NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3ß-NF-κB signalling pathways in the endothelium.

Non-muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor-α (TNF-α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose-dependent manner. Blebbistatin enhanced Akt and GSK3ß phosphorylation and inhibited NF-κB p65 nuclear translocation and IκBα degradation. These observations were similar to the effect of CHIR99021, a GSK3ß inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3ß and suppressed NF-κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF-α-stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3ß by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model.Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3ß-NF-κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders.

Target driven preclinical screening for new antimitotic chemotherapy agents.

Currently approved antimitotic therapies used in chemotherapy are microtubule-targeting agents (MTAs). Despite they achieved some level of success, they have limited efficacy as single agents, with issues of slippages and resistance, and cause significant side effects. The advances in the identification of other mitosis-related targets led to the development of new mitotic regulators aimed to perturb mitosis without interfering with microtubule dynamics in non-dividing cells trying to reduce side effects in patients. Some of these compounds like those targeted to entry and mitotic kinases, mitotic kinesins/motor proteins, and multiprotein complexes have been evaluated in vitro and in animal models, and some of them have reached clinical trials. Despite promising preclinical results, in many cases, the efficacy demonstrated by these new antimitotics was not better than current microtubule inhibitors. In this paper we review present and future strategies on the search for new antimitotic compounds based on identification of new protein targets and development of multifunctional inhibitors of mitosis in cancer cells.

Multicomponent reactions in antimitotic drug discovery.

As enabling technology, the development and application of multicomponent reactions (MCRs) are now an integral part of the work of any major medical research unit. Targeted MCR approaches focused on specific antimitotic pathways afford new solutions for the medicinal chemistry of the XXI century. In this review, the contribution of these procedures to the discovery of antimitotic drugs that are currently in clinical trials or already in the market is discussed.

Inhibition of myosin light chain kinase can be targeted for the development of new therapies against herpes simplex virus type-1 infection.

BACKGROUND: Herpes simplex virus type-1 (HSV-1) is the leading cause of infectious blindness worldwide. Through a multistep process, HSV-1 enters into naturally susceptible human corneal epithelial (HCE) cells where it establishes an optimal environment for viral replication and spread. HSV-1 employment of cytoskeletal proteins, kinases, and cell signalling pathways is crucial for the entry process. METHODS: Here we demonstrate that non-muscle myosin IIA (NM-IIA) and/or a myosin activating kinase, myosin light chain kinase (MLCK), can be targeted for the development of new and effective therapies against HSV-1. HCE cells were incubated with MLCK inhibitors ML-7 and ML-9 and NM-IIA inhibitor blebbistatin. Following the application of inhibitors, HSV-1 entry and spread to neighbouring HCE cells was evaluated. RESULTS: Upon application of MLCK inhibitors ML-7 and ML-9 and NM-IIA inhibitor blebbistatin, HSV-1 entry into HCE cells was significantly decreased. Furthermore, dramatic impairment of glycoprotein-mediated membrane fusion was seen in cells treated with MLCK inhibitors, thus establishing a role for MLCK activation in cell-to-cell fusion and multinucleated syncytial cell formation. These results also indicate that the activation of motor protein NM-IIA by MLCK is crucial for cytoskeletal changes required for HSV-1 infection of corneal cells. CONCLUSIONS: We provide new evidence that NM-IIA and MLCK can be used as effective antiviral targets against ocular herpes.

Mitosis-targeted anti-cancer therapies: where they stand.

The strategy of clinically targeting cancerous cells at their most vulnerable state during mitosis has instigated numerous studies into the mitotic cell death (MCD) pathway. As the hallmark of cancer revolves around cell-cycle deregulation, it is not surprising that antimitotic therapies are effective against the abnormal proliferation of transformed cells. Moreover, these antimitotic drugs are also highly selective and sensitive. Despite the robust rate of discovery and the development of mitosis-selective inhibitors, the unpredictable complexities of the human body's response to these drugs still herald the biggest challenge towards clinical success. Undoubtedly, the need to bridge the gap between promising preclinical trials and effective translational bedside treatment prompts further investigations towards mapping out the mechanistic pathways of MCD, understanding how these drugs work as medicine in the body and more comprehensive target validations. In this review, current antimitotic agents are summarized with particular emphasis on the evaluation of their clinical efficacy as well as their limitations. In addition, we discuss the basis behind the lack of activity of these inhibitors in human trials and the potential and future directions of mitotic anticancer strategies.

Kinetic properties and small-molecule inhibition of human myosin-6.

Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion.

Reconstructing the free-energy landscape associated to molecular motors processivity.

We propose a biochemical model providing the kinetic and energetic descriptions of the processivity dynamics of kinesin and dinein molecular motors. Our approach is a modified version of a well known model describing kinesin dynamics and considers the presence of a competitive inhibition reaction by ADP. We first reconstruct a continuous free-energy landscape of the cycle catalyst process that allows us to calculate the number of steps given by a single molecular motor. Then, we calculate an analytical expression associated to the translational velocity and the stopping time of the molecular motor in terms of time and ATP concentration. An energetic interpretation of motor processivity is discussed in quantitative form by using experimental data. We also predict a time duration of collective processes that agrees with experimental reports.

The role of non-muscle myosin IIA in aggregation and invasion of human MCF-7 breast cancer cells.

Human MCF-7/6 breast cancer cells differ from their MCF-7/AZ counterparts by their invasiveness in a number of assays in vitro, such as invasion of MCF-7 spheroids into embryonic chick heart fragments or type I collagen gels. Comparative proteomic analysis of these two variants revealed an identical pattern, except for a 230 kDa protein present in the invasive MCF-7/6 variant, but hardly detectable in the non-invasive MCF-7/AZ one. This protein appeared to be the non-muscle myosin IIA heavy chain (NMIIA), also coined MYH9. Experimental inhibition of NMIIA by reducing either its expression (via stable shRNA transduction) or its function (via the specific ATPase inhibitor blebbistatin) underpinned the decisive role of NMIIA in MCF-7 cell invasion. Inhibition of NMIIA indeed blocked the invasion of MCF-7/6 cells in three-dimensional invasion substrata such as embryonic chick heart fragments and type I collagen gels. Invasiveness of MCF-7/6 cells has been related to poor formation and compaction of aggregates, due to a functionally defective E-cadherin/catenin complex. Both genetic and pharmacological inhibition of NMIIA stimulated MCF-7/6 cell aggregation. Together, these data indicate that NMIIA is a decisive protein for MCF-7 cells to invade, indicating that this molecule is a candidate for targeted anti-invasive treatment.

The effect of NBD-Cl in nucleotide-binding of the major subunit alpha and B of the motor proteins F1FO ATP synthase and A1AO ATP synthase.

Subunit alpha of the Escherichia coli F(1)F(O) ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of alpha allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K ( d )) of 1.6 microM of bound MgATP-ATTO-647N and 2.9 microM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 microM and 55 microM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC(50)), respectively. In contrast, no effect was observed in the presence of N,N'-dicyclohexylcarbodiimide. As subunit alpha is the homologue of subunit B of the A(1)A(O) ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC(50) values of 41 microM and 42 microM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.

Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells.

The human sodium-dependent vitamin C transporter-2 (hSVCT2) plays an important role in cellular accumulation of ascorbic acid in liver cells. However, little is known about the molecular determinants that direct hSVCT2 to the cell surface in hepatocytes. We addressed this issue using live cell imaging methods to resolve the distribution and trafficking of truncated or mutated hSVCT2 constructs in a cellular model of human hepatocytes, HepG2 cells. Whereas a full-length hSVCT2-yellow fluorescent protein (YFP) fusion protein was functionally expressed at the cell surface in HepG2 cells, serial truncation and mutation analysis demonstrated an essential role for both NH(2)- and COOH-terminal sequence(s) for cell surface expression and function. Video-rate confocal imaging showed evidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles, the motility of which was impaired following disruption of microtubules using nocodazole. However, in a HepG2 cell line stably expressing hSVCT2-YFP at the cell surface, plasma membrane levels of hSVCT2 were unaffected by inhibition of microtubule-associated motor proteins; rather, surface expression of hSVCT2-YFP was increased following treatment with myosin inhibitors. Together, these results show that 1) both NH(2)- and COOH-terminal sequences are essential for proper localization of hSVCT2, 2) cell surface delivery is dependent on intact microtubules, and 3) peripheral microfilaments regulate insertion and retrieval of hSVCT2 into the plasma membrane.

Dynamics of microtubules, motor proteins and 20S proteasomes during bovine oocyte IVM.

The present study investigated the distribution of cytoplasmic dynein, dynactin and 20S proteasomes in oocytes isolated from small (<2 mm) and large (2-8 mm) follicles during IVM. Immediately after chromatin condensation (germinal vesicle (GV) breakdown), dynactin was closely associated with the chromatin and interacted with tubulin at the MI and MII spindles in oocytes recovered from large follicles. Dynactin showed perinuclear concentration. Dynein was homogeneously distributed in the cytoplasm of GV oocytes in both groups and was associated with the chromatin at the MI and MII spindle. The 20S proteasomes were found predominantly in the nucleus at the GV stage and were associated with the chromatin up to the MII stage in both groups of oocytes. The use of sodium orthovanadate, an inhibitor or phosphatase and ATPase activity, and nocodazole, a known disruptor of microtubules, affected the localisation of proteasomes in the meiotic stages. The results demonstrate the distinct dynamics of molecular motors and proteasomes during bovine oocyte IVM, their possible relationship with the developmental competence of the oocyte and the link between microtubules, their associated molecular motors and the transport of proteasomes during bovine female meiosis.

