays has shown that the furanomorphides have reduced efficacy at the mu opioid receptor compared to the orvinols.     

A large-scale procedure for the isolation of integrin GPIIb/IIIa, the human platelet fibrinogen receptor

The heterodimer GPIIb/IIIa, formed by the Ca(2+)-dependent association of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), is the major integrin at the platelet surface, where it serves as the receptor for fibrinogen and other adhesive proteins and plays a central role in platelet aggregation and in platelet adhesion to the subendothelium. Here we describe a procedure for the isolation of GPIIb/IIIa using as starting material either the whole particulate fraction, obtained by differential centrifugation after hypoosmotic lysis of glycerol-loaded platelets, or any of the fractions obtained by density gradient centrifugation of the whole particulate fraction. The procedure consists simply of differential extraction with Triton X-100 of the starting particulate fraction, anion-exchange chromatography of the 4% Triton X-100 supernatant, and size-exclusion chromatography of the GPIIb/IIIa-rich fraction retained in the ion-exchange column. The use of particulate fractions instead of whole platelets as the starting material for extraction together with differential extraction with Triton X-100 (two steps that are simple and inexpensive to perform) results in the early removal of many unwanted proteins, which otherwise would have to be removed at later stages at the expense of severely impairing the final yield of GPIIb/IIIa. Pure GPIIb/IIIa is obtained with a yield of about 48%, the highest so far reported, calculated with respect to the GPIIb and GPIIIa content in the starting particulate fraction. The final product can be stored in freeze-dried form without apparent changes in its physical and chemical properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and pharmacology of modified amidine isoxazoline glycoprotein IIb/IIIa receptor antagonists

Selective antagonism of the platelet GPIIb/IIIa receptor represents an attractive mechanism for the prevention and treatment of a number of thrombotic disease states. The antiplatelet activity of the oral GPIIb/IIIa receptor antagonists DMP 754 and DMP 802 have been disclosed. In this paper, the synthesis and biological evaluation of a series of potent N-substituted benzamidine isoxazolines are explored. The effect of benzamidine substitution on the duration of antiplatelet efficacy in dog is presented.     

C-terminal amino acid determination of the transmembrane subunits of the human platelet fibrinogen receptor, the GPIIb/IIIa complex

Glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the Ca2(+)-dependent GPIIb/IIIa complex, which acts as the fibrinogen receptor on activated platelets. GPIIb and GPIIIa are synthesized as single peptide chains. The GPIIb precursor is processed proteolytically to yield two disulphide-bonded chains, GPIIb alpha and GPIIb beta. The GPIIb/IIIa complex has two membrane attachment sites located at the C-termini of GPIIb beta and GPIIIa. The short cytoplasmic tails of GPIIb beta and/or GPIIIa become most likely associated to the cytoskeleton of activated platelets. In the present work the C-terminal amino acid residues of platelet GPIIb beta and GPIIIa have been analyzed by protein-chemical methods and compared with those predicted from cDNA analysis. We were able to confirm the positions of the C-termini in both glycoproteins and the identity of the C-terminus predicted for GPIIIa, i.e. threonine. However, glutamine, not glutamic acid as predicted for GPIIb beta from the human erythroleukemic cell line and megakaryocyte cells, was found to be the C-terminal amino acid of GPIIb beta. This indicates that the glutamic acid in the GPIIb precursor is posttranslationally modified to glutamine.     

Identification of potent,highly constrained CGRP receptor antagonists

A novel series of potent CGRP receptor antagonists containing a central quinoline ring constraint was identified. The combination of the quinoline constraint with a tricyclic benzimidazolinone left hand fragment produced an analog with picomolar potency (14, CGRP K_i = 23 pM). Further optimization of the tricycle produced a CGRP receptor antagonist that exhibited subnanomolar potency (19, CGRP K_i = 0.52 nM) and displayed a good pharmacokinetic profile in three preclinical species.     

Molecular characterization of the human platelet integrin GPIIb/IIIa and its constituent glycoproteins

Human platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer, the integrin GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Below the critical micellar concentration of Triton X100 (TtX), the three glycoproteins do not bind appreciably to TtX and form association products of large size. The size-exclusion chromatographic patterns of GPIIb, GPIIIa and GPIIb/IIIa have been obtained at 0.2% TtX, and the molecular properties of the association products and monomer fractions have been determined by analysis of the detergent bound to the glycoproteins, laser-light scattering, sedimentation velocity, and electron microscopy (TEM). The monomer of the GPIIb-TtX complex was identified by the molecular mass (M) of the glycoprotein moiety (125 +/- 15 kDa), the molecular size (9.5 +/- 1.5 nm x 11 +/- 1.5 nm) and globular shape observed by TEM. It has a molecular mass (M*) of 197 +/- 20 kDa, a sedimentation coefficient s degrees 20* of 5.8 +/- 0.1 S, a Stokes radius R s* of 6.8 +/- 0.4 nm, and a frictional ratio f*/fmin* of 1.7 +/- 0.14. The (GPIIb)n-TtX complexes are disulphide-bonded size-heterogeneous association products of GPIIb, tetramers being the smallest species found. GPIIIa has a greater propensity to self-associate than GPIIb, this tendency being lower below 1 mg GPIIIa/ml, 0.1 mM Ca2+, pH 9.0. The (GPIIIa)n-TtX complexes are noncovalent size-heterogeneous association products of GPIIIa, tetramers being the smallest form observed. The monomer of the GPIIIa-TtX complex was identified by the 103 +/- 15 kDa M determined for the glycoprotein moiety, and the 9 +/- 1.5 nm x 10 +/- 1.5 nm size and globular shape observed by TEM. It has a M* of 136 +/- 15 kDa, a s degrees 20* of 3.9 +/- 0.3 S, a Rs* of 6.4 +/- 0.5 nm, a f*/fmin* of 1.9 +/- 0.3, and, when stored at pH 7.4, has a certain tendency to form filamentous association products (20-70 nm x 2-5 nm), as observed by TEM. The GPIIb/IIIa-TtX complex in 0.2% TtX/0.1 mM Ca2+ elutes as a single monomeric fraction, as deduced from the 210 +/- 15 kDa M determined for its glycoprotein moiety and the 12 +/- 1.5 nm x 14 +/- 1.5 nm size of the globular forms observed by TEM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and pharmacological characterization of constrained analogues of Vestipitant as in vitro potent and orally active NK1 receptor antagonists

A focused exploration targeting conformationally restricted analogues of Vestipitant, resulted in the discovery of novel, in vitro potent NK₁ antagonists. In particular, two of the compounds reported exhibited a good pharmacokinetic (PK) profile and produced anxiolytic-like effects in the gerbil foot tapping (GFT) in vivo model.     

Ca2+ influx mediated through the GPIIb/IIIa complex during platelet activation

When aequorin-loaded platelets were stimulated with thrombin, the luminescence signal of aequorin showed two peaks. From experiments with 1 mM external Ca2+ or EGTA, both one-half of the first peak and the entire second peak reflected the influx of Ca2+ from the external medium, and the remaining half of the first peak reflected the mobilization of Ca2+ from its storage site. A monoclonal antibody (TM83) that recognizes the glycoprotein IIb/IIIa (GPIIb/IIIa) complex which has binding sites for fibrinogen and the synthetic peptide GRGDSP are known to inhibit fibrinogen binding and platelet aggregation. Both eliminated the second peak of intracellular free calcium ([Ca2+]i). Similar effects were observed during activation by collagen, but not during PMA activation. It was concluded that the GPIIb/IIIa complex was intimately related to a part of the Ca2+ influx during the activation of platelets.