Cailin Chen

Thrombogenic collagen-mimetic peptides: Self-assembly of triple helix-based fibrils driven by hydrophobic interactions

Collagens are integral structural proteins in animal tissues and play key functional roles in cellular modulation. We sought to discover collagen model peptides (CMPs) that would form triple helices and self-assemble into supramolecular fibrils exhibiting collagen-like biological activity without preorganizing the peptide chains by covalent linkages. This challenging objective was accomplished by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)10, as with l-phenylalanine and l-pentafluorophenylalanine in 32-mer 1a. Computational studies on homologous 29-mers 1a′–d′ (one less GPO), as pairs of triple helices interacting head-to-tail, yielded stabilization energies in the order 1a′ > 1b′ > 1c′ > 1d′, supporting the hypothesis that hydrophobic aromatic groups can drive CMP self-assembly. Peptides 1a–d were studied comparatively relative to structural properties and ability to stimulate human platelets. Although each 32-mer formed stable triple helices (CD) spectroscopy, only 1a and 1b self-assembled into micrometer-scale fibrils. Light microscopy images for 1a depicted long collagen-like fibrils, whereas images for 1d did not. Atomic force microscopy topographical images indicated that 1a and 1b self-organize into microfibrillar species, whereas 1c and 1d do not. Peptides 1a and 1b induced the aggregation of human blood platelets with a potency similar to type I collagen, whereas 1c was much less effective, and 1d was inactive (EC50 potency: 1a/1b ≫ 1c > 1d). Thus, 1a and 1b spontaneously self-assemble into thrombogenic collagen-mimetic materials because of hydrophobic aromatic interactions provided by the special end-groups. These findings have important implications for the design of biofunctional CMPs.

Mer Receptor Tyrosine Kinase Signaling Participates in Platelet Function

Objective—Recently, mice made deficient in growth arrest–specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function. Methods and Results—Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine–induced pulmonary thromoembolism and inhibited ferric chloride–induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice. Conclusion—Our data provide the first evidence that mer, presumably through activation by its ligand Gas6, participates in regulation of platelet function in vitro and platelet-dependent thrombosis in vivo.

A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody

Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164-44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6-receptor interaction.

A novel serine protease predominately expressed in macrophages.

We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with beta-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders.

Carba-nucleosides as Potent Antagonists of the Adenosine 5′-Diphosphate (ADP) Purinergic Receptor (P2Y12) on Human Platelets

Whereas the activation and aggregation of blood platelets are crucial to normal hemostasis, this ensemble is also a key factor in serious cardiovascular disorders, such as myocardial infarction, unstable angina, transient ischemic attack, stroke, peripheral arterial disease, and artherosclerosis. Indeed, abnormal thrombosis is a root cause of adverse cardiovascular events that are responsible for death and disability in humans. As a consequence, plate ACHTUNGTRENNUNGlets are targeted by clinically useful antithrombotic drugs, such as the cyclooxygenase-1 inhibitor aspirin, the GPIIb/IIIa antagonist abciximab, and the adenosine 5’-diphosphate (ADP) receptor antagonist clopidogrel. ADP is an important agonist of platelet activation and aggregation because it induces platelet shape change accompanied by the activation of fibrinogen receptors (GPIIb/IIIa). On platelets, there are three types of cell-surface receptors for ADP, which are members of the P2 purinergic class: P2X1, P2Y1, and P2Y12. [4, 5] P2Y1 and P2Y12 are G protein-coupled receptors (GPCRs), whereas P2X1 is a ligand-gated ion channel. The Gqcoupled P2Y1 receptor initiates ADP-induced platelet activation and the Gi-coupled P2Y12 receptor amplifies activation processes, including aggregation, granule secretion, and procoagulant activity, as caused by various agonists. From this perspective, antagonists of P2Y12 can be therapeutically effective by markedly inhibiting platelet function independent of the activating stimulus. Additionally, because of the restricted distribution of P2Y12 in humans, this receptor is an attractive antiplatelet target for drug discovery. The widespread clinical use of clopidogrel has demonstrated the relevance of inhibiting the platelet-specific P2Y12 receptor to prevent untoward cardiovascular events. However, clopidogrel is a prodrug that requires metabolic conversion in vivo to a highly unstable, reactive species that covalently modifies the P2Y12 receptor. [7] On account of this property, there have been observations in humans of slow onset of pharmacological action and of high interpatient variability. Thus, drug discovery efforts have been mounted to identify potent, direct acting, reversible P2Y12 antagonists, and some promising compounds have emerged, including Cangrelor (AR-C69931MX), AZD-6140, and PRT-128. In seeking suitable drug candidates in this area, we have been exploring carba-nucleoside derivatives that are structurally related to AZD-6140 as reversible P2Y12 antagonists. In this paper, we report on the synthesis and biological evaluation of novel compounds, including tetrazole-containing derivatives with high receptor affinity and excellent potency for inhibiting P2Y12-mediated effects on human platelets.

