Rimonabant is a dual inhibitor of acyl CoA:cholesterol acyltransferases 1 and 2

Acyl coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the intracellular synthesis of cholesteryl esters (CE). Both ACAT isoforms, ACAT1 and ACAT2, play key roles in the pathophysiology of atherosclerosis and ACAT inhibition retards atherosclerosis in animal models. Rimonabant, a type 1 cannabinoid receptor (CB1) antagonist, produces anti-atherosclerotic effects in humans and animals by mechanisms which are not completely understood. Rimonabant is structurally similar to two other cannabinoid receptor antagonists, AM251 and SR144528, recently identified as potent inhibitors of ACAT. Therefore, we examined the effects of Rimonabant on ACAT using both in vivo cell-based assays and in vitro cell-free assays. Rimonabant dose-dependently reduced ACAT activity in Raw 264.7 macrophages (IC₅₀ = 2.9 ± 0.38 μM) and isolated peritoneal macrophages. Rimonabant inhibited ACAT activity in intact CHO-ACAT1 and CHO-ACAT2 cells and in cell-free assays with approximately equal efficiency (IC₅₀ = 1.5 ± 1.2 μM and 2.2 ± 1.1 μM for CHO-ACAT1 and CHO-ACAT2, respectively). Consistent with ACAT inhibition, Rimonabant treatment blocked ACAT-dependent processes in macrophages, oxysterol-induced apoptosis and acetylated-LDL induced foam cell formation. From these results we conclude that Rimonabant is an ACAT1/2 dual inhibitor and suggest that some of the atherosclerotic beneficial effects of Rimonabant are, at least partly, due to inhibition of ACAT.     

Design and synthesis of N-phenylacetyl (sulfonyl) 4,5-dihydropyrazole derivatives as potential antitumor agents

A series of novel N-phenylacetyl (sulfonyl) 4,5-dihydropyrazole derivatives as potential telomerase inhibitors were synthesized. The bioassay tests show that compound 4a exhibited high activity against human gastric cancer cell SGC-7901, liver cancer Hep-G2 and human prostate PC-3 cell lines with IC₅₀ values of 21.23 ± 0.99, 29.43 ± 0.32 and 30.89 ± 1.07 μM, respectively. All title compounds were assayed for telomerase inhibition by a modified TRAP assay, the results show that compound 4a can inhibit telomerase with IC₅₀ value of 4.0 ± 0.32 μM. Docking simulation was performed to position compound 4a into the telomerase (3DU6) active site to determine the probable binding model.     

Trisubstituted ureas as potent and selective mPGES-1 inhibitors

A novel series of trisubstituted ureas has been identified as potent and selective mPGES-1 inhibitors. These compounds are selective over other prostanoid enzymes such as PGF synthase and TX synthase. This series of inhibitors was developed by lead optimization of a hit from an internal HTS campaign. Lead compound 42 is potent in A549 cell assay (IC₅₀ of 0.34 μM) and in human whole blood assay (IC₅₀ of 2.1 μM). An efficient and versatile one-pot strategy for the formation of ureas, involving a reductive amination, was developed to generate these inhibitors.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

A novel class of antagonists for the FFAs receptor GPR40

The free fatty acid receptor, GPR40, is implicated in the pathophysiology of type 2 diabetes, and is a new potential drug target for the treatment of type 2 diabetes. Its antagonist is thought to be not only a useful chemical probe for further exploring the function of GPR40 but also a lead structure for drug development. With virtual screening based on a homology model followed by a cell-based calcium mobilization assay, we found that sulfonamides are a new class of small organic antagonists for GPR40. One of the compounds, DC260126, dose-dependently inhibited GPR40-mediated Ca²⁺ elevations stimulated by linoleic acid, oleic acid, palmitoleic acid and lauric acid (IC₅₀: 6.28 ± 1.14, 5.96 ± 1.12, 7.07 ± 1.42, 4.58 ± 1.14 μM, respectively), reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells.     

Design of novel N-phenylnicotinamides as selective cyclooxygenase-1 inhibitors

A series of N-phenylnicotinamides (1-40) were designed and evaluated in vitro for their COX inhibitory activities. Most of the synthesized compounds were proved to be potent and selective inhibitors of COX-1. Compound 28 showed the most potent COX-1 inhibitory activity (COX-1 IC₅₀ = 0.68 ± 0.07 μM) and good selectivity (COX-2 IC₅₀ >100 μM). This compound may be useful as a lead compound for superior COX-1 inhibitors. On the basis of the biological results, structure-activity relationships for the COX-1-inhibitory activities of the synthesized N-phenylnicotinamides were discussed concisely.     

