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Electrophoretic mobility shift assay (EMSA) technology has been widely employed for the analysis of transcription factors such as Forkhead box protein M1 (FOXM1). However, the application of high-throughput screening (HTS) in performing, such analyses are limited as it uses time consuming electrophoresis procedure and radioisotopes. In this study, we developed a FOXM1-DNA binding domain (DBD) binding assay based on time-resolved fluorescence energy transfer (TR-FRET) that enables HTS for the inhibitors of FOXM1-DNA interaction. This assay was robust, highly reproducible and could be easily miniaturized into 384-well plate format. The signal-to-background (S/B) ratio and Z' factor were calculated as 7.46 and 0.74, respectively, via a series of optimization of the assay conditions. A pilot library screening of 1019 natural compounds was performed using the FOXM1-DBD binding assay. Five hit compounds, namely, AC1LXM, BRN5, gangaleoidin, leoidin, and roemerine were identified as the inhibitors of FOXM1. In a cell viability assay, it was demonstrated that cell proliferation of FOXM1 overexpressed cell lines was suppressed in cell lines such as MDA-MB-231 and MCF-7 by five hit compounds. These results indicate that developed FOXM1-DBD binding assay can be applied to highly efficiency HTS of compound libraries.

Proper targeting of the βPAK-interacting exchange factor (βPIX)/G protein-coupled receptor kinase-interacting target protein (GIT) complex into distinct cellular compartments is essential for its diverse functions including neurite extension and synaptogenesis. However, the mechanism for translocation of this complex is still unknown. In the present study, we reported that the conventional kinesin, called kinesin-1, can transport the βPIX/GIT complex. Additionally, βPIX bind to KIF5A, a neuronal isoform of kinesin-1 heavy chain, but not KIF1 and KIF3. Mapping analysis revealed that the tail of KIF5s and LZ domain of βPIX were the respective binding domains. Silencing KIF5A or the expression of a variety of mutant forms of KIF5A inhibited βPIX targeting the neurite tips in PC12 cells. Furthermore, truncated mutants of βPIX without LZ domain did not interact with KIF5A, and were unable to target the neurite tips in PC12 cells. These results defined kinesin-1 as a motor protein of βPIX, and may provide new insights into βPIX/GIT complex-dependent neuronal pathophysiology. [BMB Reports 2021; 54(7): 380-385].

G protein-coupled receptor kinase 5 (GRK5) has been considered as a potential target for the treatment of heart failure as it has been reported to be an important regulator of pathological cardiac hypertrophy. To discover novel scaffolds that selectively inhibit GRK5, we have identified a novel small molecule inhibitor of GRK5, KR-39038 [7-((3-((4-((3-aminopropyl)amino)butyl)amino)propyl) amino)-2-(2-chlorophenyl)-6-fluoroquinazolin-4(3H)-one]. KR-39038 exhibited potent inhibitory activity (IC50 value=0.02 μM) against GRK5 and significantly inhibited angiotensin II-induced cellular hypertrophy and HDAC5 phosphorylation in neonatal cardiomyocytes. In the pressure overload-induced cardiac hypertrophy mouse model, the daily oral administration of KR-39038 (30 mg/kg) for 14 days showed a 43% reduction in the left ventricular weight. Besides, KR-39038 treatment (10 and 30 mg/kg/ day, p.o.) showed significant preservation of cardiac function and attenuation of myocardial remodeling in a rat model of chronic heart failure following coronary artery ligation. These results suggest that potent GRK5 inhibitor could effectively attenuate both cardiac hypertrophy and dysfunction in experimental heart failure, and KR-39038 may be useful as an effective GRK5 inhibitor for pharmaceutical applications.

