Highly selective inhibition of myosin motors provides the basis of potential therapeutic application.

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the "recovery stroke" transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

Antitumor activity of an allosteric inhibitor of centromere-associated protein-E.

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.

In silico study on hydroxylated polychlorinated biphenyls as androgen receptor antagonists.

Hydroxylated polychlorinated biphenyls (HO-PCBs), major metabolites of PCBs, may have the potential to disrupt androgen hormone homeostasis. However, there is a lack of systematic investigation into the intermolecular interaction mechanism between HO-PCBs and the androgen receptor (AR). In this study, the combination of three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, and molecular dynamics (MD) simulations was performed to elucidate structural characteristics that influence the anti-androgen activity of HO-PCBs, and to provide a better understanding of the binding modes between HO-PCBs and AR. A predictive comparative molecular field analysis (CoMFA) model was developed with good robustness and predictive ability. Graphical interpretation of the model provided some insights into the structural features that affect the anti-androgen activity of HO-PCBs. The hydrogen bond interaction with Gln711, and hydrophobic interactions with residues in the hydrophobic pocket played important roles in the binding of ligand with receptor. These results are expected to be beneficial to predict anti-androgen activities of other HO-PCBs and provided possible clues for further elucidation of the binding mechanism of HO-PCBs with AR.

Molecular modeling and molecular dynamics simulation studies on the interactions of hydroxylated polychlorinated biphenyls with estrogen receptor-ß.

Endocrine-disrupting chemicals have attracted great concern. As major metabolites of polychlorinated biphenyls (PCBs), hydroxylated polychlorinated biphenyls (HO-PCBs) may disrupt estrogen hormone status because of their structural similarity to estrogen endogenous compounds. However, interactions between HO-PCBs and estrogen receptors (ERs) are not fully understood. In the present work, a molecular modeling study combining molecular docking, molecular dynamics simulations, and binding free energy calculations was performed to characterize the interactions of three HO-PCBs (4'-HO-PCB50, 2'-HO-PCB65, and 4'-HO-PCB69) having much different estrogenic activities with ERß. Docking results showed that binding between ligands and ERß was stabilized by hydrogen bond and hydrophobic interactions. The binding free energies of three ligands with ERß were calculated, and further binding free energy decomposition analysis indicated that the dominating driving force of the binding between the ligands and ERß was the van der Waals interaction. Some key residues, such as Leu298, Phe356, Gly472, His475, and Leu476, played important roles in ligand-receptor interactions by forming hydrophobic and hydrogen bond interactions with ligands. The results may be beneficial to increase understanding of the interactions between HO-PCBs and ERß.

Targeting Glutamine Metabolism with a Novel Na+/K+-ATPase Inhibitor RX108 in Hepatocellular Carcinoma.

The poor prognosis and limited therapeutic options for human hepatocellular carcinoma (HCC), the most common form of liver cancer, highlight the urgent need to identify novel therapeutic modalities. Here, we describe the antitumor activity and underlying molecular mechanisms of a novel Na+/K+-ATPase inhibitor RX108 in human HCC cells and its xenograft model. RX108 dose-dependently inhibited HCC cell proliferation in vitro and tumor growth in a xenograft mouse model, and that the inhibition was associated with induction of apoptosis. Mechanistically, RX108 significantly downregulated alanine serine cysteine transporter 2 (ASCT2) protein expression and reduced glutamine and glutamate concentration in HCC cells and tumors. In addition, RX108 exposure led to a significant decrease in cell energy metabolism in Huh7 and Hep3B cells, including decreased levels of glutathione, NADH, NADPH, and mitochondrial respiration oxygen consumption rate. Furthermore, HCC cells exhibited evidence of glutamine addiction; the antiproliferative effect of RX108 was dependent on glutamine transport. Clinically, elevated ASCT2 mRNA expression in HCCs was associated with unfavorable survival. Taken together, these findings reveal a novel approach to target glutamine metabolism through inhibiting Na+/K+-ATPase and provide a rationale for using RX108 to treat HCC in patients whose tumors express ASCT2 at high levels. RX108 is currently under clinical development.

Enzyme-Controlled Intracellular Self-Assembly of (18)F Nanoparticles for Enhanced MicroPET Imaging of Tumor.

Herein, we report the development of a new "smart" radioactive probe (i.e., 1) which can undergo furin-controlled condensation and self-assembly of radioactive nanoparticles (i.e., 1-NPs) in tumor cells and its application for enhanced microPET imaging of tumors in nude mice co-injected with its cold analog (i.e., 1-Cold). Furin-controlled condensation of 1-Cold and self-assembly of its nanoparticles (i.e., 1-Cold-NPs) in vitro were validated and characterized with HPLC, mass spectra, SEM, and TEM analyses. Cell uptake studies showed that both 1 and 1-Cold have good cell permeability. TEM images of 1-Cold-treated MDA-MB-468 cells directly uncovered that the intracellular 1-Cold-NPs were at/near the location of furin (i.e., Golgi bodies). MTT results indicated that 50 µM 1-Cold did not impose cytotoxicity to MDA-MB-468 cells up to 12 hours. MicroPET imaging of MDA-MB-468 tumor-bearing mice indicated that mice co-injected with 1 and 1-Cold showed higher uptake and longer attenuation of the radioactivity in tumors than those mice only injected with same dosage of 1. Tumor uptake ratios of 1 between these two groups of mice reached the maximum of 8.2 folds at 240 min post injection. Biodistribution study indicated that the uptake ratios of 1 in kidneys between these two groups continuously increased and reached 81.9 folds at 240 min post injection, suggesting the formation of radioactive NPs (i.e., 1-NPs) in MDA-MB-468 tumors of mice co-injected with 1 and 1-Cold. And the nanoparticles were slowly digested and secreted from the tumors, accumulating in the kidneys. Our ''smart'' probe (i.e., 1), together with the strategy of co-injection, might help researchers trace the biomarkers of interest within a longer time window.

