Randy Serafino

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.

BMS-180560, an insurmountable inhibitor of angiotensin II-stimulated responses: comparison with losartan and EXP3174

1 This study compares the activity of BMS‐180560 (2‐butyl‐4‐chloro‐1‐[[1‐[2‐(2H‐tetrazol‐5‐yl)phenyl]‐1H‐indol‐4‐yl]methyl]‐1H‐imidazole‐5‐carboxylic acid), an insurmountable angiotensin II (AII) receptor antagonist, with that of losartan and EXP3174 in functional and biochemical models of AII‐receptor activation. 2 BMS‐180560 selectively inhibited [125I]‐Sar1Ile8AII ([125I]SI‐AII) binding to rat aortic smooth muscle (RASM) cell and rat adrenal cortical AT1 receptors (Ki = 7.6 ± 1.2 and 18.4 ± 3.9 nm respectively) compared to adrenal cortical AT2 receptors (Ki = 37.6 ± 1.3 μm). The Ki values of BMS‐180560 and EXP3174, but not losartan, varied as a function of the BSA concentration used in the assays, indicating that the diacid drugs bound to albumin. 3 BMS‐180560 (3–300 nm) increased the KD of SI‐AII for RASM cell AT1 receptors. Only at high concentrations of BMS‐180560 (300 nm) were Bmax values decreased. 4 BMS‐180560 inhibited AII‐stimulated contraction of rabbit aorta with a calculated KB = 0.068 ± 0.048 nm and decreased maximal AII‐stimulated contraction at 1 nm BMS‐180560 by 75%. In the presence of 0.1% BSA, a higher KB value (5.2 ± 0.92 nm) was obtained. Losartan behaved as a competitive antagonist with a KB = 2.6 ± 0.13 nm. Contraction stimulated by endothelin‐1, noradrenaline, KC1, or the TXA2 receptor agonist U‐46619 were unaffected by BMS‐180560 (1 nm). 5 All stimulated the acidification rates of RASM cells as measured by a Cytosensor microphysiometer with an EC50 of 18 nm. Losartan (30 nm) shifted the AII concentration‐effect curves in a competitive manner whereas BMS‐180560 (0.01 and 0.1 nm) decreased the maximum responses by 60 and 75% respectively. Inhibition by losartan and BMS‐180560 could be reversed following washout although recovery took longer for BMS‐180560. 6 In [3H]‐myoinositol‐labelled RASM cells, losartan (30 and 200 nm), shifted the EC50 for AII‐stimulated [3H]‐inositol monophosphate formation to higher values, with no change in the maximal response. By contrast, EXP3174 (0.1 to 1 nm) decreased the maximal response in a concentration‐dependent manner (17–55%). BMS‐180560 (3 and 10 nm) increased the EC50 for AII and decreased the maximum response by 30 and 80% respectively. The inhibition by EXP3174 and BMS‐180560 could be reversed by inclusion of losartan (200 nm) indicating that the inhibition was not irreversible. 7 In conclusion, BMS‐180560 is a potent, specific, predominantly competitive, reversible AII receptor antagonist, which displays insurmountable receptor antagonism. At concentrations of BMS‐180560 which have no effect on receptor number, BMS‐180560 produced insurmountable antagonism of AII‐stimulated second messenger formation, extracellular acidification, and smooth muscle contraction.

Discovery of Novel, Induced-Pocket Binding Oxazolidinones as Potent, Selective, and Orally Bioavailable Tankyrase Inhibitors

Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein levels and elevates β-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1 and 2) may reduce the levels of β-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with improved pharmacokinetic properties (39, 40).

Development of Novel Dual Binders as Potent, Selective, and Orally Bioavailable Tankyrase Inhibitors

Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting β-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.

Binding and signal transduction of the cloned vascular angiotensin II (AT1a) receptor cDNA stably expressed in Chinese hamster ovary cells

The vascular angiotensin (A) II receptor cDNA (AT1a) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-AT1a. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT1 receptor. Specific binding of sarcosine1(-)[125I]tyrosine4-isoleucine8-AII ([125I]SI-AII) to CHO-AT1a membranes reached equilibrium after 1 h at 25 degrees C and was consistently greater than 95% of total binding. Saturation binding analyses demonstrated [125I]SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (KD) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [125I]SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII > AII > losartan > AI >> PD 123,177. AII (1 microM) treatment of CHO-AT1a cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 microM) did not effect adenylate cyclase activity in CHO-AT1a cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10 microM losartan. These results indicate that CHO-AT1a cells express functional AT1a receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-AT1a cells should provide a useful model for studies of AT1a receptor domains which are critical to signaling pathways.

Endothelin analogs which distinguish vasoconstrictor and vasodilator ETB receptors

[Pen 1,11, Nle7, Glu9, Ala18]-Sarafotoxin S6b (BMS-184696) and [Pen1,11, Nle7, Glu9, Leu18]-sarafotoxin S6b (BMS-184697) were synthesized with the aim of preparing ETB receptor antagonists. BMS-184696 was a potent ETA antagonist, an extremely potent vasoconstrictor ETB agonist, and a non-competitive vasodilator ETB antagonist with no agonist activity. BMS-184697 was a potent ETA antagonist, a potent vasoconstrictor ETB agonist, and a vasodilator ETB agonist with moderate potency. The ability of BMS-184696 to activate the vasoconstrictor ETB receptor but not the vasodilator ETB receptor, despite having high affinity binding to the vasodilator ETB receptor as evidenced by its antagonist activity, strongly suggests the existence of ETB receptor subtypes.

Structure-activity studies of endothelin leading to novel peptide ETA antagonists

With the goal of producing receptor antagonists, numerous monocyclic and bicyclic endothelin analogs were prepared and tested for vasoconstrictor activity, receptor affinity and functional antagonist activity. Bis-penicillamine endothelin analogs containing Ala or Asn at position 18 were functional antagonists, with Ki values of 20-40 nM but KB values of about 1 microM (e.g., [Pen1,11, Nle7, Ala18]-endothelin-1, Ki = 42 nM, KB = 1.2 microM). While these peptides are antagonists at the ETA receptor, they appear to be at least partial agonists at another receptor subtype.