Motivation Blockade of the human ether-à-go-go-related gene (hERG) channel by small compounds causes a prolonged QT interval that can lead to severe cardiotoxicity and is a major cause of the many failures in drug development. Thus, evaluating the hERG-blocking activity of small compounds is important for successful drug development. To this end, various computational prediction tools have been developed, but their prediction performances in terms of sensitivity and negative predictive value (NPV) need to be improved to reduce false negative predictions. Results We propose a computational framework, DeepHIT, which predicts hERG blockers and non-blockers for input compounds. For the development of DeepHIT, we generated a large-scale gold-standard dataset, which includes 6632 hERG blockers and 7808 hERG non-blockers. DeepHIT is designed to contain three deep learning models to improve sensitivity and NPV, which, in turn, produce fewer false negative predictions. DeepHIT outperforms currently available tools in terms of accuracy (0.773), MCC (0.476), sensitivity (0.833) and NPV (0.643) on an external test dataset. We also developed an in silico chemical transformation module that generates virtual compounds from a seed compound, based on the known chemical transformation patterns. As a proof-of-concept study, we identified novel urotensin II receptor (UT) antagonists without hERG-blocking activity derived from a seed compound of a previously reported UT antagonist (KR-36676) with a strong hERG-blocking activity. In summary, DeepHIT will serve as a useful tool to predict hERG-induced cardiotoxicity of small compounds in the early stages of drug discovery and development. Availability and implementation https://bitbucket.org/krictai/deephit and https://bitbucket.org/krictai/chemtrans Supplementary information Supplementary data are available at Bioinformatics online.

Melanin-concentrating hormone receptor 1 (MCHR1) has been a target for appetite suppressants, which are helpful in treating obesity. However, it is challenging to develop an MCHR1 antagonist because its binding site is similar to that of the human Ether-à-go-go-Related Gene (hERG) channel, whose inhibition may cause cardiotoxicity. Most drugs developed as MCHR1 antagonists have failed in clinical development due to cardiotoxicity caused by hERG inhibition. Machine learning-based prediction models can overcome these difficulties and provide new opportunities for drug discovery. In this study, we identified KRX-104130 with potent MCHR1 antagonistic activity and no cardiotoxicity through virtual screening using two MCHR1 binding affinity prediction models and an hERG-induced cardiotoxicity prediction model. In addition, we explored other possibilities for expanding the new indications for KRX-104130 using a transcriptome-based drug repositioning approach. KRX-104130 increased the expression of low-density lipoprotein receptor (LDLR), which induced cholesterol reduction in the gene expression analysis. This was confirmed by comparison with gene expression in a nonalcoholic steatohepatitis (NASH) patient group. In a NASH mouse model, the administration of KRX-104130 showed a protective effect by reducing hepatic lipid accumulation, liver injury, and histopathological changes, indicating a promising prospect for the therapeutic effect of NASH as a new indication for MCHR1 antagonists.

This study explored the radioprotective effects and possible underlying mechanisms of KR-31831 against radiation-induced injury in a mouse model. KR-31831 (30 and 50 mg/kg) was administered to mice 24 h and 30 min before exposure to a single lethal or sublethal dose of whole-body irradiation (WBI) (7 or 4 Gy, respectively). These animals were then evaluated for changes in mortality, various hematological and biochemical parameters, and histological features in response to these treatments. In addition, RNA sequencing was used to profile the radiation-induced transcriptomic response in the bone marrow cells. The results showed that KR-31831 dose-dependently prolonged the 30-day survival period and prevented damage to radiation-sensitive organs, such as the intestine and testis, in response to WBI. Damage to the hematopoietic system was also notably improved in the KR-31831-treated mice, as evidenced by an increase in bone marrow and peripheral blood cells, as well as recovery of the histopathological characteristics of the bone marrow. These protective effects were achieved, at least in part, via the suppression of radiation-induced increases in apoptotic cell death and erythropoietin levels in the plasma. Furthermore, the gene expression profiles of the bone marrow cells of the WBI-treated mice suggested that KR-31831 upregulates the expression of the genes involved in regulating apoptosis and modulating the immune response, both of which are required for protecting the bone marrow. These results suggest the potential therapeutic efficacy of KR-31831 for protection against radiation-induced injury.

