No abstract

We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.

Ischemia resulting from myocardial infarction (MI) promotes VEGF expression, leading to vascular permeability (VP) and edema, a process that we show here contributes to tissue injury throughout the ventricle. This permeability/edema can be assessed noninvasively by MRI and can be observed at the ultrastructural level as gaps between adjacent endothelial cells. Many of these gaps contain activated platelets adhering to exposed basement membrane, reducing vessel patency. Following MI, genetic or pharmacological blockade of Src preserves endothelial cell barrier function, suppressing VP and infarct volume, providing long-term improvement in cardiac function, fibrosis, and survival. To our surprise, an intravascular injection of VEGF into healthy animals, but not those deficient in Src, induced similar endothelial gaps, VP, platelet plugs, and some myocyte damage. Mechanistically, we show that quiescent blood vessels contain a complex involving Flk, VE-cadherin, and β β-catenin that is transiently disrupted by VEGF injection. Blockade of Src prevents disassociation of this complex with the same kinetics with which it prevents VEGF-mediated VP/edema. These findings define a molecular mechanism to account for the Src requirement in VEGF-mediated permeability and provide a basis for Src inhibition as a therapeutic option for patients with acute MI. Cheresh is on the Scientific Advisory Board of TargeGen but is not an employee, board member, or a recipient of research funding from the company. TargeGen is developing small molecule therapies for use in treating ischemic diseases; however, these molecules are independent from those described in the current manuscript. Citation for this article:

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC 50 ¼ 6 nM), FLT3 (IC 50 ¼ 25 nM) and RET (IC 50 ¼ 17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC 50 ¼ 169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC 50 of B200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations. Leukemia (2007) IntroductionAcquisition of somatic mutations such as JAK2V617F 1 results in constitutive activation of JAK-STAT signaling, which is thought to play a primary role in the pathogenesis of myeloproliferative disorders (MPD) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In normal hematopoiesis, ligand-induced activation of a spectrum of hematopoietic cytokine receptors, including receptors for erythropoietin, thrombopoietin (MPL) and granulocyte colony stimulating factor, converges upon Janus kinase 2 (JAK2). The importance of JAK2 in hematopoiesis has been demonstrated in mice that are genetically deficient in JAK2, and have severe defects in erythropoiesis. 2 Dysregulated JAK-STAT signaling may be important in JAK2V617F-negative MPD as well, in that other activating JAK2 alleles have been identified in these patients, including JAK2 exon 12 mutations, JAK2D620E, JAK2DIREED, in addition to activating mutations in MPL at position W515. [3][4][5][6] Expression of JAK2V617F in vivo in a murine bone marrow transplant assay results in a phenotype resembling PV; 7,8 in contrast, MPLW515L expression in a similar assay results in a PMF-like phenotype. 9 Inhibition of JAK2 with small molecule 'tool' compounds that lack potential for clinical development, and that are not selective among JAK family members, induce apoptotic cell death in hematopoietic cell lines transformed either with JAK2V617F 10 or with MPLW515L, 9 and, similarly, may decrease the hematocrit in mouse models of JAK2V617F-induced disease. 8 MPD are currently not captured in the surveillance, epidemiology and end results (SEER) database or other cancer registries, but incidence of PV, ET and PMF has been estimated in the 1-5/100 000 per year range. 11 Because of relatively long survival after diagnosis, it has been estimated that the prevalence of MPD is on the order of 80 000-100 000 cases in the United States, significantly higher than that of BCR-ABLpositive chronic myeloid leukemia (CML). Although the MPD are relatively indolent, most patients ultimately develop...

The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection.