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Background Acute oral toxicity of drug candidates can lead to drug development failure; thus, predicting the acute oral toxicity of small compounds is important for successful drug development. However, evaluation of the acute oral toxicity of small compounds considered in the early stages of drug discovery is limited because of cost and time. Here, we developed a computational framework, PredAOT, that predicts the acute oral toxicity of small compounds in mice and rats. Methods PredAOT is based on multiple random forest models for the accurate prediction of acute oral toxicity. A total of 6226 and 6238 compounds evaluated in mice and rats, respectively, were used to train the models. Results PredAOT has the advantage of predicting acute oral toxicity in mice and rats simultaneously, and its prediction performance is similar to or better than that of existing tools. Conclusion PredAOT will be a useful tool for the quick and accurate prediction of the acute oral toxicity of small compounds in mice and rats during drug development.

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.

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 .

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.

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.

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.

G-protein-coupled receptor kinase (GRK)-2 and -5 are emerging therapeutic targets for the treatment of cardiovascular disease. In our efforts to discover novel small molecules to inhibit GRK-2 and -5, a class of compound based on 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine was identified as a novel hit by high throughput screening campaign. Structural modification of parent benzoxazole scaffolds by introducing substituents on phenyl displayed potent inhibitory activities toward GRK-2 and -5.

Salvianolic acid A, an active compound present in Salvia miltiorrhiza, is a phenolic carboxylic acid derivative, ((2R)-3-(3,4-Dihydroxyphenyl)-2-[(E)-3-[2-[(E)-2-(3,4-dihydroxyphenyl) ethenyl]-3,4-dihydroxyphenyl] prop-2-enoyl]oxypropanoic acid). The present study was performed to investigate the underlying mechanisms of anti-inflammatory effects with salvianolic acid A, specially focused on nuclear factor κB (NF-κB) signaling pathway by targeting the IκB kinase β (IKKβ). The effect of salvianolic acid A for IKKβ activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The underlying mechanisms of salvianolic acid A were examined using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. IKKβ studies based on IMAP-TR-FRET showed that salvianolic acid A possesses a potent IKKβ inhibitory activity with Ki value of 3.63 μM in an ATP-noncompetitive manner. Pretreatment with salvianolic acid A (10, 30 μM) decreased LPS-induced expression of iNOS and COX-2, thereby inhibiting production of nitric oxide and prostaglandin E(2), respectively. In addition, salvianolic acid A (10, 30 μM) also attenuated the LPS-induced IκBα phosphorylation and degradation, and NF-κB translocation. These results suggest that salvianolic acid A modulates NF-κB-dependent inflammatory pathways through IKKβ inhibition and these anti-inflammatory effects will aid in understanding the pharmacology and mode of action of salvianolic acid A.

Compounds containing 2-arybenzimidazole ring systems linked to arylpiperidines were synthesized and evaluated as MCH-R1 antagonists. The results of structure–activity relationship studies led to the identification of compound 4c as a potent MCH-R1 antagonist (IC50 = 1 nM). This compound also has good metabolic stability, and favorable pharmacokinetic and brain penetration properties. However 4c was found to be potent inhibitor of the hERG potassium channel.

The time-resolved fluorescence technique based on melanin-concentrating hormone (MCH) receptor subtype-1 (MCH-1 receptor) binding assay was adopted to carry out a bioassay-guided fractionation of the methanol extract of Morus alba leaves. This fractionation and purification led to the isolation of two compounds identified as pheophorbide a methyl ester and 132(S)-hydroxypheophorbide a methyl ester. These active pheophorbides exhibited potent inhibitory activity in binding of europium-labeled MCH to the human recombinant MCH-1 receptor (IC50 value; 4.03 and 0.33 ?M, respectively). Besides binding activity, the pheophorbides inhibited MCH-mediated extracellular signal-regulated kinase (ERK) phosphorylation in Chinese hamster ovary cells expressing human MCH-1 receptor. These results suggest that pheophorbide a methyl ester and 132(S)-hydroxypheophorbide a methyl ester act as modulators of MCH-1 receptor and MCH-mediated ERK signaling. Copyright © 2009 John Wiley & Sons, Ltd.

Control of NF-kappaB release through the inhibition of IKKbeta has been identified as a potential target for the treatment of inflammatory and autoimmune diseases. We have employed structure based virtual screening scheme to identify lead like molecule from ChemDiv database. Homology models of IKKbeta enzyme were developed based on the crystal structures of four kinases. The efficiency of the homology model has been validated at different levels. Docking of known inhibitors library revealed the possible binding mode of inhibitors. Besides, the docking sequence analyses results indicate the responsibility of Glu172 in selectivity. Structure based virtual screening of ChemDiv database has yielded 277 hits. Top scoring 75 compounds were selected and purchased for the IKKbeta enzyme inhibition test. From the combined approach of virtual screening followed by biological screening, we have identified six novel compounds that can work against IKKbeta, in which 1 compound had highest inhibition rate 82.09% at 10 microM and IC(50) 1.76 microM and 5 compounds had 25.35-48.80% inhibition.

Blocking of poly(ADP-ribose) polymerase (PARP)-1 has been expected to protect the heart from ischemia-reperfusion injury. We have recently identified a novel and orally active PARP-1 inhibitor, KR-33889 [2-[methoxycarbonyl(4-methoxyphenyl)-methylsulfanyl]-1H-benzimidazole-4-carboxylic acid amide], and its major metabolite, KR-34285 [2-[carboxy(4-methoxyphenyl)methylsulfanyl]-1H-benzimidazole-4-carboxylic acid amide]. KR-33889 potently inhibited PARP-1 activity with an IC(50) value of 0.52 +/- 0.10 microM. In H9c2 myocardial cells, KR-33889 (0.03-30 microM) showed a resistance to hydrogen peroxide (2 mM)-mediated oxidative insult and significantly attenuated activation of intracellular PARP-1. In anesthetized rats subjected to 30 min of coronary occlusion and 3 h of reperfusion, KR-33889 (0.3-3 mg/kg i.v.) dose-dependently reduced myocardial infarct size. KR-34285, a major metabolite of KR-33889, exerted similar patterns to the parent compound with equi- or weaker potency in the same studies described above. In separate experiments for the therapeutic time window study, KR-33889 (3 mg/kg i.v.) given at preischemia, at reperfusion or in both, in rat models also significantly reduced the myocardial infarction compared with their respective vehicle-treated group. Furthermore, the oral administration of KR-33889 (1-10 mg/kg p.o.) at 1 h before occlusion significantly reduced myocardial injury. The ability of KR-33889 to inhibit PARP in the rat model of ischemic heart was confirmed by immunohistochemical detection of poly(ADP-ribose) activation. These results indicate that the novel PARP inhibitor KR-33889 exerts its cardioprotective effect in in vitro and in vivo studies of myocardial ischemia via potent PARP inhibition and also suggest that KR-33889 could be an attractive therapeutic candidate with oral activity for several cardiovascular disorders, including myocardial infarction.

The vascular relaxant effects on isolated rat aorta of amlodipine camsylates (S-, R-enantiomer, and R/ S-racemate), were evaluated and compared with that of S-amlodipine besylate. Furthermore, antihypertensive effects were measured in spontaneously hypertensive rat (SHR). The S-amlodipine camsylate concentration-dependently inhibited Ca2+-induced contraction of rat aorta with a very slow onset of action (reached its maximum at 3.5h; ED50: 1.50 ± 0.24 nM), having a potency 2-fold higher than those of R/S-amlodipine camsylate (ED50: 3.36 ± 0.91 nM) and similar to those of S-amlodipine besylate (ED50: 1.44 ± 0.14 nM), whereas the R-amlodipine camsylate has 590-fold lower vasorelaxant activity (ED50: 886.4 ± 49.7 nM). In SHR, orally administered S-amlodipine camsylate produced a dose-dependent and long-lasting (>10 h) antihypertensive effect (ED20: 0.89 mg/kg), with a potency 2-fold higher than those of R/S-amlodipine camsylate (ED20: 1.82 mg/kg) and similar to those of S-amlodipine besylate (ED20: 0.71 mg/kg). In contrast, the R-amlodipine camsylate has no effect even-though administrated high concentration 10 mg/kg. These results suggest that S-amlodipine camsylate has the potency and long-lasting antihypertensive activity as single enantiomer drug, and its antihypertensive effect is not significantly different to that of S-amlodipine besylate.

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.

The arising critical implications of G-protein-coupled receptor kinase 2 and 5 (GRK2 and 5) in heart failure have been attracting attention and inhibitors of GRK2 and 5 were considered as a novel therapeutic strategy to prevent and treat heart failure disease. Despite this large therapeutic potential, to date few GRK2 inhibitors have been identified and GRK5 inhibitors in public have been unknown. In our efforts to discover a novel scaffold with potent GRK2 and GRK5 inhibitory activities, we found that a series of 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amines have been identified as a new class of GRK2 and 5 kinase inhibitor. Structural modification of parent benzoxazole scaffolds by introducing substituents on phenyl displayed potent inhibitory activities toward GRK2 and 5. This research highlight discusses the processing and findings of the recent study.

Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. In our efforts to discover novel small molecules to inhibit EphA2, a class of compound based on 4-substituted quinazoline containing 7-(morpholin-2-ylmethoxy) group was identified as a novel hit by high throughput screening campaign. Structural modification of parent quinazoline scaffolds by introducing substituents on aniline displayed potent inhibitory activities toward EphA2.

Kamolonol (7-[[(1R,2R,4R,4aS,5R,8aS)-4-hydroxy-1,2,4a,5-tetramethyl-6-oxo-3,4,5,7,8,8a-hexahydro-2H-naphthalen-1-yl]methoxy]chromen-2-one) is a sesquiterpene coumarin and an active component of gum extracts from Ferula assafoetida. The aim of this study was to investigate the anti-fibrotic and anti-cellular hypertrophic effects of kamolonol, and further to explore its possible mechanism. Kamolonol (3–30 μM) significantly inhibited stress fiber formation induced by angiotensin II (Ang II) in rat heart-derived H9c2 cells. Furthermore, kamolonol (3–30 μM) showed a potent inhibitory effect on Ang II-induced cellular hypertrophy in H9c2 cells. Next, a Rho-associated kinase (ROCK) activity was measured because actin stress fiber formation and/or cellular hypertrophy are usually induced by the activation of ROCK. Rho-associated kinase 2 (ROCK2) studies using a time-resolved fluorescence resonance energy transfer (TR-FRET) showed that kamolonol possesses a potent ROCK2 inhibitory activity with IC50 values of 2.27 μM, and has an ATP-competitive inhibitory mode. In validation study, pretreatment of kamolonol (3–30 μM) for 2 h decreased the Ang II-induced phosphorylation of myosin phosphatase 1 (MYPT1) and myosin light chain 2 (MLC2). Taken together, these results indicate that kamolonol suppresses Ang II-induced stress fiber formation and cellular hypertrophy, and propose that one mechanism underlying these anti-fibrotic and anti-cellular hypertrophic effects involves inhibition of the ROCK-MLC pathway.

The discovery and optimization of novel pyrrolo[3,4-b]pyridin-7(6H)-one MCH-R1 antagonists are described. A systematic SAR study probing the effects of aryl-, benzyl- and arylthio-substituents at the 2-position of the pyrrolo[3,4-b]pyridin-7(6H)-ones led to identification of the 2-[(4-fluorophenyl)thio] derivative 7b as a highly potent MCH-R1 antagonist. This compound also has favorable pharmacokinetic properties along with a high metabolic stability and a minimal impact on CYP isoforms and hERG.

The arising critical implications of Rho kinase signaling in cardiovascular diseases have been attracting attention in the pharmacological potential of Rho kinase inhibitors. We identified a novel inhibitor of Rho kinase (2-(1H-indazole-5-yl)amino-4-methoxy-6-piperazino triazine; DW 1865) and characterized its effects in biochemical, cellular, tissue and animal based assays. DW 1865 potently inhibited the kinase activity of both Rho kinase 1 and Rho kinase 2 in vitro, and behaved as an ATP-competitive inhibitor. Interestingly, DW1865 was 10 times more potent in inhibiting Rho kinase activities than fasudil as a selective Rho kinase inhibitor. The activity of DW1865 was shown to be highly selective for Rho kinase in the panel assay of 13 other kinases. In the isolated vascular tissue study, DW1865 exerted vasorelaxation in phenylephrine- or 5-hydroxytriptamine-induced contraction in a concentration-dependent manner manner. In spontaneously hypertensive rats, administration of DW1865 caused a significant and dose-related reduction in blood pressure. Furthermore, DW1865 blocked angiotensin II-induced stress fiber formation and cellular hypertrophy in rat heart-derived H9c2 cells. Taken together, these results suggest that DW1865 is a highly selective and potent Rho kinase inhibitor that will alleviate the pathophysiological actions of Rho kinase such as stress fiber formation, cellular hypertrophy, and hypertension.

