Jeffrey T. Finer

Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure

A small molecule improves cardiac function by accelerating the transition of myosin into a force-producing state. 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.

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.

Reflections of a lucid dreamer: optical trap design considerations.

Publisher Summary This chapter discusses the optical trap design considerations. The optical trap technique can be used to constrain and move small particles in solution using a light microscope and laser beam. Trapping size scales and sensitivity are well suited for studying the mechanical properties of single cells, organelles, and even molecules. The chapter describes considerations involved in the planning and implementation of an optical trapping microscope for high-resolution force and displacement measurements of trapped particles. These molecules will bind to and move the actin filament, allowing measurement of their mechanical properties at the single molecule level. The chapter illustrates the observations that these beads with nanometer resolution use active feedback loops to suppress bead diffusion by rapid trap deflection, and observethe specimen by using brightfield and fluorescent imaging simultaneously.

In vitro methods for measuring force and velocity of the actin-myosin interaction using purified proteins.

Publisher Summary This chapter describes in vitro methods for measuring force and velocity of the actin–myosin interaction, using purified proteins. Myosin is a class of molecular motor that causes unidirectional movement of actin filaments, using the chemical energy obtained from the hydrolysis of ATP. In vitro motility assays provide an important approach to investigate myosin function, using only a small number of purified components. One intrinsic property of the myosin enzyme is its step size, which is defined as the average distance that a myosin moves an actin filament per ATP hydrolyzed. A tightly coupled model for myosin action depends on a one-to-one relationship between the release of ATP hydrolysis products and a force producing conformational change in myosin while bound to actin. The value of the step size could help to differentiate between these different models of myosin function. There is a minimum length of actin filament, dependent on the density of myosin on the surface, for continuous movement in the in vitro motility assay. Filaments longer than minimum length move continuously at the maximum speed, whereas shorter filaments dissociate from the surface in the presence of ATP. Another key to good movement in the in vitro motility assay is the quality of the myosin used. The in vitro motility assay can be extended to allow the measurement of force on single actin filaments by combining it with the technique of optical trapping.

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.

An infrastructure for high-throughput microscopy: instrumentation, informatics, and integration.

High-throughput, image-based cell assays are rapidly emerging as valuable tools for the pharmaceutical industry and academic laboratories for use in both drug discovery and basic cell biology research. Access to commercially available assay reagents and automated microscope systems has made it relatively straightforward for a laboratory to begin running assays and collecting image-based cell assay data, but doing so on a large scale can be more challenging. Challenges include process bottlenecks with sample preparation, image acquisition, and data analysis as well as day-to-day assay consistency, managing unprecedented quantities of image data, and fully extracting useful information from the primary assay data. This chapter considers many of the decisions needed to build a robust infrastructure that addresses these challenges. Infrastructure components described include integrated laboratory automation systems for sample preparation and imaging, as well as an informatics infrastructure for multilevel image and data analysis. Throughout the chapter we describe a variety of strategies that emphasize building processes that are scaleable, highly efficient, and rigorously quality controlled.