Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present.

Structural Development of Salicylanilide-Based SPAK Inhibitors as Candidate Antihypertensive Agents.

Hypertension is an important target for drug discovery. We have focused on the with-no-lysine kinase (WNK)-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal cascade as a potential target, and we previously developed a screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling. Herein we used this system to examine the structure-activity relationship (SAR) of salicylanilide derivatives as SPAK kinase inhibitors.

CSE1L promotes nuclear accumulation of transcriptional coactivator TAZ and enhances invasiveness of human cancer cells.

The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial-mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation.

Activation of β2-adrenergic receptor signals suppresses mesenchymal phenotypes of oral squamous cell carcinoma cells.

Metastasis is a primary reason related to the mortality of oral squamous cell carcinoma (OSCC) patients. A program called epithelial-mesenchymal transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium-derived carcinoma. During EMT, epithelial cancer cells acquire motile mesenchymal phenotypes and detach from primary tumors.

Development of Androgen-Antagonistic Coumarinamides with a Unique Aromatic Folded Pharmacophore.

First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide () and bicalutamide (), are effective for most prostate cancer patients, but resistance often appears after several years due to the mutation of AR. Second-generation AR antagonists are effective against some of these castration-resistant prostate cancers, but their structural variety is still limited. In this study, we designed and synthesized 4-methyl-7-(-alkyl-arylcarboxamido)coumarins as AR antagonist candidates and evaluated their growth-inhibitory activity toward androgen-dependent SC-3 cells.

Chemical Screening of Nuclear Receptor Modulators.

Nuclear receptors are ligand-inducible transcriptional factors that control multiple biological phenomena, including proliferation, differentiation, reproduction, metabolism, and the maintenance of homeostasis. Members of the nuclear receptor superfamily have marked structural and functional similarities, and their domain functionalities and regulatory mechanisms have been well studied. Various modulators of nuclear receptors, including agonists and antagonists, have been developed as tools for elucidating nuclear receptor functions and also as drug candidates or lead compounds.

Structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK inhibitors blocking WNK kinase signaling.

We report here structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK (STE20/SPS1-related proline/alanine-rich kinase) inhibitors. Abnormal activation of the signal cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK and NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension, and therefore inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for antihypertensive drugs. Based on the structure of lead compound 2, we examined the SAR of N-(4-phenoxyphenyl)benzamide derivatives, and developed compound 20l as a potent SPAK inhibitor.

Characterization of a novel compound that promotes myogenesis via Akt and transcriptional co-activator with PDZ-binding motif (TAZ) in mouse C2C12 cells.

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in the regulation of cell proliferation and differentiation. TAZ activity changes in response to the cellular environment such as mechanic and nutritional stimuli, osmolarity, and hypoxia. To understand the physiological roles of TAZ, chemical compounds that activate TAZ in cells are useful as experimental reagents.

Prostaglandin E and its receptor EP2 trigger signaling that contributes to YAP-mediated cell competition.

Cell competition is a biological process by which unfit cells are eliminated from "cell society." We previously showed that cultured mammalian epithelial Madin-Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by "normal" MDCK cells. However, the molecular mechanism underlying the elimination of active YAP-expressing cells was unknown.

Structural development of a type-1 ryanodine receptor (RyR1) Ca-release channel inhibitor guided by endoplasmic reticulum Ca assay.

Type-1 ryanodine receptor (RyR1) is a calcium-release channel localized on sarcoplasmic reticulum (SR) of the skeletal muscle, and mediates muscle contraction by releasing Ca from the SR. Genetic mutations of RyR1 are associated with skeletal muscle diseases such as malignant hyperthermia and central core diseases, in which over-activation of RyR1 causes leakage of Ca from the SR. We recently developed an efficient high-throughput screening system based on the measurement of Ca in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor.

Class IIb HDAC Inhibition Enhances the Inhibitory Effect of Am80, a Synthetic Retinoid, in Prostate Cancer.

Combination therapy is often an effective strategy to treat cancer. In this study, we examined the growth-inhibitory effects of Am80 (tamibarotene), a specific retinoic acid receptor (RAR) α/β agonist, in combination with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), or a DNA methyl transferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine, on androgen receptor (AR)-positive and AR-negative prostate cancer cell lines (LNCaP and PC-3, respectively). We found that the combination therapy of SAHA and Am80 showed an enhanced growth-inhibitory effect on LNCaP cells.