Baicalein is a flavonoid (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran4-one) and an active principle in Scutellaria baicalensis. The present study was performed to investigate the mechanisms underlying the anti-fibrotic effects of baicalein with a focus on Rho kinase (ROCK) inhibition. The effect of baicalein on ROCK activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The underlying mechanisms of baicalein were examined using angiotensin II-stimulated H9c2 cells. Rho kinase (ROCK1 and ROCK2) studies using IMAP-TR-FRET showed that baicalein possesses potent ROCK inhibitory activity with IC50 values of 6.55 and 2.82 µM, respectively. Pretreatment with baicalein (for 2 h) concentration-dependently decreased the angiotensin II-induced phosphorylation of myosin phosphatase (MYPT) and myosin light chain (MLC). Furthermore, baicalein also concentration-dependently suppressed actin stress fiber formation in angiotensin II-stimulated H9c2 cells. These results suggest that baicalein potently inhibits ROCK and that by so doing it modulates actin stress fiber formation. These anti-fibrotic effects of baicalein explain, at least in part, its pharmacology and mode of action.

Regulation of NF-κB activation through the inhibition of IKKβ has been identified as a promising target for the treatment of inflammatory and autoimmune disease such as rheumatoid arthritis. In order to develop novel IKKβ inhibitors, we performed high throughput screening toward around 8000 library compounds, and identified a hit compound containing rhodanine moiety. We modified the structure of hit compound to obtain potent and selective IKKβ inhibitors. Throughout hit-to-lead studies, we have discovered optimized compounds which possess blocking effect toward NF-κB activation and TNFα production in cell as well as inhibition activity against IKKβ. Among them, compound 3q showed the potent inhibitory activity against IKKβ, and excellent selectivity over other kinases such as p38α, p38β, JNK1, JNK2, and JNK3 as well as IKKα.

A novel series of 4-arylphthalazin-1(2H)-one linked to arylpiperidines were synthesized and evaluated as MCH-R1 antagonists. The results of an extensive SAR study probing the effects of substituents on the 4-arylphthalazin-1(2H)-one C-4 aryl group led to the identification of the 4-(3,4-difluorophenyl) derivative as a highly potent MCH-R1 inhibitor with an IC50 = 1 nM. However, further investigations showed that this substance has unacceptable pharmacokinetic properties including a high clearance and volume of distribution.

A serine–threonine kinase IKK-2 plays an important role in activation of NF-κB through phosphorylation of the inhibitor of NF-κB (IκB). As NF-κB is a major transcription factor that regulates genes responsible for cell proliferation and inflammation, development of selective IKK-2 inhibitors has been an important area of anti-inflammatory and anti-cancer research. In this study, to obtain active and selective IKK-2 inhibitors, various substituents were introduced to a piperidinyl aminopyrimidine core structure. The structure–activity relationship study indicated that hydrogen, methanesulfonyl, and aminosulfonyl groups substituted at the piperidinylamino functionality provide high inhibitory activity against IKK-2. Also, morpholinosulfonyl and piperazinosulfonyl group substituted at the aromatic ring attached to the aminopyrimidine core significantly increased the inhibitory activity of the resulting derivatives. In particular, compound 17 with the aromatic piperazinosulfonyl substituent showed the most potent (IC50 = 1.30 μM) and selective (over other kinases such as p38α, p38β, JNK1, JNK2, JNK3, and IKK-1) inhibitory activity against IKK-2.

Background Nuclear factor kappa B (NF-κB) is a chief nuclear transcription factor that controls the transcription of various genes; and its activation is tightly controlled by Inhibitor kappa B kinase (IKK). The irregular transcription of NF-κB has been linked to auto-immune disorders, cancer and other diseases. The IKK complex is composed of three units, IKKα, IKKβ, and the regulatory domain NEMO, of which IKKβ is well understood in the canonical pathway. Therefore, the inhibition of IKKβ by drugs forms the molecular basis for anti-inflammatory drug research. Results The ligand- and structure-based virtual screening (VS) technique has been applied to identify IKKβ inhibitors from the ChemDiv database with 0.7 million compounds. Initially, a 3D-QSAR pharmacophore model has been deployed to greatly reduce the database size. Subsequently, recursive partitioning (RP) and docking filters were used to screen the pharmacophore hits. Finally, 29 compounds were selected for IKKβ enzyme inhibition assay to identify a novel small molecule inhibitor of IKKβ protein. Conclusions In the present investigation, we have applied various computational models sequentially to virtually screen the ChemDiv database, and identified a small molecule that has an IC50 value of 20.3μ M. This compound is novel among the known IKKβ inhibitors. Further optimization of the hit compound can reveal a more potent anti-inflammatory agent.

