Corticotropin releasing hormone receptor 2 antagonist, RQ-00490721, for the prevention of pressure overload-induced cardiac dysfunction.

BACKGROUND: G protein-coupled receptors (GPCRs) regulate the pathological and physiological functions of the heart. GPCR antagonists are widely used in the treatment of chronic heart failure. Despite therapeutic advances in the treatments for cardiovascular diseases, heart failure is a major clinical health problem, with significant mortality and morbidity. Corticotropin releasing hormone receptor 2 (CRHR2) is highly expressed in cardiomyocytes, and cardiomyocyte-specific deletion of the genes encoding CRHR2 suppresses pressure overload-induced cardiac dysfunction. This suggests that the negative modulation of CRHR2 may prevent the progression of heart failure. However, there are no systemic drugs against CRHR2. FINDINGS: We developed a novel, oral, small molecule antagonist of CRHR2, RQ-00490721, to investigate the inhibition of CRHR2 as a potential therapeutic approach for the treatment of heart failure. In vitro, RQ-00490721 decreased CRHR2 agonist-induced 3', 5'-cyclic adenosine monophosphate (cAMP) production. In vivo, RQ-00490721 showed sufficient oral absorption and better distribution to peripheral organs than to the central nervous system. Oral administration of RQ-00490721 inhibited the CRHR2 agonist-induced phosphorylation of cAMP-response element binding protein (CREB) in the heart, which regulates a transcription activator involved in heart failure. RQ-00490721 administration was not found to affect basal heart function in mice but protected them from pressure overload-induced cardiac dysfunction. INTERPRETATION: Our results suggest that RQ-00490721 is a promising agent for use in the treatment of chronic heart failure.

Discovery and Characterization of Allosteric WNK Kinase Inhibitors.

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

Interferon regulatory factors (IRFs) repress transcription of the chicken ovalbumin gene.

Although the ovalbumin (Ov) gene has served as a model to study tissue-specific, steroid hormone-induced gene expression in vertebrates for decades, the mechanisms responsible for regulating this gene remain elusive. Ov is repressed in non-oviduct tissue and in estrogen-deprived oviduct by a strong repressor site located from -130 to -100 and designated CAR for COUP-TF adjacent repressor. The goal of this study was to identify the CAR binding protein(s). A transcription factor database search revealed that a putative interferon-stimulated response element (ISRE), which binds interferon regulatory factors (IRFs), is located in this region. Gel mobility shift assays demonstrated that the protein(s) binding to the CAR site is recognized by an IRF antibody and that mutations in the ISRE abolish that binding. In hopes of identifying the IRF(s) responsible for the tissue-specific regulation of Ov, mRNA levels for IRFs-4, -8, and -10 were measured in seven tissues from chicks treated with or without estrogen. PCR experiments showed that both IRF-8 and -10 are expressed in all chick tissues tested whereas IRF-4 has a much more limited expression pattern. Transfection experiments with OvCAT (chloramphenicol acetyltransferase) reporter constructs demonstrated that both IRF-4 and IRF-10 are capable of repressing the Ov gene even in the presence of steroid hormones and that nucleotides in the ISRE are required for repression. These experiments indicate that the repressor activity associated with the CAR site is mediated by IRF family members and suggest that IRF members also repress Ov in non-oviduct tissues.

Tumor growth inhibition by synthetic and expressed siRNA targeting focal adhesion kinase.

Focal adhesion kinase (FAK) was first identified as a viral Src substrate, and substantial experimental data have significantly correlated the elevated FAK expression in human tumor cells with an increased cell adhesion and invasion potential. However, studies investigating the role of FAK in cell proliferation have been limited. Recently, a technique known as RNA interference (RNAi) was successfully adapted to mammalian cells to decrease specifically the expression of targeted cellular genes. In this study, we investigated the role of FAK in cell proliferation, adhesion, and migration by using small interfering RNA (siRNA) technique. Firstly, we constructed a plasmid library expressing short hairpin RNAs (shRNAs) targeting FAK and selected clones substantially suppressing FAK expression in HeLa and HT1080 cells. We then studied the function of FAK in the highly invasive human prostate cancer cell line, PC3M, and mouse breast cancer cell line 4T1, by using selected shRNA clones (#40 and #42) and siRNAs chemically synthesized following the target sequences of #40 and #42. We demonstrated that the decrease of FAK protein expression by treatment with shRNA/siRNA targeting FAK inhibited cell adhesion on a fibronectin/laminin-coated plate, cell migration in a haptotactic migration assay, and cell proliferation in vitro. Furthermore, it suppressed tumor growth in vivo in heterotopic/orthotopic mice models. These results support our hypothesis that FAK plays a crucial role in tumor formation and growth in vivo by regulation of cell adhesion and proliferation by FAK-dependent signals.

Human alkaline phosphatase expression and secretion into chicken eggs after in vivo gene electroporation in the oviduct of laying hens.

In vivo gene electroporation was used to examine whether or not a recombinant protein is synthesized in the chicken oviduct and subsequently secreted into eggs. A plasmid DNA containing a secretion form of the human alkaline phosphatase gene was injected into mucosa of the chicken magnum. Immediately, in vivo gene electroporation was conducted. The human alkaline phosphatase activity in the oviduct mucosa increased and reached its peak at 2 days posttransfection, followed by a sharp decrease to a negligible level at 4 days posttransfection. In the egg white, the alkaline phosphatase activity showed a similar change to that in the magnum mucosa except for a delay of 4 days. The present results imply that in vivo gene electroporation method in the oviduct may serve as a rapid production system of recombinant proteins into chicken eggs.