Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca2+ 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 Ca2+ 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. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

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. Structural design and development based on our previous hit compound, aryloxybenzanilide derivative 2, and the veterinary anthelmintic closantel (3) led to the discovery of compound 10 a as a potent SPAK inhibitor with reduced toxicity. Compound 10 a decreased the phosphorylation level of NCC in mouse kidney in vivo, and appears to be a promising lead compound for a new class of antihypertensive drugs.

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. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells.

Activation of ß2-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. Recent lines of evidence have suggested that EMT confers cancer cells with tumor-initiating ability. Therefore, selective targeting of EMT would lead to the development of effective therapeutic agents. In this study, using a chemical biology approach, we identified isoxsuprine, a ß2-adrenergic receptor (ß2-AR) agonist as a low-molecular-weight compound that interferes with the acquisition of mesenchymal phenotypes of oral cancer cells. Treatment of multiple types of oral cancer cells with isoxsuprine led to the downregulation of mesenchymal cell markers that was accompanied by reduced cell motility. Similar inhibitory effects were also observed for isoprenaline, a non-selective ß-adrenergic receptor (ß-AR) agonist. In addition, inhibition of cell migration upon treatment with isoxsuprine was reverted by a non-selective ß-AR antagonist, propranolol, and the CRISPR/Cas9 system-mediated deletion of the ß2-AR gene, suggesting that the effects exerted by isoxsuprine involved signals mediated by ß2-AR. In addition, in a subcutaneous xenograft model of oral cancer cells, the administration of isoxsuprine effectively suppressed primary tumor growth, suggesting ß2-AR signals to be a promising cancer therapeutic target for treatment of OSCC.

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. Many assay systems are currently available to evaluate the modulation of nuclear receptor functions, and are useful as screening tools in the discovery and development of new modulators. In this review, we cover the chemical screening methods for nuclear receptor modulators, focusing on assay methods and chemical libraries for screening. We include some recent examples of the discovery of nuclear receptor modulators.

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

First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide (2a) and bicalutamide (3), 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-(N-alkyl-arylcarboxamido)coumarins as AR antagonist candidates and evaluated their growth-inhibitory activity toward androgen-dependent SC-3 cells. Coumarinamides with a secondary amide bond did not show inhibitory activity, but their N-methylated derivatives exhibited AR-antagonistic activity. Especially, 19b and 31b were more potent than the lead compound 7b, which was comparable to hydroxyflutamide (2b). Conformational analysis showed that the inactive coumarinamides with a secondary amide bond have an extended structure with a trans-amide bond, while the active N-methylated coumarinamides have a folded structure with a cis-amide bond, in which the two aromatic rings are placed face-to-face. Docking study suggested that this folded structure is important for binding to AR. Selected coumarinamide derivatives showed AR-antagonistic activity toward LNCaP cells with T877A AR, and they had weak progesterone receptor (PR)-antagonistic activity. The folded coumarinamide structure appears to be a unique pharmacophore, different from those of conventional AR antagonists.

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. Compounds 20l is a promising candidate for a new class of antihypertensive drugs.

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. Kaempferol, TM-25659, and ethacridine are reported as TAZ activators. However, as each TAZ activator has a distinct property in cellular functions, additional TAZ activators are awaiting. We screened for TAZ activators and previously reported IB008738 as a TAZ activator that promotes myogenesis in C2C12 cells. In this study, we have characterized IBS004735 that was obtained in the same screening. IBS004735 also promotes myogenesis in C2C12 cells, but is not similar to IBS008738 in the structure. IBS004735 activates TAZ via Akt and has no effect on TAZ phosphorylation, which is the well-described key modification to regulate TAZ activity. Thus, we introduce IBS004735 as a novel TAZ activator that regulates TAZ in a yet unidentified mechanism.

Prostaglandin E2 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. Here, we used high-throughput chemical compound screening to identify cyclooxygenase-2 (COX-2) as a key molecule triggering cell competition. Our work shows that COX-2-mediated PGE2 secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase-cyclic AMP-PKA pathway that, in the presence of active YAP, induces E-cadherin internalization leading to apical extrusion. Thus, COX-2-induced PGE2 appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.

Structural development of a type-1 ryanodine receptor (RyR1) Ca2+-release channel inhibitor guided by endoplasmic reticulum Ca2+ 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 Ca2+ 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 Ca2+ from the SR. We recently developed an efficient high-throughput screening system based on the measurement of Ca2+ in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor. Here, we designed and synthesized a series of quinolone derivatives based on 1 as a lead compound. Derivatives bearing a long alkyl chain at the nitrogen atom of the quinolone ring and having a suitable substituent at the 7-position of quinolone exhibited potent RyR1 channel-inhibitory activity. Among the synthesized compounds, 14h showed more potent activity than dantrolene, a known RyR1 inhibitor, and exhibited high RyR1 selectivity over RyR2 and RyR3. These compounds may be promising leads for clinically applicable RyR1 channel inhibitors.