Synthesis and topoisomerase II inhibitory and cytotoxic activity of oxiranylmethoxy- and thiiranylmethoxy-chalcone derivatives

In order to find potential anticancer drug candidate targeting topoisomerases enzyme, we have designed and synthesized oxiranylmethoxy- and thiiranylmethoxy-retrochalcone derivatives and evaluated their pharmacological activity including topoisomerases inhibitory and cytotoxic activity. Of the compounds prepared compound 25 showed comparable or better cytotoxic activity against cancer cell lines tested. Compound 25 inhibited MCF7 (IC₅₀: 0.49 ± 0.21 μM) and HCT15 (IC₅₀: 0.23 ± 0.02 μM) carcinoma cell growth more efficiently than references. In the topoisomerases inhibition test, all the compounds were inactive to topoisomerase I but moderate inhibitors to topoisomerase II enzyme. Especially, compound 25 inhibited topoisomerase II activity with comparable extent to etoposide at 100 μM concentrations. Correlation between cytotoxicity and topoisomerase II inhibitory activity implies that compound 25 can be a possible lead compound for anticancer drug impeding the topoisomerase II function.     

Design strategies to target crystallographic waters applied to the Hsp90 molecular chaperone

A series of novel and potent small molecule Hsp90 inhibitors was optimized using X-ray crystal structures. These compounds bind in a deep pocket of the Hsp90 enzyme that is partially comprised by residues Asn51 and Ser52. Displacement of several water molecules observed crystallographically in this pocket using rule-based strategies led to significant improvements in inhibitor potency. An optimized inhibitor (compound 17) exhibited potent Hsp90 inhibition in ITC, biochemical, and cell-based assays (K_d = 1.3 nM, K_i = 15 nM, and cellular IC₅₀ = 0.5 μM).     

Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is primarily responsible for intracellular biosynthesis of active glucocorticoid, and its tissue-specific dysregulation has been implicated in the development of metabolic syndromes. We have developed a cell-based assay for measuring 11β-HSD1 activities using murine skeletal muscle cell line C2C12. We found that the messenger RNA (mRNA) expression of 11β-HSD1 increased on differentiation with enhanced enzyme activity as determined by homogeneous time-resolved fluorescence (HTRF) assay. Carbenoxolone, a well-known 11β-HSD1 inhibitor, exhibited an IC₅₀ value similar to that in in vitro microsomal assay (IC₅₀ = 0.3 μM). Unlike in vitro microsomal assay, cosubstrate NADPH was not required in the cell-based assay, indicating that viable cells might provide a sufficient amount of endogenous NADPH to catalyze the enzymatic conversion of inactive cortisone to active cortisol. Treatment of C2C12 myotubes with cortisone concentration dependently transactivated and transrepressed glutamine synthase and interleukin-6, respectively, which were abrogated by carbenoxolone or RU-486 (mifepristone), a glucocorticoid receptor antagonist. Accordingly, a newly designed cell-based assay using differentiated skeletal muscle cells would be useful for high-throughput screening of 11β-HSD1 inhibitors as well as for understanding the molecular mechanisms of glucocorticoid action.     

A novel class of dual mPGES-1/5-LO inhibitors based on the α-naphthyl pirinixic acid scaffold

Dual inhibition of microsomal prostaglandin E₂ synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) represents a promising strategy in the development of novel anti-inflammatory drugs targeting the arachidonic acid cascade. Herein, a class of α-naphthyl pirinixic acids is characterized as dual mPGES-1/5-LO inhibitors. Systematic structural variation was focused on the lipophilic backbone of the scaffold and yielded detailed structure-activity relationships (SAR) with compound 16 (IC₅₀ mPGES-1 = 0.94 μM; IC₅₀ 5-LO = 0.1 μM) showing the most favorable in vitro pharmacological profile.     

Cytotoxic and antioxidant activities of diterpenes and sterols from the Vietnamese soft coral Lobophytum compactum

Two new diterpenes, lobocompactols A (1) and B (2), and five known compounds (3-7) were isolated from the methanol extract of the soft coral Lobophytum compactum using combined chromatographic methods and identified based on NMR and MS data. Each compound was evaluated for cytotoxic activity against A549 (lung) and HL-60 (acute promyelocytic leukemia) human cancer cell lines. Among them, compound 5 exhibited strong cytotoxic activity against the A549 cell line with an IC₅₀ of 4.97 ± 0.06 μM. Compounds 3, 4, and 7 showed moderate activity with IC₅₀ values of 23.03 ± 0.76, 31.13 ± 0.08, and 36.45 ± 0.01 μM, respectively. The cytotoxicity of 5 on the A549 cells was comparable to that of the positive control, mitoxantrone (MX). All compounds exhibited moderate cytotoxicity against the HL-60 cell line, with IC₅₀ values ranging from 17.80 ± 1.43 to 59.06 ± 2.31 μM. Their antioxidant activity was also measured using oxygen radical absorbance capacity method, compounds 1 and 2 exhibiting moderate peroxyl radical scavenging activity of 1.4 and 1.3 μM Trolox equivalents, respectively, at a concentration of 5 μM.     

