3-Substituted-4-hydroxycoumarin as a new scaffold with potent CDK inhibition and promising anticancer effect: Synthesis,molecular modeling and QSAR studies

A new series of 3-substituted-4-hydroxycoumarin derivatives was designed, synthesized, and evaluated for CDK inhibiting and anticancer activities. All the synthesized target compounds showed remarkably high affinity and selectivity towards CDK1B, compared to flavopiridol, with Ki values in the low nanomolar range (Ki = 0.35–0.88 nM). Most of them elicited considerable inhibiting effect against CDK9T1 (Ki = 3.26–23.45 nM). Moreover, all the target compounds were tested in vitro against eighteen types of human tumor cell lines. The hydrazone 3a, N-phenylpyrazoline derivative 6b and 2-aminopyridyl-3-carbonitrile derivative 8c were the most potent anticancer agents against MCF-7 breast cancer cell line (IC₅₀ = 0.21, 0.21 and 0.23 nM, respectively). The target compounds 3a, 6b and 8c were further evaluated in MCF-7 breast cancer mouse xenograft model and showed in vivo efficacy at 10 mg/kg dose. The docking study confirmed a unique binding mode in the active site of CDK1B with better score than flavopiridol. Quantitative structure activity relationship study was done and revealed a highly predictive power R² of 0.81.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21Waf1/Cip1 mediated inhibition of Hh-Gli signaling

Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC₅₀ value was noted in cells treated with the combination of resveratrol (3 μM) and curcumin (3 μM) in comparison to 30 μM of resveratrol or curcumin alone. Resveratrol + curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21^Waf/Cip1 in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21^Waf/Cip1 knockout cells suggests this combination caused apoptosis through p21^Waf/Cip1. Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2  ^Waf/Cip1 mediated inhibition of Hedgehog-Gli cascade.     

Design,synthesis and biological activity of 3-oxoamino-benzenesulfonamides as selective and reversible LSD1 inhibitors

Lysine specific demethylase 1 (LSD1) is a flavin-dependent amine oxidase that selectively removes one or two methyl groups from H3 at Lys4 and is recognized as a promising therapeutic target for cancer and other diseases. Here, a series of 3-oxoamino-benzenesulfonamides were synthesized and evaluated for their inhibitory activity against LSD1. Compounds 7b and 7h showed the most potent inhibition with the IC₅₀ values of 9.5 and 6.9 μM, respectively. Furthermore, the LSD1 inhibition of 7b and 7h were reversible and selective. Docking study presented the possible binding mode between 7b, 7h and the LSD1 active site. Taken together, 3-oxoamino-benzenesulfonamides may represent a new class of reversible LSD1 inhibitors and 7b and 7h were two hit compounds deserved further structural optimization.     

The anthraquinone emodin inhibits the non-exported FIKK kinase from Plasmodium falciparum

The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC₅₀ = 2 μM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

Synthesis of pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates as potential anticancer agents

A series of pyrazolo[1,5-a]pyrimidine linked 2-aminobenzothizole conjugates (6a–t) were synthesized and evaluated for their anticancer activity against five human cancer cell lines. Among them two compounds 6p and 6m showed significant anticancer activity with IC₅₀ values ranging from 2.01 to 7.07 and 1.94–3.46 μM, respectively. Moreover, cell cycle arrest in G₂/M and reduction in Cdk1 expression level were observed upon treatment of these compounds and they also induced caspase-3 dependent apoptosis. This was further confirmed by staining as well as DNA fragmentation analysis.     

Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroA_A.sp. A phylogenetic analysis revealed that AroA_A.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The K_M for PEP and IC₅₀ [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroA_A.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC₅₀ [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroA_A.sp. The specific activity of the wild-type AroA_A.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.     