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/ß 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. Further studies with various HDAC isotype-selective inhibitors showed that SAHA and KD5170 (a selective class I and II HDAC inhibitor) each increased the RARα protein level in LNCaP cells. Our results indicate that the target of the enhancing effect belongs to the Class IIb HDACs, especially HDAC6. Dual targeting of Class IIb HDAC and RARα may be a candidate therapeutic strategy for prostate cancer.

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. We previously reported a novel (benzoylaminophenoxy)phenol derivative 10a, which is structurally distinct from conventional AR antagonists. Here, we systematically examined the structure-activity relationship of (benzoylaminophenoxy)phenol derivatives on the inhibitory activity on the prostate cancer cell proliferations. We found that the 4-[4-(benzoylamino)phenoxy]phenol backbone is important for anti-prostate cancer activity. Introduction of a small substituent at the 2 position of the central benzene ring (B ring) increases the activity. Among the synthesized compounds, 19a and 19b exhibited the most potent inhibitory activity toward dihydrotestosterone-induced proliferation of several androgen-dependent cell lines, SC-3 (wild-type AR), LNCaP (T877A AR), and 22Rv1 (H874Y AR), but interestingly also inhibited proliferation of AR-independent PC-3 cells. These compounds, which have a different pharmacophore from conventional AR antagonists, are promising drug candidates for the treatment of 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. SGs accumulate apoptosis-related molecules and play anti-apoptotic roles. Thus, hypoxia-induced SG formation contributes to drug resistance in cancer cells. For this reason, inhibition of SG formation is expected to be beneficial in cancer therapy. To prove this concept, chemical reagents that inhibit SG formation are required as experimental tools. We searched for chemical compounds that suppress SG formation and identified that ß-estradiol, progesterone, and stanolone (hereafter described as EPS) inhibit SG formation in human cervical cancer HeLa cells. As it turned out, EPS block PKR but not PERK, thus fail to suppress SG formation in most cancer cells, where SGs are formed via PERK. Nevertheless, in this study, we used HeLa cells as a model and demonstrated that EPS block hypoxia-induced SG formation in HeLa cells and consequently reduce drug resistance that HeLa cells acquire under hypoxia. Our findings support that inhibition of SG formation is a useful method to control cancers.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-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 Ca2+ 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 Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

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. METHODS: We derived mouse gastric cancer (GC) cell lines from DGC of the DCKO mice demonstrating enhanced tumourigenic activity in immunodeficient mice and acquired tolerance to cytotoxic anti-cancer agents. RESULTS: We performed a synthetic lethal screening of 1535 annotated chemical compounds, and identified 27 candidates selectively killing the GC cell lines. The most potent drug mestranol, an oestrogen derivative, and other oestrogen receptor modulators specifically attenuated cell viability of the GC cell lines by inducing apoptosis preceded by DNA damage. Moreover, mestranol could significantly suppress tumour growth of the GC cells subcutaneously transplanted into nude mice, consistent with longer survival time in the female DCKO mice than in the male. Expectedly, human E-cadherin-mutant and -low gastric cancer cells showed higher susceptibility to oestrogen drugs in contrast to E-cadherin-intact ones in vitro and in vivo. CONCLUSIONS: These findings may lead to the development of novel therapeutic strategies targeting DGC.

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. YAP1 inhibitors are expected to be useful in cancer therapy. On the other hand, in certain cancers, YAP1 upregulates p73-dependent gene transcription and behaves as a tumor suppressor. Moreover, as YAP1 regulates self-renewal and differentiation of tissue stem cells and plays an important role in tissue homeostasis, YAP1 activators may contribute to the regenerative medicine. With this in our mind, we screened for YAP1 activators by using human retinal pigment epithelial ARPE-19 cells expressing the TEAD-responsive fluorescence reporter under the coexpression of YAP1. From an extensive chemical compound library (n = 18,606) 47 candidate YAP1 activators were identified. These compounds were characterized to determine whether this assay provides bona fide YAP1 activators. Importantly, one YAP1 activator was effective against the human multiple myeloma IM-9 cells and chronic myeloid leukemia K562 cells.Implications: YAP1 activation limits growth, induces apoptosis, and may be useful at suppressing hematological cancers. Mol Cancer Res; 16(2); 197-211. ©2017 AACR.

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. In this study, we developed novel inhibitors of this signal cascade from the 9-aminoacridine lead compound 1, one of the hit compounds obtained by screening our chemical library for WNK-SPAK binding inhibitors. Among the synthesized acridine derivatives, several acridine-3-amide and 3-urea derivatives, such as 10 (IC50: 6.9µM), 13 (IC50: 2.6µM), and 20 (IC50: 4.8µM), showed more potent inhibitory activity than the lead compound 1 (IC50: 15.4µM). Compounds 10 and 20 were confirmed to inhibit phosphorylation of NCC in vivo.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy.

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 µM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

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. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.