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308 307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from −14.0 to −17.09 kcal mol⁻¹ were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CL^pro. Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CL^pro expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC₅₀ ranged from 38.57 ± 2.41 to 101.38 ± 3.27 μM. Two strong 3CL^pro inhibitors were further identified as competitive inhibitors of 3CL^pro with K_i values of 9.11 ± 1.6 and 9.93 ± 0.44 μM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL^pro were also identified.     

Identification of new inhibitors of protein kinase R guided by statistical modeling

We report the identification of new, structurally diverse inhibitors of interferon-induced, double-stranded RNA-activated protein kinase (PKR) using a combined experimental and computational approach. A training set with which to build a predictive statistical model was generated by screening a set of 80 known Ser/Thr kinase inhibitors against recombinant human PKR, resulting in the identification of 28 compounds from 18 chemical classes with <0.1 μM ? IC₅₀ ? 20 μM. The model built with this data was used to screen a database of 5 million commercially available compounds in silico to identify candidate inhibitors. Testing of 128 structurally diverse candidates resulted in the confirmation of 20 new inhibitors from 11 chemical classes with 2 μM ? IC₅₀ ? 20 μM. Testing of 34 analogs in the newly identified pyrimidin-2-amine active series provided initial SAR. One newly identified inhibitor, N-[2-(1H-indol-3-yl)ethyl]-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-amine (compound 51), inhibited intracellular PKR activation in a dose-dependent manner in primary mouse macrophages without evident toxicity at effective concentrations.     

Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate

Copper(II) complexes [Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H₂satp) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near − 0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (~ 1.85 μ_B) are avid DNA binders giving K_b values within 1.0 × 10⁵ − 8.0 × 10⁵ M^− 1. Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC₅₀ = 8.3(± 1.0) μM) in visible light, while showing lower dark toxicity (IC₅₀ = 17.2(± 1.0) μM). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC₅₀ = 30.0(± 1.0) μM in dark), while retaining its photocytotoxicity (IC₅₀ = 8.0(± 1.0) μM).     

Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease

Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC₅₀ <100 μM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds revealed a competitive model, with sub-micromolar K_i value for the best inhibitor (7). Unfortunately, an in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxication by 7.     

Characterization of Tightly Associated Smooth Muscle Myosin-Myosin Light-Chain Kinase-Calmodulin Complexes

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ∼ 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at ∼ 1 nM free [Ca²⁺]. There were two MLCK pools that bound unphosphorylated SMM with K_d ∼ 10 and 0.2 μM and phosphorylated SMM with K_d ∼ 20 and 0.2 μM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca²⁺ and phosphorylation of SMM occurred at a pCa₅₀ of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.     

Kamolonol suppresses angiotensin II-induced stress fiber formation and cellular hypertrophy through inhibition of Rho-associated kinase 2 activity

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 Ferulaassafoetida. 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.     

Discovery of quinolinone derivatives as potent FLT3 inhibitors

Recently some fms-like tyrosine kinase 3 (FLT3) inhibitors have shown good efficacy in acute myeloid leukemia (AML) patients. In an effort to develop anti-leukemic drugs, we investigated quinolinone derivatives as novel FLT3 inhibitors. Two substituted quinolinones, KR65367 and KR65370 were subjected to FLT3 kinase activity assay and showed potent inhibition against FLT3 kinase activity in vitro, with IC₅₀ of 2.7 and 0.57 nM, respectively. As a measure of selectivity, effects on the activity of other kinases were also tested. Both compounds have negligible activity against Met, Ron, epidermal growth factor receptor, Aurora A, Janus kinase 2, and insulin receptor; with IC₅₀ greater than 10 μM. KR compounds showed strong growth inhibition in MV4;11 AML cells and increased the apoptotic cell death in flow cytometric analyses. A decrease in STAT5 phosphorylation by KR compounds was observed in MV4;11 cells. Furthermore, in vitro evaluation of compounds structurally related to KR65367 and KR65370 showed a good structure-activity relationship.     

Production of soluble Neprilysin by endothelial cells

A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC₅₀ values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.