The concerted nature between three catalytic subunits driving the F1 rotary motor.

F(1), a rotational molecular motor, shows strong cooperativity during ATP catalysis when driving the rotation of the central gamma subunit surrounded by the alpha(3)beta(3) subunits. To understand how the three catalytic beta subunits cooperate to drive rotation, we made a hybrid F(1) containing one or two mutant beta subunits with altered catalytic kinetics and observed its rotations. Analysis of the asymmetric stepwise rotations elucidated a concerted nature inside the F(1) complex where all three beta subunits participate to rotate the gamma subunit with a 120 degrees phase. In addition, observing hybrid F(1) rotations at various solution conditions, such as ADP, P(i) and the ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) provides additional information for each elementary event. This novel experimental system, which combines single molecule observations and biochemical methods, enables us to dynamically visualize the catalytic coordination inside active enzymes and shed light on how biological machines provide unidirectional functions and rectify information from stochastic reactions.

The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the epsilon subunit.

The chloroplast-type F(1) ATPase is the key enzyme of energy conversion in chloroplasts, and is regulated by the endogenous inhibitor epsilon, tightly bound ADP, the membrane potential and the redox state of the gamma subunit. In order to understand the molecular mechanism of epsilon inhibition, we constructed an expression system for the alpha(3)beta(3)gamma subcomplex in thermophilic cyanobacteria allowing thorough investigation of epsilon inhibition. epsilon Inhibition was found to be ATP-independent, and different to that observed for bacterial F(1)-ATPase. The role of the additional region on the gamma subunit of chloroplast-type F(1)-ATPase in epsilon inhibition was also determined. By single molecule rotation analysis, we succeeded in assigning the pausing angular position of gamma in epsilon inhibition, which was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for ATP binding. These results suggest that the epsilon subunit of chloroplast-type ATP synthase plays an important regulator for the rotary motor enzyme, thus preventing wasteful ATP hydrolysis.

Inhibition of kinesin motor proteins by adociasulfate-2.

Kinesin motor proteins are involved in cell division and intracellular transport of vesicles and organelles, and as such, they play a role in neurological disease, cancer, and developmental disorders. Inhibitors of kinesin would be valuable as probes of cell physiology and as potential therapeutics. Adociasulfate-2 (AS-2) is the only known natural product inhibitor of kinesins, but its mechanism of action is unknown. We utilized kinetic studies, dynamic light scattering, and transmission electron microscopy to investigate the inhibitory action of AS-2. Our data suggest that AS-2 is not a classical 1:1 inhibitor. Instead, a rodlike aggregate that mimics microtubules is complexed with kinesin and inhibits its ATPase activity. An intriguing implication of this hypothesis is that aggregates of a chiral natural product can have interesting and biologically relevant properties. This mode of action might represent one way in which a small molecule can disrupt a protein-protein interaction.

The globular tail domain of myosin Va functions as an inhibitor of the myosin Va motor.

The actin-activated ATPase activity of full-length mammalian myosin Va is well regulated by Ca2+, whereas that of truncated myosin Va without the C-terminal globular tail domain (GTD) is not. Here, we have found that exogenous GTD is capable of inhibiting the actin-activated ATPase activity of GTD-deleted myosin Va. A series of truncated constructs of myosin Va further showed that the entire length of the first coiled-coil (coil-1) of the tail domain is critical for GTD-dependent regulation of myosin Va and that deletion of 58 residues from the C-terminal end of coil-1 markedly hampered regulation. Negative staining electron microscopy revealed that GTD-deleted myosin Va formed a "Y"-shaped structure, which was converted to a triangular shape, similar to the structure of full-length myosin Va in the inhibited state, by addition of exogenous GTD. In contrast, the triangular shape was not observed when the C-terminal 58 residues of coil-1 were deleted, even in the presence of exogenous GTD. Based on these results, we propose a model for the formation of the inhibited state of myosin Va. GTD binds to the C-terminal end of coil-1. The neck-tail junction of myosin Va is flexible, and the long neck enables the head domain to reach the GTD associated with the end of coil-1. Once the head interacts with the GTD, the triangular inhibited conformation is stabilized. Consistent with this model, we found that shortening of the neck of myosin Va by two IQ motifs abolished the regulation by GTD, whereas regulation was partially restored by shortening of coil-1 by an amount comparable to that of the two IQ motifs.

