The Irreversible Covalent Fibroblast Growth Factor Receptor Inhibitor PRN1371 Exhibits Sustained Inhibition of FGFR after Drug Clearance.

An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site.

Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors.

Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncology indications.

Prolonged and tunable residence time using reversible covalent kinase inhibitors.

Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d.

High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888.

Background: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition.

Rapamycin inhibits VEGF-induced microvascular hyperpermeability in vivo.

Objective: To test the hypothesis that rapamycin inhibits induced microvascular hyperpermeability directly in vivo.

Methods: Male golden Syrian hamsters (80-120 g) were treated with either rapamycin (at 0.1, 0.

EXEL-7647 inhibits mutant forms of ErbB2 associated with lapatinib resistance and neoplastic transformation.

Purpose: Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical efficacy for kinase-targeted therapeutics. We report the prospective discovery of ErbB2 mutations that confer resistance to the small-molecule inhibitor lapatinib.

Experimental Design: We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for survival and growth.

Genetic variations in vascular endothelial growth factor and endothelial nitric oxide synthase and their contributions to human disease.

This review addresses a rapidly growing area of vascular biology, i.e. genomic variations in vascular genes that underlie different human phenotypes.

Receptor-mediated basic fibroblast growth factor signaling regulates cyclic pressure-induced human endothelial cell proliferation.

Vascular endothelial cells sense and respond to pressure by molecular mechanism(s) which, to date, remain poorly understood. The present study investigated basic fibroblast growth factor (bFGF) signaling as a putative mechanotransduction pathway involved in the proliferative responses of human umbilical vein endothelia cells (HUVECs) to 60/20 mm Hg cyclic pressure at 1 Hz for 24 h. Under these conditions, the enhanced proliferative response of these HUVECs was not associated with an increased synthesis/release of bFGF, but involved rapid (within 30 min from the onset of exposure to pressure) tyrosine phosphorylation of the bFGF receptor, FGFR-2.

Stanniocalcin: no longer just a fish tale.

Stanniocalcin was originally described as a hormone with calcitonin-like actions in fish. During the last decade, mammalian forms of stanniocalcin have been identified, and this discovery has led to important advances in our understanding of this enigmatic polypeptide hormone. This review briefly covers some early studies on stanniocalcin in fish and then provides a more in-depth look at some of the more intriguing, new aspects of its functions in mammals.

Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells.

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high-density oligonucleotide arrays, representing approximately 22000 genes.

Stanniocalcin 1 is an autocrine modulator of endothelial angiogenic responses to hepatocyte growth factor.

Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g.

A novel interleukin-17 receptor-like protein identified in human umbilical vein endothelial cells antagonizes basic fibroblast growth factor-induced signaling.

We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells. One gene identified by this approach encodes a type I transmembrane receptor that shares sequence homology with the intracellular domain of members of the interleukin-17 (IL-17) receptor family. Real-time quantitative PCR and Northern analyses revealed that this gene is highly expressed in human umbilical vein endothelial cells and in several highly vascularized tissues such as kidney, colon, skeletal muscle, heart, and small intestine.

Branching out: a molecular fingerprint of endothelial differentiation into tube-like structures generated by Affymetrix oligonucleotide arrays.

The process of endothelial differentiation into a network of tube-like structures with patent lumens requires an integrated program of gene expression. To identify genes upregulated in endothelial cells during the process of tube formation, RNA was prepared from several different time points (0, 4, 8, 24, 40, and 48 hours) and from three different experimental models of human endothelial tube formation: in collagen gels and fibrin gels driven by the combination of PMA (80), bFGF (40 ng/ml) and bFGF (40 ng/ml) or in collagen gels driven by the combination of HGF (40 ng/ml) and VEGF (40 ng/ml). Gene expression was evaluated using Affymetrix Gene Chip oligonucleotide arrays.

Vascular endothelial growth factor-induced genes in human umbilical vein endothelial cells: relative roles of KDR and Flt-1 receptors.

Objective: This study evaluated the relative roles of the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 in the mediation of altered gene expression elicited by VEGF.