In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC₅₀ values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC₅₀ values of 50.8 and 40.2 μM, while the other active compounds did not significantly affect CYP1A2 activity with IC₅₀ values more than 100 μM. K_i values further determined for eupatorin and sinensetin were 46.4 and 35.2 μM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations     

Role of AKAP 149–PKA–PDE4A complex in cell survival and cell differentiation processes

The cellular localization of A-kinase anchoring proteins (AKAPs), protein kinase A (PKAs) and phosphodiesterases (PDEs) is a key step to the spatiotemporal regulation of the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP). In this paper the cellular distribution of the mitochondrial AKAP 149–PKA–PDE4A complex and its implications in the cell death induced by YTX treatment, a known PDE modulator, was studied. K-562 cell line was incubated with YTX for 24 or 48 h. Under these conditions AKAP 149, PKA and type-4A PDE (PDE4A) levels were measured in the cytosol, in the plasma membrane and in the nucleus. Apoptotic hallmarks were also measured after the same conditions. In addition, YTX effect on cell viability was checked after AKAP 149 and PDE4A silencing. The results obtained show a decrease in AKAP 149–PKA–PDE4A levels in cytosol after YTX exposure. 24 h after the toxin addition, the complex expression increased in the plasma membrane and after 48 h in the nucleus domain. Furthermore Bcl-2 levels were decreased and the expression of caspase 3 together with caspase 8 activity were increased after 24 h of toxin incubation but not after 48 h. These results suggest apoptotic cell death at 24 h and a non-apoptotic cell death after 48 h. When AKAP 149 and PDE4A were silenced YTX did not induce cellular death. In summary, AKAP 149–PKA–PDE4A complex localization is related with YTX effect in K-562 cell line. When this complex is mainly located in the plasma membrane apoptosis is activated while when the complex is in the nuclear domain non-apoptotic cellular death or cellular differentiation is activated. Therefore AKAP 149–PKA–PDE4A distribution and integrity have a key role in cellular survival.     

Discovery of 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methylpyridin-2-yl)-benzamide as a novel glucokinase activator (GKA) for the treatment of type 2 diabetes mellitus

Novel heteroaryl-containing benzamide derivatives were synthesized and screened using an in vitro assay measuring increases in glucose uptake and glucokinase activity stimulated by 10 mM glucose in rat hepatocytes. From a library of synthesized compounds, 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methyl pyridin-2-yl)-benzamide (19e) was identified as a potent glucokinase activator with assays demonstrating an EC₅₀ of 315 nM and the induction of a 2.23 fold increase in glucose uptake. Compound 19e exhibited a glucose AUC reduction of 32% (50 mg/kg) in an OGTT study with C57BL/6J mice compared to 28% for metformin (300 mg/kg). Single treatment of the compound in C57BL/J6 and ob/ob mice elicited basal glucose lowering activity, while in a two-week repeated dose study with ob/ob mice, the compound significantly decreased blood glucose levels with no evidence of hypoglycemia risk. In addition, 19e exhibited favorable pharmacokinetic parameters in mice and rats and excellent safety margins in liver and testicular toxicity studies. Compound 19e was therefore selected as a development candidate for the potential treatment of type 2 diabetes.     

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10⁻¹⁰, 10⁻⁹, and 10⁻⁸ M incubated with oocytes co-cultured with follicular hemisections for 15 h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10⁻⁷, 10⁻⁵, and 10⁻³ M blocked this effect (P < 0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200 μM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P < 0.05). Only the oocytes’ cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6 h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.     

Biomechanics of simulated versus natural cross-country sit skiing

The purpose of this study was to investigate the biomechanics of cross-country sit-skiing in simulated and natural skiing. Thirteen international level athletes participated in a ski ergometer test (simulated conditions) and a test on snow in a ski-tunnel (natural conditions) using their personal sit-ski. Tests in both conditions were performed at individual maximal speed. When comparing the two conditions the main results were: (1) maximal speed in simulated conditions was lower (p < 0.05) but correlated well with the natural condition (r = 0.79, p < 0.001); (2) no differences in pole force variables were found; peak force (r = 0.77, p < 0.01) and average force (r = 0.78, p < 0.01) correlated well; (3) recovery time and time to peak did not differ and time to impact correlated with each other (r = 0.88, p < 0.01); (4) no differences were found in peak electromyography (EMG) and average EMG for Triceps, Pectoralis, and Erector Spinae; Rectus Abdominis did not differ in peak. EMG peak and average EMG of all muscles were correlated between the two conditions (r = 0.65–0.94; p < 0.05–0.01). Although some differences were observed, this study demonstrated that technical skill proficiency in natural and simulated cross-country skiing is comparable from a force production and muscle activation perspective.     

