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.

Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models.

The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

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.

Small-molecule WNK inhibition regulates cardiovascular and renal function.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

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.

Regulation of expression of the mouse adiponectin gene by the C/EBP family via a novel enhancer region.

Adiponectin plays protective roles against the development of insulin resistance and atherosclerosis. To clarify the regulation of adiponectin gene expression, reporter gene assay by using the several truncated mouse adiponectin 5'-flanking regions was performed after the differentiation of 3T3-L1 preadipocytes. The results indicated that a novel mouse adiponectin enhancer exists in the-2228 to -2066 region. Nuclear proteins from the differentiated adipocytes bound to two DNA fragments, namely, -2153 to -2114 and -2093 to -2054. Both fragments had a common motif, CACAATGC, which was similar to the CCAAT/enhancer binding protein (C/EBP) binding motif. A gel mobility shift assay with anti-C/EBPs antibodies showed that C/EBP alpha, beta, and delta bound to this motif. These data provide the first evidence that the transcriptional activity of the mouse adiponectin gene during adipocyte differentiation is enhanced by the motif in a novel adiponectin enhancer region, via the recruitment of the C/EBPs.

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.

An oviduct-specific and enhancer-like element resides at about -3000 in the chicken ovalbumin gene.

The chicken ovalbumin (Ov) gene is one of the best models to study tissue-specific gene regulation because it is only expressed in the oviduct. In this paper, a tissue-specific element was characterized by 5'-flanking region deletion in combination with in vivo gene electroporation (EP). Plasmids with varying lengths of truncated Ov 5'-flanking region fused to the Renilla luciferase reporter gene were transfected in vivo into oviduct, muscle, and pancreas. A chicken oviduct-specific and enhancer-like region (designated COSE) was implicated between -3100 and -2800. The COSE showed up-regulation of gene expression in oviduct, but not in muscle or in pancreas. The COSE region was further characterized by gel mobility shift assays using nuclear extracts from oviduct, pancreas and liver. With the region from -2900 to -2851, designated T2, there were two distinct protein-DNA complexes: one found only in oviduct extract and the other detected only in pancreas and liver. These data suggest a model where the regulation of Ov gene expression in the oviduct and non-oviduct tissues is exerted at least in part by the presence of protein modulators that bind to the COSE element.

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.