Methods And Results: We used mutants of VEGF selective for the KDR and Flt-1 receptors to differentiate gene expression patterns mediated by wild-type VEGF (VEGFwt) in human umbilical vein endothelial cells. RNA was extracted from cells treated for 24 hours with 1 nmol/L of each ligand, and gene expression was monitored by using oligonucleotide arrays (Affymetrix U95A).

Time course of skeletal muscle repair and gene expression following acute hind limb ischemia in mice.

DNA microarrays were used to measure the time course of gene expression during skeletal muscle damage and regeneration in mice following femoral artery ligation (FAL). We found 1,289 known sequences were differentially expressed between the FAL and control groups. Gene expression peaked on day 3, and the functional cluster "inflammation" contained the greatest number of genes.

VEGF-C mediates cyclic pressure-induced endothelial cell proliferation.

Mechanical forces modulate endothelial cell functions through several mechanisms including regulation of gene transcription. In the present study, gene transcription by human umbilical vein endothelial cells (HUVEC) either maintained under control pressure (that is, standard cell culture conditions equivalent to 0.15 mmHg sustained hydrostatic pressure) or exposed to 60/20 mmHg sinusoidal pressures at 1 Hz were compared using Affymetrix GeneChip microarrays to identify cellular/molecular mechanisms associated with endothelial cell responses to cyclic pressure.

Stanniocalcin 1 alters muscle and bone structure and function in transgenic mice.

Fish stanniocalcin (STC) inhibits uptake of calcium and stimulates phosphate reabsorption. To determine the role of the highly homologous mammalian protein, STC-1, we created and characterized transgenic mice that express STC-1 under control of a muscle-specific promoter. STC-1 transgenic mice were smaller than wild-type littermates and had normal growth plate cartilage morphology but increased cartilage matrix synthesis.

In silico data filtering to identify new angiogenesis targets from a large in vitro gene profiling data set.

The objective of this study was to use gene expression data from well-defined cell culture models, in combination with expression data from diagnostic samples of human diseased tissues, to identify potential therapeutic targets and markers of disease. Using Affymetrix oligonucleotide array technology, we identified a common profile of genes upregulated during endothelial morphogenesis into tubelike structures in three in vitro models of angiogenesis. Rigorous data selection criteria were used to identify a list of over 1,000 genes whose expression was increased more than twofold over baseline at either 4, 8, 24, 40 or 50 h.

Regulation of endothelial cell proliferation and apoptosis by cyclic pressure.

The present study investigated the proliferative and apoptotic responses of human umbilical vein endothelial cells (HUVECs) to well-defined, sinusoidal pressures (60/20, 100/60, and 140/100 mm Hg/mm Hg) at 1 Hz for up to 24 h under Media 199 containing either 1% FBS and 0.04% bovine brain extract (BBE) (low serum/growth factor conditions) or 10% FBS and 0.4% BBE (normal serum/growth factor conditions).

Gene expression profiling in silico: relative expression of candidate angiogenesis associated genes in renal cell carcinomas.

Recent advances in gene expression profiling have led to the development of comprehensive databases which can be queried in various manners. In the present report, we have taken a list of genes previously associated with angiogenesis, either in in vivo or in in vitro models, and queried a commercial database established by GeneLogic to determine the relative expression of these candidate genes in normal kidneys and in renal cell carcinomas (RCC). We identified a number of genes, including CXCR4, matrix metalloproteinase 9, thrombospondin 2, and vascular endothelial growth factor, that were highly expressed in RCC versus normal tissue.

A dominant-negative p65 PAK peptide inhibits angiogenesis.

PAK1 is a protein kinase downstream of the small GTPases Rac and Cdc42 that previous work has implicated in endothelial cell migration via modulation of cell contraction. The first proline-rich region of PAK that binds to an SH3 domain from the adapter protein NCK was responsible for these dominant-negative effects. To test the role of PAK in angiogenesis, we prepared a peptide in which the proline-rich region was fused to the polybasic sequence from the HIV Tat protein to facilitate entry into cells.

Microcirculation down under.

The Seventh World Congress for Microcirculation, organized by the Australian and New Zealand Microcirculation Society, was held on 19-22 August 2001 in Sydney, New South Wales, Australia.