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

Abstract

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 (IC 50 = 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.M yosins comprise a family of ATP-dependent motor proteins capable of producing directed force via interaction with their track, the F-actin filament. Force production by these motors powers numerous cellular processes such as muscle contraction, intracellular transport, and cell migration and division (1). Several myosins have also been linked to genetic disorders where either gain or loss of motor function can lead to disease. These motor proteins represent promising targets for the development of drugs modulating force production in cells, tissues, and muscle (2-4). Here we report a selective, smallmolecule inhibitor of smooth muscle myosin (SMM) able to induce muscle relaxation. This mechanism of action has potential relevance for many diseases where smooth muscle contractility is central to the pathophysiology, such as asthma (5, 6) and chronic obstructive pulmonary disease (7).Smooth muscle contractility can be activated through different pathways. Existing airway smooth muscle relaxants, such as β-adrenergic agonists and muscarinic antagonists, ultimately inhibit the activity of SMM. However, they do so via specific upstream signaling pathways. Direct inhibition of SMM contractility has the advantage of relaxing contracted smooth muscle regardless of the molecular stimulus driving it. Moreover, application of SMM inhibitors to the airway provides a means of selectively modulating contractility of these tissues by delivering a high local concentration of drug. We thus set about identifying selective inhibitors of SMM that can effectively relax muscle in vivo, leading to the discovery of a highly selective, small-molecule inhibitor, CK-2018571 (CK-571).The detailed inhibitory mechanism of CK-571 was elucidated by a combination of in vitro characterization of the step in which the drug traps the motor and determination of the high-resolution structure of SMM cocrystallized with CK-571. The drug targets an intermediate state that occurs during the recovery stroke, the large conformational rearrangement that enables repriming of the motor. Blocking this critical transiti...

Allosteric Modulation of Cardiac Myosin Dynamics by Omecamtiv Mecarbil

“…This finding is in agreement with the emerging model that CLD rotation is stabilised by the closure of Switch 2 upon ATP binding rather than being induced by it, so that it can occur at least partially before the changes in the nucleotide binding site take place [12,43,46]. This would also explain the recent observation that myosin can be trapped by inhibitors in intermediate states of the recovery stroke without closure of Switch 2 [12]. Moreover, a decoupling between Switch 2 and lever arm motions is consistent with the recent observation of a new structural state of Myosin VI where a large conformational change of Switch 2 does not produce a corresponding change in the lever arm position [47].…”

“…The motions observed here, while not representing an actual transition to the pre-powerstroke state and while they might have been enhanced by the absence of the Regulatory Domain, suggest a conformational selection scenario for the recovery stroke, where the type of motions involved in the stroke are already partially sampled by the CLD before the binding of ATP. This finding is in agreement with the emerging model that CLD rotation is stabilised by the closure of Switch 2 upon ATP binding rather than being induced by it, so that it can occur at least partially before the changes in the nucleotide binding site take place [12,43,46]. This would also explain the recent observation that myosin can be trapped by inhibitors in intermediate states of the recovery stroke without closure of Switch 2 [12].…”

“…Structural information on the binding site is available only for some of these inhibitors. Blebbistatin [11] binds to the 50-K cleft between the U50K and L50K subdomains, while the smooth muscle myosin inhibitor CK-571 has been recently shown to bind to a pocket between the relay and SH1 helices [12]. Interestingly, CK-571 uses a unique inhibition mechanism where the drug stabilises a previously unknown intermediate step along the recovery stroke and prevents myosin to reach the pre-powerstroke state, thus hindering the ATP hydrolysis [12].…”

An intermediate along the recovery stroke of myosin VI revealed by X-ray crystallography and molecular dynamics

“…Defective myosins were found to be implicated in severe pathologies in humans such as hypertrophic cardiomyopathy (2) and deafness (3), while others, including myosin VI, were shown to have a role in cancer cell proliferation and metastasis (4). Recent studies highlighted the therapeutic potential of small-molecule inhibitors (5) and activators (6)(7)(8) targeting myosin, demonstrating that a detailed knowledge of the force production mechanism in this motor family would facilitate the rational design of drug candidates.…”

Mavacamten stabilizes the auto-inhibited state of two-headed cardiac myosin

“…We detected free phosphate in solution as described in [46]. 12.5 M cardiac HMM (25 M heads) was manually mixed with 20 M ATP (Sigma-Aldrich), allowed to incubate for 5.0 seconds and quenched with 0.6 M perchloric acid, and detected with malachite green as described in [47].…”