IkappaB kinase (IKK) is critical in proinflammatory cytokine-induced IkappaBalpha phosphorylation and subsequent activation of the nuclear transcription factor NF-kappaB complex. The activated NF-kappaB plays a major role in the pathogenesis of a number of human disorders, such as rheumatic and chronic inflammatory diseases. The inhibition of NF-kappaB activation by small molecule inhibitors that targets IKKbeta may provide a pharmacological basis for interfering with these acute processes. To date, only three inhibitors have passed preclinical trials; on the other hand, identifying novel IKKbeta inhibitors could evolve as potential candidates to meet the clinical requirements in the future. In the present work, we have employed a virtual screening (VS) method to identify novel compounds. The VS scheme is comprised of pharmacophore filtering and, subsequently, receptor based screening. The VS scheme was applied to the databases of 1.04 million compounds to identify three novel compounds that can inhibit the IKKbeta at a micro molar range. Moreover, these compounds can be raised into a potential anti-inflammatory drug candidate after optimizing and passing several phases of clinical trials.

The present study was performed to evaluate the cardiovascular effects of ethanolic extract from the root bark of Ulmus macrocarpa (RBUM) in rats. The effects of RBUM on the vascular response of isolated rat aorta and the blood pressure of spontaneously hypertensive rats (SHRs) were evaluated. In addition, its antioxidant activity in H9c2 cells was investigated. In the free radical scavenging assay using 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH), RBUM exhibited significant scavenging activity with an EC50 value of 14.3 microg/ml. RBUM also induced resistance to hydrogen peroxide-mediated oxidative insult in H9c2 myocardial cells. In isolated rat aortic preparations, RBUM exhibited potent vascular relaxant effect with an EC50 value of 1.9 microg/ml. This relaxation was significantly inhibited by denudation of the endothelial layer, pretreatment with NG-nitro-L-arginine methyl ester (10 microM), raising extracellular K+ (45 mM), and pretreatment with tetraethylammonium (10 mM). In an antihypertensive study with SHRs, long-term administration with RBUM (100 mg/kg) for 42 d decreased systolic blood pressure (approximately 20 mmHg). In SHRs after 42 d of treatment, RBUM recovered aortic relaxation to acetylcholine and sodium nitroprusside, and attenuated lipid peroxidation in liver of SHRs. These results suggest that chronic treatment with RBUM exerts antihypertensive effects in SHRs, and its direct vasorelaxant and antioxidant properties may contribute to reduce elevated blood pressure.

We evaluated the cardioprotective effects of 4-cyano (benzo[b]thiophene-2-carbonyl)guanidine (KR-33028), a recently developed inhibitor of the Na+/H+ exchanger (NHE), on hypoxia-induced H9c2 cell death and on perfused rat hearts subjected to ischemia/reperfusion. KR-33028 inhibited in a concentration-dependent manner the recovery from acidosis induced by an NH4Cl prepulse in PS120 fibroblast cells expressing the human NHE-1 isoform (IC50: 2.59 microM). Treatment with KR-33028 (1-10 microM) significantly decreased hypoxia-induced necrotic cell death and apoptotic cell death in H9c2 cells. KR-33028 significantly inhibited hypoxia-induced increases in cytosolic and mitochondrial Ca2+ level and cytochrome c release, and recovered hypoxia-induced Delta psi(m) reduction. In the perfused rat hearts subjected to 30 min of ischemia and 30 min of reperfusion, KR-33028 (1-10 microM) improved cardiac contractility, decreased lactate dehydrogenase release, and increased content of tissue ATP, creatine phosphate and glycogen in a concentration-dependent manner. In addition, KR-33028 did not produce significant acute or subacute toxicity in the rats at doses tested. Our results suggest that a novel NHE-1 inhibitor KR-33028 possesses potent cardioprotective effects with minimal toxicity and that the effects may be mediated by inhibition of intracellular Ca2+ overload and mitochondrial cell death pathway.