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 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 (ATP) 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.
Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-proteincoupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K d of 60 nM, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1-and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu 291 ) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu 291 , because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu 291 . In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu 291, from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu 291 to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.Chemokines are a large family of small proteins that mediate attraction of leukocytes to inflammatory sites (1-3). The chemokine family shares a common pattern of disulfide bonds and a common overall tertiary structure as shown in solution or crystallographically determined structures (4 -6). The chemokine family is divided into four subfamilies based on the number of residues between the first and second cysteine. Among the chemokines, the CC chemokine monocyte chemoattracant-1 (MCP-1) 1 has received a great deal of attention because of its involvement in diseases. MCP-1 expression is elevated in the inflamed synovium of rheumatoid arthritis, and its expression is reduced by anti-arthritic drugs (7,8). Other work has shown that MCP-1 is elevated in asthmatic patients; the am...
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.entromere-associated protein-E (CENP-E; kinesin-7) is a kinetochore-associated kinesin motor protein with an essential and exclusive role in metaphase chromosome alignment and satisfaction of the mitotic checkpoint (1). CENP-E is a likely candidate to integrate the mechanics of kinetochore-microtubule interaction with the mitotic checkpoint signaling machinery responsible for restraining cell-cycle progression into anaphase. CENP-E is a large dimeric protein consisting of an N-terminal kinesin motor domain tethered to a globular C-terminal domain through an extended coiled-coil rod domain (2, 3). The C-terminal, noncatalytic region of CENP-E is not only sufficient to specify localization to kinetochores, but it also mediates interaction of CENP-E with the serine/threonine kinase BubR1, a key effector of mitotic checkpoint signaling that forms complexes with the checkpoint proteins Cdc20, Bub3, and Mad2 to inhibit the ubiquitin ligase activity of the anaphase promoting complex APC/C CDC20 (4-7). The combined interaction of CENP-E with microtubules and a key regulator of APC/C CDC20 has led to the hypothesis that CENP-E functions as the key kinetochore microtubule receptor responsible for silencing mitotic checkpoint signal transduction after capture of spindle microtubules. This hypothesis was further strengthened by the finding that CENP-E could stimulate the kinase activity of BubR1 in a microtubule-sensitive manner (8, 9). In vitro, the addition of CENP-E to BubR1 resulted in a stimulation of BubR1 kinase activity. The addition of microtubules suppressed this stimulatory activity, an effect thought to be mediated by the CENP-E kinesin motor domain. Although the importance of CENP-E interaction with BubR1 and the role of BubR1-mediated phosphorylation in mitotic checkpoint function remain unclear, CENP-E remains a prominent candidate to play a key role in mitotic checkpoint signal transduction.Depletion of CENP-E from ...
Limited neuromuscular input results in muscle weakness in neuromuscular disease either because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission1. We developed a small molecule fast skeletal troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neuromuscular input is diminished secondary to a neuromuscular disease. Binding selectively to the fast skeletal troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards as does the force-frequency relationship of a nerve-muscle pair. In vitro and in vivo, CK-2017357 increases the production of force at sub-maximal stimulation rates. Importantly, we show that sensitization of the fast skeletal troponin complex to calcium improves muscle force and grip strength immediately after single doses of CK-2017357 in a model of neuromuscular disease, myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.
Background-Therapy for chronic systolic heart failure (sHF) has improved over the past 2 decades, but the armamentarium of drugs is limited and consequently sHF remains a leading cause of death and disability. In this investigation, we examined the effects of a novel cardiac myosin activator, omecamtiv mecarbil (formerly CK-1827452) in 2 different models of heart failure. Methods and Results-Two different models of sHF were used: (1) pacing-induced sHF after myocardial infarction (MI-sHF) and (2) pacing-induced sHF after 1 year of chronic pressure overload left ventricular hypertrophy (LVH-sHF).Omecamtiv mecarbil increased systolic function in sHF dogs, chronically instrumented to measure LV pressure, wall thickness, and cardiac output. Omecamtiv mecarbil, infused for 24 hours, induced a sustained increase without desensitization (PϽ0.05) in wall thickening (25Ϯ6.2%), stroke volume (44Ϯ6.5%) and cardiac output (22Ϯ2.8%), and decreased heart rate (15Ϯ3.0%). The major differences between the effect of omecamtiv mecarbil on cardiac function and the effect induced by a catecholamine, for example, dobutamine, is that omecamtiv mecarbil did not increase LV dP/dt but rather increased LV systolic ejection time by 26Ϯ2.9% in sHF. Another key difference is that myocardial O 2 consumption (MVO 2 ), which increases with catecholamines, was not significantly affected by omecamtiv mecarbil. Conclusions-These results demonstrate that chronic infusion of the cardiac myosin activator, omecamtiv mecarbil, improves LV function in sHF without the limitations of progressive desensitization and increased MVO 2. This unique profile may provide a new therapeutic approach for patients with sHF. (Circ Heart Fail. 2010;3:522-527.)
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