In silico investigations of anti-androgen activity of polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) have attracted great concern as global environmental pollutants and representative endocrine disruptors. In this work, a molecular model study combining three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, and molecular dynamics (MD) simulations was performed to explore the structural requirement for the anti-androgen activities of PCBs and to reveal the binding mode between the PCBs and androgen receptor (AR). The best comparative molecular similarity indices analysis (CoMSIA) model, obtained from receptor-based alignment, shows leave-one-out cross-validated correlation coefficient (q(2)) of 0.665 and conventional correlation coefficient (R(2)) of 0.945. The developed model has a highly predictive ability in both internal and external validation. Furthermore, the interaction mechanisms of PCBs to AR were analyzed by molecular docking and MD simulation. Molecular docking indicated that all the PCBs in the data set docked in a hydrophobic pocket. The Binding free energies calculated by Molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) not only exhibited a good correlation with the experimental activity, but also could explain the activity difference of the studied compounds. The binding free energy decomposition analysis indicates that the van der Waals interaction is the major driving force for the binding process.

Molecular docking, molecular dynamics simulation, and structure-based 3D-QSAR studies on the aryl hydrocarbon receptor agonistic activity of hydroxylated polychlorinated biphenyls.

The binding interactions between hydroxylated polychlorinated biphenyls (HO-PCBs) and the aryl hydrocarbon receptor (AhR) are suspected of causing toxic effects. To understand the binding mode between HO-PCBs and AhR, and to explore the structural characteristics that influence the AhR agonistic activities of HO-PCBs, the combination of molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR), and molecular dynamics (MD) simulations was performed. Using molecular docking, the HO-PCBs were docked into the binding pocket of AhR, which was generated by homology modeling. Comparative molecular similarity index analysis (CoMSIA) models were subsequently developed from three different alignment rules. The optimum 3D-QSAR model showed good predictive ability (q(2)=0.583, R(2)=0.913) and good mechanism interpretability. The statistical reliability of the CoMSIA model was also validated. In addition, molecular docking and MD simulations were applied to explore the binding modes between the ligands and AhR. The results obtained from this study may lead to a better understanding of the interaction mechanism between HO-PCBs and AhR.

Molecular docking, molecular dynamics simulation, and structure-based 3D-QSAR studies on estrogenic activity of hydroxylated polychlorinated biphenyls.

Hydroxylated polychlorinated biphenyls (HO-PCBs), major metabolites of PCBs, have been reported to present agonist or antagonist interactions with estrogen receptor α (ERα) and induce ER-mediated responses. In this work, a multistep framework combining molecular docking, molecular dynamics (MD) simulations, and structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were performed to explore the influence of structural features on the estrogenic activities of HO-PCBs, and to investigate the molecular mechanism of ERα-ligand interactions. The CoMSIA (comparative molecular similarity indices analysis) model was developed from the conformations obtained from molecular docking. The model exhibited statistically significant results as the cross-validated correlation coefficient q² was 0.648, the non-cross-validated correlation coefficient r² was 0.968, and the external predictive correlation coefficient r(pred)² was 0.625. The key amino acid residues were identified by molecular docking, and the detailed binding modes of the compounds with different activities were determined by MD simulations. The binding free energies correlated well with the experimental activity. An energetic analysis, MM-GBSA energy decomposition, revealed that the van der Waals interaction was the major driving force for the binding of compounds to ERα. The hydrogen bond interactions between the ligands and residue His524 help to stabilize the conformation of ligands at the binding pocket. These results are expected to be beneficial to predict estrogenic activities of other HO-PCB congeners and helpful for understanding the binding mechanism of HO-PCBs and ERα.

Cardiac myosin activation: a potential therapeutic approach for systolic heart failure.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Discovery of omecamtiv mecarbil the first, selective, small molecule activator of cardiac Myosin.

We report the design, synthesis, and optimization of the first, selective activators of cardiac myosin. Starting with a poorly soluble, nitro-aromatic hit compound (1), potent, selective, and soluble myosin activators were designed culminating in the discovery of omecamtiv mecarbil (24). Compound 24 is currently in clinical trials for the treatment of systolic heart failure.

Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Arrays of self-assembled monolayers for studying inhibition of bacterial adhesion.

This paper describes a simple and convenient method for the rapid screening of potential inhibitors of bacterial adhesion and for the quantitative evaluation of the efficacy of the inhibitors using arrays of self-assembled monolayers (SAMs) of alkanethiolates on gold that are presented on a 96-well microtiter plate. The SAMs present mixtures of alpha-D-mannopyranoside (a ligand that promotes the adhesion of uropathogenic Escherichia coli by binding to the FimH proteins on the tip of type 1 pili), and tri(ethylene glycol) moieties (organic groups that resist nonspecific adsorption of proteins and cells). The SAMs provide surfaces for studies of adhesion of uropathogenic E. coli to specific ligands; they also provide excellent resistance to nonspecific adhesion. Using arrays of mannoside-presenting SAMs, inhibitors of bacterial adhesion were easily screened by observing the number of bacteria that adhered to the surface of the SAMs in the presence of inhibitor. The potency of the inhibitor was quantified by measuring the percentage of inhibition as a function of the concentration of the inhibitor. The properties of SAMs, when combined with the convenience and standardization of a microtiter plate, make arrays of SAMs a versatile tool that can be applied to high-throughput screening of inhibitors of bacterial, viral, and mammalian cell adhesion and of strongly binding ligands for proteins.