The synthesis and biological evaluation as potential urotensin-II receptor antagonists of a series of 5-arylfuran-2-carboxamide derivatives 1, bearing a 4-(3-chloro-4-(piperidin-4-yloxy)benzyl)piperazin-1-yl group, are described. The results of a systematic SAR investigation of furan-2-carboxamides with C-5 aryl groups possessing a variety of aryl ring substituents led to identification of the 3,4-difluorophenyl analog 1y as a highly potent UT antagonist with an IC50 value of 6 nM. In addition, this substance was found to display high metabolic stability, and low hERG inhibition and cytotoxicity, and to have an acceptable PK profile.

Motivation Poor metabolic stability leads to drug development failure. Therefore, it is essential to evaluate the metabolic stability of small compounds for successful drug discovery and development. However, evaluating metabolic stability in vitro and in vivo is expensive, time-consuming and laborious. In addition, only a few free software programs are available for metabolic stability data and prediction. Therefore, in this study, we aimed to develop a prediction model that predicts the metabolic stability of small compounds. Results We developed a computational model, PredMS, which predicts the metabolic stability of small compounds as stable or unstable in human liver microsomes. PredMS is based on a random forest model using an in-house database of metabolic stability data of 1917 compounds. To validate the prediction performance of PredMS, we generated external test data of 61 compounds. PredMS achieved an accuracy of 0.74, Matthew’s correlation coefficient of 0.48, sensitivity of 0.70, specificity of 0.86, positive predictive value of 0.94 and negative predictive value of 0.46 on the external test dataset. PredMS will be a useful tool to predict the metabolic stability of small compounds in the early stages of drug discovery and development. Availability and implementation The source code for PredMS is available at https://bitbucket.org/krictai/predms, and the PredMS web server is available at https://predms.netlify.app. Supplementary information Supplementary data are available at Bioinformatics online.

Motivation Identification of blood–brain barrier (BBB) permeability of a compound is a major challenge in neurotherapeutic drug discovery. Conventional approaches for BBB permeability measurement are expensive, time-consuming and labor-intensive. BBB permeability is associated with diverse chemical properties of compounds. However, BBB permeability prediction models have been developed using small datasets and limited features, which are usually not practical due to their low coverage of chemical diversity of compounds. Aim of this study is to develop a BBB permeability prediction model using a large dataset for practical applications. This model can be used for facilitated compound screening in the early stage of brain drug discovery. Results A dataset of 7162 compounds with BBB permeability (5453 BBB+ and 1709 BBB-) was compiled from the literature, where BBB+ and BBB- denote BBB-permeable and non-permeable compounds, respectively. We trained a machine learning model based on Light Gradient Boosting Machine (LightGBM) algorithm and achieved an overall accuracy of 89%, an area under the curve (AUC) of 0.93, specificity of 0.77 and sensitivity of 0.93, when 10-fold cross-validation was performed. The model was further evaluated using 74 central nerve system compounds (39 BBB+ and 35 BBB-) obtained from the literature and showed an accuracy of 90%, sensitivity of 0.85 and specificity of 0.94. Our model outperforms over existing BBB permeability prediction models. Availabilityand implementation The prediction server is available at http://ssbio.cau.ac.kr/software/bbb.

Drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (hERG). The blockage leads to long QT syndrome (LQTS), which is a severe life-threatening cardiac side effect. Therefore, a virtual screening method to predict drug-induced hERG-related cardiotoxicity could facilitate drug discovery by filtering out toxic drug candidates. In this study, we generated a reliable hERG-related cardiotoxicity dataset composed of 2130 compounds, which were carried out under constant conditions. Based on our dataset, we developed a computational hERG-related cardiotoxicity prediction model. The neural network model achieved an area under the receiver operating characteristic curve (AUC) of 0.764, with an accuracy of 90.1%, a Matthews correlation coefficient (MCC) of 0.368, a sensitivity of 0.321, and a specificity of 0.967, when ten-fold cross-validation was performed. The model was further evaluated using ten drug compounds tested on guinea pigs and showed an accuracy of 80.0%, an MCC of 0.655, a sensitivity of 0.600, and a specificity of 1.000, which were better than the performances of existing hERG-toxicity prediction models. The neural network model can predict hERG-related cardiotoxicity of chemical compounds with a high accuracy. Therefore, the model can be applied to virtual high-throughput screening for drug candidates that do not cause cardiotoxicity. The prediction tool is available as a web-tool at http://ssbio.cau.ac.kr/CardPred .

The pathophysiological implications of binding of urotensin II (U-II) to urotensin II receptor (UT) in pulmonary arterial hypertension (PAH) have been proposed recently. Besides high expression of U-II in experimental models and patients with PAH, U-II has been shown to increase proliferation of pulmonary vascular smooth muscle cells and inflammatory responses, which were critical for PAH pathophysiology. However, the direct role of the urotensinergic system in the pathogenesis of PAH is yet to be understood. The aim of the present study was to determine whether a novel UT antagonist, KR36676, attenuates the pathophysiological progression of PAH in an animal model of PAH. PAH was induced by a single subcutaneous injection of monocrotaline (MCT, 60mg/kg) in rats. All the animals received KR36676 (30mg/kg/day) or vehicle by oral gavage. Three weeks after MCT-injection, changes in hemodynamic parameters, extent of right ventricular hypertrophy, fibrosis and pulmonary vascular remodeling, and degree of protein expression were determined. Oral administration of KR36676 effectively decreased the MCT-induced increase in right ventricular systolic pressure, hypertrophy and fibrosis. Furthermore, wall thickness of pulmonary arterioles, proliferation of pulmonary vascular cells, and inflammatory response significantly decreased in the KR36676-treated group following MCT injection compared to that in the MCT-treated vehicle group. These preventive effects of KR36676 are mediated, at least in part, by suppression of ERK1/2 and NF-κB signaling pathways. The novel UT antagonist, KR36676, effectively prevented MCT-induced PAH progression and pulmonary vascular remodeling in rat model. Our findings support the therapeutic efficacy of UT antagonist in PAH prevention and elucidate the possible underlying mechanisms of action.

Urotensin II (UII) is a neural hormone that induces cardiac hypertrophy and may be involved in the pathogenesis of cardiac remodeling and heart failure. Hypertrophy has been linked to histone deacetylase 5 (HDAC5) phosphorylation and nuclear factor κB (NF-κB) translocation, both of which are predominantly mediated by G protein-coupled receptor kinase 5 (GRK5). In the present study, we found that UII rapidly and strongly stimulated nuclear export of HDAC5 and nuclear import of NF-κB in H9c2 cells overexpressing the urotensin II receptor (H9c2UT). Hence, we hypothesized that GRK5 and its signaling pathway may play a role in UII-mediated cellular hypertrophy. H9c2UT cells were transduced with a GRK5 small hairpin RNA interference recombinant lentivirus, resulting in the down-regulation of GRK5. Under UII stimulation, reduced levels of GRK5 in H9c2UT cells led to suppression of UII-mediated HDAC5 phosphorylation and activation of the NF-κB signaling pathway. In contrast, UII-mediated activations of ERK1/2 and GSK3α/β were not affected by down-regulation of GRK5. In a cellular hypertrophy assay, down-regulation of GRK5 significantly suppressed UII-mediated hypertrophy of H9c2UT cells. Furthermore, UII-mediated cellular hypertrophy was inhibited by amlexanox, a selective GRK5 inhibitor, in H9c2UT cells and neonatal cardiomyocytes. Our results suggest that GRK5 may be involved in a UII-mediated hypertrophic response via activation of NF-κB and HDAC5 at least in part by ERK1/2 and GSK3α/β-independent pathways.