The relationship of corticospinal excitability with pain,motor performance and disability in subjects with chronic wrist/hand pain

There is a growing body of evidence of changes in corticospinal excitability associated with musculoskeletal disorders, however there is a lack of knowledge of how these changes relate to measures of pain, motor performance and disability. An exploratory study was performed utilizing Transcranial Magnetic Stimulation to investigate differences in corticospinal excitability in the Abductor Pollicis Brevis (APB) between 15 pain-free subjects and 15 subjects with chronic wrist/hand pain and to determine how corticospinal excitability was associated with measures of pain (visual analog scale, AUSCAN™), hand motor performance (isometric and key pinch strength, Purdue Pegboard Test), disability (AUSCAN™), and spinal motoneuronal excitability. Input–output curves demonstrated increased corticospinal excitability of the APB in the affected hand of subjects with chronic pain (p < 0.01). Changes in corticospinal excitability were significantly correlated with pain intensity (r = 0.77), disability (r = 0.58), and negatively correlated with motoneuronal excitability (r = −0.57). Corticospinal excitability in subjects with heterogeneous injuries of the wrist/hand was associated with disability and pain.     

Torque-onset determination: Unintended consequences of the threshold method

Background: Compared with visual torque-onset-detection (TOD), threshold-based TOD produces onset bias, which increases with lower torques or rates of torque development (RTD). Purpose: To compare the effects of differential TOD-bias on common contractile parameters in two torque-disparate groups. Methods: Fifteen boys and 12 men performed maximal, explosive, isometric knee-extensions. Torque and EMG were recorded for each contraction. Best contractions were selected by peak torque (MVC) and peak RTD. Visual-TOD-based torque-time traces, electromechanical delays (EMD), and times to peak RTD (tRTD) were compared with corresponding data derived from fixed 4-N m- and relative 5%MVC-thresholds. Results: The 5%MVC TOD-biases were similar for boys and men, but the corresponding 4-N m-based biases were markedly different (40.3 ± 14.1 vs. 18.4 ± 7.1 ms, respectively; p < 0.001). Boys–men EMD differences were most affected, increasing from 5.0 ms (visual) to 26.9 ms (4 N m; p < 0.01). Men’s visually-based torque kinetics tended to be faster than the boys’ (NS), but the 4-N m-based kinetics erroneously depicted the boys as being much faster to any given %MVC (p < 0.001). Conclusions: When comparing contractile properties of dissimilar groups, e.g., children vs. adults, threshold-based TOD methods can misrepresent reality and lead to erroneous conclusions. Relative-thresholds (e.g., 5% MVC) still introduce error, but group-comparisons are not confounded.     

Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5, anti-RSV and anti-Sb-1 agents

A library of eighty-six assorted benzimidazole derivatives was screened for antiviral activity against a panel of ten RNA and DNA viruses.Fifty-two of them displayed different levels of activity against one or more viruses, among which CVB-5, RSV, BVDV and Sb-1 were the most frequently affected. In particular, fourteen compounds exhibited an EC₅₀ in the range 9–17 μM (SI from 6 to >11) versus CVB-5, and seven compounds showed an EC₅₀ in the range 5–15 μM (SI from 6.7 to ⩾20) against RSV, thus resulting comparable to or more potent than the respective reference drugs (NM108 and ribavirin). Most of these compounds derive from 2-benzylbenzimidazole, but also other molecular scaffolds [as 1-phenylbenzimidazole (2), 2-trifluoromethylbenzimidazole (69), dihydropyrido[3′,2′:4,5]imidazo[1,2-a][1,4]benzodiazepin-5-one (3), dibenzo[c,e]benzimidazo[1,2-a]azepine (22), and 2-(tetrahydropyran-2-yl)benzimidazole (81, 82 and 86)] are related to interesting levels of activity against these or other viruses (BVDV, Sb-1). Thus, these scaffolds (some of which, so far unexplored), represent valid starting points to develop more efficient agents against pathologies caused by CVB-5, RSV, BVDV and Sb-1 viruses.