Arterial antithrombotic activity of rivaroxaban, an orally active factor Xa inhibitor, in a rat electrolytic carotid artery injury model of thrombosis.

Rivaroxaban, an oral, direct factor Xa inhibitor, has been approved in several countries for thromboprophylaxis after elective hip or knee arthroplasty based on favorable benefit-risk profile and improved efficacy compared to enoxaparin in reducing the composite of symptomatic and asymptomatic deep vein thrombosis, nonfatal pulmonary embolism, and all-cause mortality. Given the potential therapeutic utility of factor Xa inhibition in arterial thrombosis, we evaluated the antithrombotic activity of rivaroxaban in a model of arterial thrombosis in anesthetized rats in which thrombotic occlusion was induced by electrolytic injury of the carotid artery. Rivaroxaban, 0.3, 1 or 3 mg/kg, enoxaparin, 10 mg/kg, or vehicle were infused intravenously to anesthetized rats and time to occlusion as well as coagulation parameters monitored following carotid electrolytic injury. Although the lowest dose of rivaroxaban (0.3 mg/kg) did not prolong occlusion time compared to vehicle, rivaroxaban at 1 or 3 mg/kg prevented occlusion in all vessels during the 30-min observation period (median occlusion time >30 min), which was greater than that following a single dose of enoxaparin infused at a dose of 10 mg/kg (median time to occlusion = 21.6 min). Rivaroxaban was also effective following oral dosing at 3 mg/kg. Rivaroxabans antithrombotic activity was paralleled by dose-dependent increases in prothrombin time (PT) and activated clotting time (ACT) without significant changes in activated partial thromboplastin time. Rivaroxaban also markedly increased Russells viper venom time (RVVT) and decreased thrombin–antithrombin complex concentrations at all doses. These findings support the potential utility of rivaroxaban in arterial thrombotic disorders such as acute coronary syndrome, stroke and peripheral arterial disease.

Thrombin Receptor Modulators: Medicinal Chemistry, Biological Evaluation, and Clinical Application

The serine protease a-thrombin maintains haemostasis through its coagulant, anticoagulant, and platelet-activator functions. This enzyme also has important cellular effects involving cell proliferation, cytokine and growth factor release, and tissue remodeling, which are mediated by G-protein coupled receptors known as protease-activated receptors (PARs). Thrombin can activate three of the four PAR family members, and PAR1 is the primary thrombin-responsive receptor in human cells. PAR1 plays an important role during the response to tissue injury and the associated inflammatory processes. The blockade of PAR1 offers a new approach for treating various disorders that depend on thrombin generation, including thrombosis and restenosis. Antagonists of PAR1 will interrupt thrombin’s receptor function, but not thrombin’s proteolytic activity, thereby providing an alternative means to attenuate the pathological effects of thrombin. This chapter deals with the topic of PAR1, with the key medicinal chemistry, pharmacology, and clinical aspects of PAR1 antagonists, and with the topic of PAR4. The full potential of PAR1 antagonists has yet to be realized commercially, but the promise of novel therapeutics is reflected by two antiplatelet PAR1 antagonists in advanced human clinical trials.

6-Benzhydryl-4-amino-quinolin-2-ones as Potent Cannabinoid Type 1 (CB1) Receptor Inverse Agonists and Chemical Modifications for Peripheral Selectivity

A novel series of 6-benzhydryl-4-amino-quinolin-2-ones was discovered as cannabinoid type 1 receptor (CB1R) inverse agonists based on the high-throughput screening hit, compound 1a. Structure-activity relationships were studied to improve in vitro/in vivo pharmacology and restrict distribution to the peripheral circulation. We adopted several strategies such as increasing topological polar surface area, incorporating discrete polyethylene glycol side chains, and targeting P-glycoprotein (P-gp) to minimize access to the brain. Compound 6a is a P-gp substrate and a potent and highly selective CB1R inverse agonist, demonstrating excellent in vivo metabolic stability and a low brain to plasma ratio. However, brain receptor occupancy studies showed that compound 6a may accumulate in brain with repeat dosing. This was evidenced by compound 6a inhibiting food intake and inducing weight loss in diet-induced obese mice. Thus, a strategy based on P-gp efflux may not be adequate for peripheral restriction of the disclosed quinolinone series.