The angiogenic function of nucleolin is mediated by vascular endothelial growth factor and nonmuscle myosin.

Nucleolin, originally described as a nuclear protein, was recently found to be expressed on the surface of endothelial cells during angiogenic. However, the functions of cell-surface nucleolin in angiogenic remain mysterious. Here we report that upon endothelial cells adhering to extracellular matrix components, vascular endothelial growth factor (VEGF) mobilizes nucleolin from nucleus to cell surface. Functional blockage or down-regulation of the expression of cell-surface nucleolin in endothelial cells significantly inhibits the migration of endothelial cells and prevents capillary-tubule formation. Moreover, nonmuscle myosin heavy chain 9 (MyH9), an actin-based motor protein, is identified as a nucleolin-binding protein. Subsequent studies reveal that MyH9 serves as a physical linker between nucleolin and cytoskeleton, thus modulating the translocation of nucleolin. Knocking down endogenous MyH9, specifically inhibiting myosin activity, or overexpressing functional deficient MyH9 disrupts the organization of cell-surface nucleolin and inhibits its angiogenic function. These studies indicate that VEGF, extracellular matrix, and intracellular motor protein MyH9 are all essential for the novel function of nucleolin in angiogenic.

High mobility of carboxyl-terminal region of bacterial chemotaxis phosphatase CheZ is diminished upon binding divalent cation or CheY-P substrate.

In Escherichia coli chemotaxis, the CheZ phosphatase catalyzes the removal of the phosphoryl group from the signaling molecule, CheY. The cocrystal structure of CheZ with CheY x BeF3- x Mg2+ (a stable analogue of CheY-P) revealed that CheZ is a homodimer with a multidomain, nonglobular structure. To explore the effects of CheZ/CheY complex formation on CheZ structure, the rotational dynamics of the different structural domains of CheZ [the four-helix bundle, the N-terminal helix, the C-terminal helix, and the putative disordered linker between the C-terminal helix and the bundle] were evaluated. To monitor dynamics of the different regions, fluorescein probes were covalently attached at various locations on CheZ through reaction with engineered cysteine residues and the rotational behavior of the fluoresceinated derivatives were assessed using steady state fluorescence anisotropy. Anisotropy measurements at various solution viscosities (Perrin plot analysis) demonstrated large differences in global rotational motion for fluorophores located on different regions. Rotational correlation times for probes located on the four-helix bundle and the N-terminal helix agreed well with theoretical values predicted for a protein the size and shape of the four-helix bundle. However, the rotational correlation times of probes located on the linker and the C-terminal helix were 8-20x lower, indicating rapid motion independent of the bundle. The anisotropies of probes located on the linker and the C-terminal helix increased in the presence of divalent cation (Mg2+, Ca2+, or Mn2+) in a saturable fashion, consistent with a binding event (Kd approximately 1-4 mM) that results in decreased mobility. The anisotropies of probes located on the C-terminal helix and the C-terminal portion of the linker increased further as a result of binding CheY-P. In light of the recently available structural data and the high independent mobility of the C-terminus demonstrated here, we interpret the CheY-P-dependent increase in anisotropy to be a consequence of decreased mobility of the C-terminal region due to binding interactions with CheY-P, and not to the formation of higher order aggregates of the CheZ2(CheY-P)2 complex.

Application of molecular modeling to analysis of inhibition of kinesin motor proteins of the BimC subfamily by monastrol and related compounds.

Application of molecular modeling approaches has potential to contribute to rational drug design. These approaches may be especially useful when attempting to elucidate the structural features associated with novel drug targets. In this study, molecular docking and molecular dynamics were applied to studies of inhibition of the human motor protein denoted HsEg5 and other homologues in the BimC subfamily. These proteins are essential for mitosis, so compounds that inhibit their activity may have potential as anticancer therapeutics. The discovery of a small-molecule cell-permeable inhibitor, monastrol, has stimulated research in this area. Interestingly, monastrol is reported to inhibit the human and Xenopus forms of Eg5, but not those from Drosophila and Aspergillus. In this study, homology modeling was used to generate models of the Xenopus, Drosophila, and Aspergillus homologues, using the crystal structure of the human protein in complex with monastrol as a template. A series of known inhibitors was docked into each of the homologues, and the differences in binding energies were consistent with reported experimental data. Molecular dynamics revealed significant changes in the structure of the Aspergillus homologue that may contribute to its relative insensitivity to monastrol and related compounds.