A dual readout assay based on fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) exhibits many advantages over single assay technology in terms of screening quality and efficiency. In this study, we developed a dual readout assay combining FP and TR-FRET to identify ribosomal S6 kinase 1 (RSK1) inhibitors. This dual readout assay can monitor both FP and TR-FRET signals from a single RSK1 kinase reaction by using the immobilized metal affinity for phosphochemical (IMAP)-based assay. The Z' value and signal to background (S/B) ratio were 0.85 and 4.0 using FP, and 0.79 and 10.6 using TR-FRET, which led to performance of a pilot library screening against the drug repositioning set consisting of 2320 compounds with a reasonable reproducibility. From this screening, we identified 16 compounds showing greater than 50% inhibition against RSK1 for both FP and TR-FRET; 6 compounds with greater than 50% inhibition only for FP; and 4 compounds with greater than 50% inhibition only for TR-FRET. In a cell-based functional assay to validate the hit compounds, 10 compounds identified only in a single assay had little effect on the RSK-mediated phosphorylation of liver kinase B1, whereas 5 compounds showing greater than 80% inhibition for both FP and TR-FRET reduced the phosphorylation of liver kinase B1. These results demonstrate that the dual readout assay can be used to identify hit compounds by subsequently monitoring both FP and TR-FRET signals from one RSK1 reaction.

Inhibition of IKK-β (inhibitor of nuclear factor kappa-B kinase subunit beta) has been broadly documented as a promising approach for treatment of acute and chronic inflammatory diseases, cancer, and autoimmune diseases. Recently, we have identified a novel class of thiazolidine-2,4-diones as structurally novel modulators for IKK-β. Herein, we report a hit optimization study via analog synthesis strategy aiming to acquire more potent derivative(s), probe the structure activity relationship (SAR), and get reasonable explanations for the elicited IKK-β inhibitory activities though an in silico docking simulation study. Accordingly, a new series of eighteen thiazolidine-2,4-dione derivatives was rationally designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as noteworthy IKK-β potential modulators. Successfully, new IKK-β potent modulators were obtained, including the most potent analog up-to-date 7m with IC50 value of 260 nM. A detailed structure activity relationship (SAR) was discussed and a mechanistic study for 7m was carried out indicating its irreversible inhibition mode with IKK-β (Kinact value = 0.01 (min−1). Furthermore, the conducted in silico simulation study provided new insights for the binding modes of this novel class of modulators with IKK-β.

DITMD (1, 3- Dioxolo[4,5-g] isoquinolinium 5, 6, 7, 8- tetrahydro- 4- methoxy- 6, 6- dimethyl- 5- [2- oxo- 2- (2-pyridinyl)ethyl] - iodide) is a natural product-like compound with a hydrocotarnine moiety. The aim of this study was to investigate the anticancer effects of DITMD including mitotic arrest, apoptosis, radiosensitization, and to further explore its possible mechanism. DITMD (3−30 µM) induced an obvious cell cycle delay at G2/M transition and apoptosis in HeLa cells. In a validation study, DITMD caused chromosome alignment defects and accumulation of mitotic markers such as polo-like kinase 1, cyclin B1, and phospho-histone H3. DITMD pre-treatment for 11 h also significantly decreased the cells’ survival after X-ray irradiation. In mechanism studies, DITMD inhibited the polo-box domain of polo-like kinase 1 but not the conserved kinase domain. Molecular modeling also suggests that DITMD binds at the phosphate group recognition site and inhibits the action on phospho-peptide ligands. In addition, DITMD was analyzed as a PLHSpT competitive inhibitor with an IC50 value of 2.1 μM and exhibited good selectivity against 105 distinct kinases. Taken together, these results indicate that DITMD induced chromosome alignment defects, apoptosis and radio-sensitization, and suggest that one mechanism underlying these anticancer effects involves inhibiting the polo-box domain-dependent functions of polo-like kinase 1.

