Jacquelynn J. Cook

Disintegrins: a family of integrin inhibitory proteins from viper venoms.

Abstract Disintegrins represent a new class of low molecular weight, RGD-containing, cysteine-rich peptides isolated from the venom of various snakes. They interact with the β 1 and β 3 families of integrins and their potency is at least 500–2000 times higher than short RGDX peptides. Analysis of the amino acid sequences of 14 different disintegrins suggests that the RGD sequence, in the spatial configuration determined by the appropriate pairing of the cysteine residues, functions as a cell recognition site. However, certain nonconserved amino acids appear to modify the activity of disintegrins, their specificity for various receptors, and their ability to compete specifically with various ligands.

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Pharmacological Properties of MK-3207, a Potent and Orally Active Calcitonin Gene-Related Peptide Receptor Antagonist

Calcitonin gene-related peptide (CGRP) has long been hypothesized to play a key role in migraine pathophysiology, and the advent of small-molecule antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and migraine efficacy. 2-[(8R)-8-(3,5-Difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2′-oxo-1,1′,2′,3-tetrahydrospiro[indene-2,3′-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) represents the third CGRP receptor antagonist to display clinical efficacy in migraine trials. Here, we report the pharmacological characterization of MK-3207, a potent and orally bioavailable CGRP receptor antagonist. In vitro, MK-3207 is a potent antagonist of the human and rhesus monkey CGRP receptors (Ki = 0.024 nM). In common with other CGRP receptor antagonists, MK-3207 displays lower affinity for CGRP receptors from other species, including canine and rodent. As a consequence of species selectivity, the in vivo potency was assessed in a rhesus monkey pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging. MK-3207 produced a concentration-dependent inhibition of dermal vasodilation, with plasma concentrations of 0.8 and 7 nM required to block 50 and 90% of the blood flow increase, respectively. The tritiated analog [3H]MK-3207 was used to study the binding characteristics on the human CGRP receptor. [3H]MK-3207 displayed reversible and saturable binding (KD = 0.06 nM), and the off-rate was determined to be 0.012 min−1, with a t1/2 value of 59 min. In vitro autoradiography studies on rhesus monkey brain slices identified the highest level of binding in the cerebellum, brainstem, and meninges. Finally, as an index of central nervous system penetrability, the in vivo cerebrospinal fluid/plasma ratio was determined to be 2 to 3% in cisterna magna-ported rhesus monkeys.

Binding of glycoprotein IIIa-derived peptide 217-231 to fibrinogen and von Willebrand factors and its inhibition by platelet glycoprotein IIb/IIIa complex

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) IIIa 217-231 and similar portions of other beta subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIIa 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIIIa 217-231(Y) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPIIb/IIIa displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of 125I-GPIIIa 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPIIb/IIIa complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.

Identification of an Orally Bioavailable, Potent, and Selective Inhibitor of GlyT1.

Amalgamation of the structure-activity relationship of two series of GlyT1 inhibitors developed at Merck led to the discovery of a clinical candidate, compound 16 (DCCCyB), which demonstrated excellent in vivo occupancy of GlyT1 transporters in rhesus monkey as determined by displacement of a PET tracer ligand.

Comparison of the In Vivo Distribution of Four Different Annexin A5 Adducts in Rhesus Monkeys

Annexin A5 has been used for the detection of apoptotic cells, due to its ability to bind to phosphatidylserine (PS). Four different labeled Annexin A5 adducts were evaluated in rhesus monkey, with radiolabeling achieved via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Of these adducts differing conjugation methods were employed which resulted in nonspecific radiolabeling (AxA5-I), or site-specific radiolabeling (AxA5-II). A nonbinding variant of Annexin A5 was also evaluated (AxA5-IINBV), conjugation here was site specific. The fourth adduct examined had both specific and nonspecific conjugation techniques employed (AxA5-IImDOTA). Blood clearance for each adduct was comparable, while appreciable uptake was observed in kidney, liver, and spleen. Significant differences in uptake of AxA5-I and AxA5-II were observed, as well as between AxA5-II and AxA5-IINBV. No difference between AxA5-II and AxA5-IImDOTA was observed, suggesting that conjugating DOTA nonspecifically did not affect the in vivo biodistribution of Annexin A5.

Characterization of an antibody raised against reduced glycoprotein IIIa of human platelets

A polyclonal antibody against reduced and vinylpyridylethylated human glycoprotein IIIa was raised in rabbits. Its reactivity with reduced GPIIIa was about 500 times higher than that of the antibody against native GPIIIa. The lowest amounts of purified reduced and native GPIIIa recognized by the antibody against reduced GPIIIa were 25 and 400 ng, respectively. The antibody did not recognize native GPIIIa (about 1-2 micrograms) in platelet extracts and chymotryptic degradation products of GPIIIa. It inhibited ADP-induced platelet aggregation but it did not inhibit fibrinogen binding to ADP-stimulated platelets. Our experiments suggest that the antigenicity of GPIIIa (beta 3 integrin) depends on the conformation of the molecule determined by numerous S-S bridges between cysteine residues.

Studies on the Localization of Fibrinogen Binding Sites on Platelet Glycoprotein IIIa

The domains of various integrins involved in the binding to adhesive proteins are poorly characterized. The approaches to localize ligand binding sites on the adhesive receptors include studies on the genetic characterization of the defective β 2 subunits in leukocyte adhesion deficiency and the defective β 1 subunit in Glanzmann thrombasthenia, studies on the chemical and photoaffinity crosslinking of the adhesive ligands to the receptors, proteolytic degradation of the integrins, attempts to recognize binding sites with monoclonal antibodies, and studies on the synthetic peptides derived from glycoprotein IIIa (GPIIIa).