Structure-activity relationship of novel (benzoylaminophenoxy)phenol derivatives as anti-prostate cancer agents.

The androgen receptor (AR) is a ligand-inducible transcription factor belonging to the nuclear receptor superfamily, and is a target molecule for development of drugs to treat prostate cancer. However, AR antagonists in clinical use, such as flutamide (3a) and bicalutamide (4), encounter resistance after several years of hormone therapy, predominantly due to mutations of AR. Thus, although some new-generation AR antagonists have been developed, novel types of AR antagonists are still required to treat drug-resistant prostate cancer.

Chemical compounds that suppress hypoxia-induced stress granule formation enhance cancer drug sensitivity of human cervical cancer HeLa cells.

In eukaryotic cells, when exposed to certain types of stress including hypoxia, eIF2α is phosphorylated by several kinases including protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK). Subsequently, protein translation is stopped and stress granules (SGs) are formed. Cancer cells form SGs under hypoxia.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca Measurement to Identify Inhibitors of Ryanodine Receptor Ca-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca concentrations in the endoplasmic reticulum (ER) ([Ca]).

Identification of selective inhibitors for diffuse-type gastric cancer cells by screening of annotated compounds in preclinical models.

Background: Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and poor patient prognosis. We have previously established an E-cadherin/p53 double conditional knockout (DCKO) mouse line as the first genetically engineered one, which morphologically and molecularly recapitulates human DGC. In this study, we explored low-molecular-weight drugs selectively eliminating mouse and human DGC cells.

Novel YAP1 Activator, Identified by Transcription-Based Functional Screen, Limits Multiple Myeloma Growth.

Yes-associated protein 1 (YAP1) interacts with numerous transcription factors, including TEA-domain family proteins (TEAD) and p73. YAP1 is negatively regulated by the tumor suppressor Hippo pathway. In human cancers, the deregulation of the Hippo pathway and YAP1 gene amplification lead to the activation of YAP1, which induces epithelial-mesenchymal transition (EMT) and drug resistance.

Development of WNK signaling inhibitors as a new class of antihypertensive drugs.

Pseudohypoaldosteronism type II (PHAII) is characterized by hyperkalemia and hypertension despite a normal glomerular filtration rate. Abnormal activation of the signal cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK (STE20/SPS1-related proline/alanine-rich kinase) and NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension. Thus, inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for a new class of antihypertensive drugs.

The mevalonate pathway regulates primitive streak formation via protein farnesylation.

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors.

Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation.

All trans-retinoic acid analogs promote cancer cell apoptosis through non-genomic Crabp1 mediating ERK1/2 phosphorylation.

All trans retinoic acid (atRA) is one of the most potent therapeutic agents, but extensive toxicity caused by nuclear RA receptors (RARs) limits its clinical application in treating cancer. AtRA also exerts non-genomic activities for which the mechanism remains poorly understood. We determine that cellular retinoic acid binding protein 1 (Crabp1) mediates the non-genomic activity of atRA, and identify two compounds as the ligands of Crabp1 to rapidly and RAR-independently activate extracellular signal regulated kinase 1/2 (ERK1/2).

A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs on Early Embryogenesis.

Mammalian fetal development is easily disrupted by exogenous agents, making it essential to test new drug candidates for embryotoxicity and teratogenicity. To standardize the testing of drugs that might be used to treat pregnant women, the U.S.

Design and synthesis of 4-benzyl-1-(2H)-phthalazinone derivatives as novel androgen receptor antagonists.

The androgen receptor (AR) plays important roles in multiple physiological functions, including differentiation, growth, and maintenance of male reproductive organs, and also has effects on hair and skin. In this paper, we report the synthesis of nonsteroidal AR antagonists having a 4-benzyl-1-(2H)-phthalazinone skeleton. Among the synthesized compounds, 11c with two ortho-substituents on the phenyl group potently inhibited SC-3 cell proliferation (IC50: 0.

A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells.

The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1-10 and GFP11 cells).