In a search for novel molecules to treat inflammatory disorders, we identified several compounds with inhibitory action against the IKK2 enzyme using in silico methods. Based on the virtual hit of compounds 1 and 2, a novel series of 2-phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-one derivatives was designed, synthesized, and evaluated for IKK2 inhibitory activity. Among the synthesized derivatives, compounds 17f and 19f showed good IKK2 inhibitory potency, which have 4-carboxaminophenyl on the 2-furan ring and a methoxy group on the phenylimino moiety at the 2-position of the core structure. The most potent compound was 2-(2,4-dimethoxyphenyl)imino-5((5(4-carboxaminophenyl)furan-2-yl)methylene)thiazolidin-4-one (19f, IC50 = 0.94 μM), which represents a synergic effect of the two virtual hit compounds against IKK2. We also identified compounds showing inhibitory activities against interleukin (IL)-17, CCK-8, and tumor necrosis factor-alpha (TNF-α), which are NF-κB-dependent pro-inflammatory cytokine mediators.

Background and Purpose Blockade of the actions of urotensin-II (U-II) mediated by the urotensin (UT) receptor should improve cardiac function and prevent cardiac remodelling in cardiovascular disease. Here, we have evaluated the pharmacological properties of the recently identified UT receptor antagonist, 2-(6,7-dichloro-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-methyl-N-(2-(pyrrolidin-1-yl)-1-(4-(thiophen-3-yl)phenyl) ethyl)acetamide (KR36676). Experimental Approach Pharmacological properties of KR36676 were studied in a range of in vitro assays (receptor binding, calcium mobilization, stress fibre formation, cellular hypertrophy) and in vivo animal models such as cardiac hypertrophy induced by transverse aortic constriction (TAC) or myocardial infarction (MI). Key Results KR36676 displayed high binding affinity for the UT receptor (Ki: 0.7 nM), similar to that of U-II (0.4 nM), and was a potent antagonist at that receptor (IC50: 4.0 nM). U-II-induced stress fibre formation and cellular hypertrophy were significantly inhibited with low concentrations of KR36676 (≥0.01 μM). Oral administration of KR36676 (30 mg·kg−1) in a TAC model in mice attenuated cardiac hypertrophy and myocardial fibrosis. Moreover, KR36676 restored cardiac function and myocyte size in rats with MI-induced cardiac hypertrophy. Conclusions and Implications A highly potent UT receptor antagonist exerted anti-hypertrophic effects not only in infarcted rat hearts but also in pressure-overloaded mouse hearts. KR36676 could be a valuable pharmacological tool in elucidating the complicated physiological role of U-II and UT receptors in cardiac hypertrophy.

Members of a series of benzo[b]thiophene-2-carboxamide derivatives, possessing an N-(1-(3-bromo-4-(piperidin-4-yloxy)benzyl)piperidin-4-yl) group, were synthesized and evaluated as urotensin-II receptor antagonists. The results show that these substances have potent UT binding affinities. Observations made in a systematic SAR investigation of the effects of a variety of substituents (R(1) and R(2)) at the 5- and 6-positions in the benzo[b]thiophene-2-carboxamide moiety on UT binding affinities led to identification of the 5-cyano analog 7f as a highly potent UT antagonist with an IC50 value of 25nM. Despite having a good metabolic stability, 7f is a potent inhibitor of CYP isozyme and displays an unsuitable PK profile.

