John T. Hunt

Venous smooth muscle contains vasoconstrictor ETB-like receptors

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.

Preclinical antitumor activity of BMS-599626, a pan-HER kinase inhibitor that inhibits HER1/HER2 homodimer and heterodimer signaling.

Purpose: The studies described here are intended to characterize the ability of BMS-599626, a small-molecule inhibitor of the human epidermal growth factor receptor (HER) kinase family, to modulate signaling and growth of tumor cells that depend on HER1 and/or HER2. Experimental Design: The potency and selectivity of BMS-599626 were assessed in biochemical assays using recombinant protein kinases, as well as in cell proliferation assays using tumor cell lines with varying degrees of dependence on HER1 or HER2 signaling. Modulation of receptor signaling was determined in cell assays by Western blot analyses of receptor autophosphorylation and downstream signaling. The ability of BMS-599626 to inhibit receptor heterodimer signaling in tumor cells was studied by receptor coimmunoprecipitation. Antitumor activity of BMS-599626 was evaluated using a number of different xenograft models that represent a spectrum of human tumors with HER1 or HER2 overexpression. Results: BMS-599626 inhibited HER1 and HER2 with IC50 of 20 and 30 nmol/L, respectively, and was highly selective when tested against a broad panel of diverse protein kinases. Biochemical studies suggested that BMS-599626 inhibited HER1 and HER2 through distinct mechanisms. BMS-599626 abrogated HER1 and HER2 signaling and inhibited the proliferation of tumor cell lines that are dependent on these receptors, with IC50 in the range of 0.24 to 1 μmol/L. BMS-599626 was highly selective for tumor cells that depend on HER1/HER2 and had no effect on the proliferation of cell lines that do not express these receptors. In tumor cells that are capable of forming HER1/HER2 heterodimers, BMS-599626 inhibited heterodimerization and downstream signaling. BMS-599626 had antitumor activity in models that overexpress HER1 (GEO), as well as in models that have HER2 gene amplification (KPL4) or overexpression (Sal2), and there was good correlation between the inhibition of receptor signaling and antitumor activity. Conclusions: BMS-599626 is a highly selective and potent inhibitor of HER1 and HER2 kinases and inhibits tumor cell proliferation through modulation of receptor signaling. BMS-599626 inhibits HER1/HER2 receptor heterodimerization and provides an additional mechanism of inhibiting tumors in which receptor coexpression and heterodimerization play a major role in driving tumor growth. The preclinical data support the advancement of BMS-599626 into clinical development for the treatment of cancer.

Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors

A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

Synergy between chemotherapeutic agents and CTLA-4 blockade in preclinical tumor models

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) binding agent, has proven to be an effective monotherapy for metastatic melanoma and has shown antitumor activity in trials when administered with other therapeutic agents. We hypothesized that the combination of ipilimumab with chemotherapeutic agents, such as ixabepilone, paclitaxel, etoposide, and gemcitabine, may produce therapeutic synergy based on distinct but complementary mechanisms of action for each drug and unique cellular targets. This concept was investigated using a mouse homolog of ipilimumab in preclinical murine tumor models, including SA1N fibrosarcoma, EMT-6 mammary carcinoma, M109 lung carcinoma, and CT-26 colon carcinoma. Results of CTLA-4 blockade in combination with one of various chemotherapeutic agents demonstrate that synergy occurs in settings where either agent alone was not effective in inducing tumor regression. Furthermore, when combined with CTLA-4 blockade, ixabepilone, etoposide, and gemcitabine elicited prolonged antitumor effects in some murine models with induction of a memory immune response. Future investigations are warranted to determine which specific chemo-immunotherapy combinations, if any, will produce synergistic antitumor effects in the clinical setting.

Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase.

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner.

Solution conformation of a cyclic pentapeptide endothelin antagonist : comparison of structures obtained from constrained dynamics and conformational search

The structure of a cyclic pentapeptide, cyclo‐(d‐Trp‐d‐Asp‐l‐Pro‐d‐Val‐l‐Leu), that has high selectivity for the endothelin ETAA receptor has been determined by NMR spectroscopy using constrained molecular dynamics and conformational search procedures. Structures obtained using two methods of refinement, namely (i) constrained molecular dynamics; and (ii) systematic searches of conformational space for optimal satisfaction of distance constraints, were compared to those obtained from systematic searches of conformational space without NMR data. The two different procedures of refinement produce similar conformations that are consistent with the NMR distance constraints. Conformational searches for optimal energy without any NMR distance constraints produced several low‐energy structures, two of which have essentially the same backbone as those structures derived from distance‐constrained procedures and one of these even reproduces several side‐chain positions well. The pentapeptide backbone consists of a linked γ and β‐turn conformation, with the leucine and tryptophan as corner residues of the type II β‐turn. The side chains are highly ordered both in aqueous solvent and in dimethyl sulfoxide. In aqueous media the leucine side chain is directed towards the indole ring, presumably to reduce the non‐polar surface exposure, producing unusual upfield shifts for the methyls (and particularly Hγ). This structural feature was reproduced in one of the structures obtained from conformational searches performed without NMR data. Exhaustive conformational searches appear to provide an alternative method for structure generation for cyclic peptides.

Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor

Recently a thrombin receptor with a unique mechanism of activation was cloned from a megakaryocyte-like cell line (Vu et al., Cell 64:1057-1068, 1991). Thrombin cleaves a portion of this receptor creating a new N-terminus that acts as a tethered-ligand to activate the receptor. A thrombin receptor activating peptide (SFLLRNPNDKYEPF) homologous to the new N-terminus was shown to activate platelets. We synthesized this peptide and demonstrated that it desensitized platelets to activation by low concentrations of alpha-thrombin but not gamma-thrombin. We also synthesized a thrombin exosite inhibitor (BMS 180742) that inhibited platelet aggregation induced by low, but not high, concentrations of alpha-thrombin. In contrast, a thrombin active site inhibitor, N alpha-(2-naphthylsulfonyl-glycyl)-D,L-amidinophenylalanylpiperi dide, competitively inhibited thrombin-induced platelet aggregation. We conclude that thrombin-induced platelet activation is mediated by at least two pathways: one activated by low concentrations of alpha-thrombin and blocked by a thrombin exosite inhibitor that appears to be coupled to the tethered-ligand thrombin receptor, and another that is stimulated by higher concentrations of alpha-thrombin and by gamma-thrombin and does not require the thrombin exosite for activation. Both pathways are blocked by a thrombin active site inhibitor.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors.

Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

Solid phase synthesis of phosphinic acid endothelin converting enzyme inhibitors

Abstract We have synthesized a series of phosphinic acids by solid phase peptide synthesis where we have explored effect on inhibition of changes in the P 2 ′ binding site. The most potent compounds show inhibition of ECE similar to phosphoramidon.

Synthesis, SAR, and Evaluation of 4-[2,4-Difluoro-5-(cyclopropylcarbamoyl)phenylamino]pyrrolo[2,1-f][1,2,4]triazine-based VEGFR-2 kinase inhibitors

Introduction of the 2,4-difluoro-5-(cyclopropylcarbamoyl)phenylamino group at the C-4 position of the pyrrolo[2,1-f][1,2,4] triazine scaffold led to the discovery of a novel sub-series of inhibitors of VEGFR-2 kinase activity. Subsequent SAR studies on the 1,3,5-oxadiazole ring appended to the C-6 position of this new sub-family of pyrrolotriazines resulted in the identification of low nanomolar inhibitors of VEGFR-2. Antitumor efficacy was observed with compound 37 against L2987 human lung carcinoma xenografts in athymic mice.