The kinase known as IKK-β activates NF-κB signaling pathway leading to expression of several genes contributing to inflammation, immune response, and cell proliferation. Modulation of IKK-β kinase activity could be useful for treatment and management of such diseases. Starting from a discovered weakly active hit compound, twenty four thiazolidinedione-scaffold based chemical entities belonging to five series have been designed, synthesized and evaluated as potential IKK-β modulators. Among them, compounds 6q, 6r and 6u showed low micromolar IC50 values while compounds 6v, 6w, and 6x elicited submicromolar IC50 values equal to 0.4, 0.7 and 0.9 μM respectively. These submicromolar IC50 values are 243, 139 and 105 folds the value of the reported IC50 of the starting hit compound. Kinetic study of compounds 6v and 6w confirmed this class of modulators as irreversible inhibitors. LPS-treated RAW 264.7 macrophages proved the anti-inflammatory activity of compounds 6q and 6v. Assay of hERG inhibition demonstrated a safe profile of compound 6q suggesting it as a lead for further development of IKK-β modulators.

Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z' factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z' factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.

The preparation and SAR profile of thieno[3,2-b]pyridinyl urea derivatives as novel and potent urotensin-II receptor antagonists are described. An activity optimization study, probing the effects of substituents on thieno[3,2-b]pyridinyl core and benzyl group of the piperidinyl moiety, led to the identification of p-fluorobenzyl substituted thieno[3,2-b]pyridinyl urea 6n as a highly potent UT antagonist with an IC50 value of 13nM. Although 6n displays good metabolic stability and low hERG binding activity, it has an unacceptable oral bioavailability.

The popular screening method for urotensin (UT) receptor antagonists is to measure the intracellular calcium concentration with a calcium-sensitive fluorescent dye. This assay format has an inherent limitation on the problem related to the fluorescence interference as it involves fluorescent dyes. In the present study, a label-free assay for the screening of UT receptor antagonists was developed by using dynamic mass redistribution (DMR) assay based on label-free optical biosensor. The addition of urotensin II (UII) stimulated a DMR profile to HEK293 cells stably expressing the human UT receptor (HEK293UT cells) but not on parental cells. The EC50 value of UII in label-free assay was 4.58 nM, which is very similar to that in conventional calcium mobilization assay (4.15 nM). Compared with the calcium mobilization assay for UII (Z′ factor, 0.77), the current label-free assay presented improved Z′ factor (0.81), with a relatively similar S/B ratio (28.0 and 25.6, respectively). The known high-affinity UT receptor antagonists, SB657510, GSK562590, and urantide, exhibited comparable IC50 values but rather less potent in the DMR assay than in calcium mobilization. Our DMR assay was able to present various functional responses, including inverse agonism in SB657510 and GSK1562590 as well as partial agonism in urantide. Moreover, the DMR assay exerted the stable antagonist window upon the minimal agonist stimulus. These results suggest that the label-free cell-based UT receptor assay can be applicable to evaluate the various functional activities of UT receptor-related drug candidates.

Polymyxin B is widely used antibiotic in the clinic for resistant Gram-negative infections. In addition, polymyxin B-immobilized hemoperfusion cartridge has been used for endotoxin removal therapy in patients with septic shock. The aim of this study was to investigate the anti-fibrotic and anti-cellular hypertrophic effects of polymyxin B, and further to explore its possible mechanism. Polymyxin B (3, 10 μM) significantly inhibited stress fiber formation induced by angiotensin II (Ang II) in rat heart-derived H9c2 cells. Furthermore, polymyxin B (1 - 10 μM) showed a potent inhibitory effect on Ang II-induced cellular hypertrophy in H9c2 cells. Under the mechanism study, the inhibitory activities of polymyxin B against kinases involved in cellular hypertrophy such as AKT1, CAMK, GRK5, GSK3β, MLCK, PKC, PKD2, AMPK, ROCK2, p70S6K, SGK1were evaluated. Polymyxin B possesses a potent G protein related kinase 5 (GKR5) inhibitory activity with IC50 value of 1.1 μM, and has an ATP non-competitive inhibitory mode. Taken together, these results indicate that polymyxin B alleviates Ang II-induced stress fiber formation and cellular hypertrophy, and propose that one mechanism underlying these effects involves inhibition of the GRK5 pathway.