Actin and myosin are responsible for many forms of cell movement. Actin-myosin interactions are critical for muscle contraction, and they support cytoskeletal rearrangements in non-muscle cells during processes like cytokinesis, cell signaling, endocytosis, and cell adhesion and migration. Given that these processes underlie many cell functions, altering the dynamics of cell contractility and cytoskeletal rearrangement with modulators that target actin or myosin is an attractive approach for the treatment of many diseases. Small molecules that target actin-myosin dynamics are in clinical trials for the treatment of cardiovascular conditions like hypertrophic cardiomyopathy and heart failure. Others are proposed to halt cancer metastasis, benefit neurological conditions such as autism and Alzheimer’s disease, or improve neuronal function after traumatic spinal cord injury.

Jump to actin and myosin modulators

Actin and Myosin

Actin is a monomeric, globular protein that forms thin filaments in muscle cells and polymerizes into microfilaments in the cytoskeleton. It serves as a track for myosins, a large family of molecular motor proteins that form thick filaments in muscle cells and convert chemical energy from ATP into mechanical energy for cell movement. Most myosins are classified as class II myosins (myosin II), which are further grouped by tissue expression. Muscle myosin II isoforms are expressed in skeletal, cardiac, and smooth muscle cells in a tissue-specific manner whereas non-muscle myosin II is ubiquitously expressed.

Contractile Assemblies in Muscle and Non-Muscle Cells

The cyclical engagement of actin and myosin drives muscle contraction and plays a key role in cytoskeletal rearrangements in non-muscle cells. The sliding filament model describes how actin and myosin are thought to coordinate muscle contraction (Figure 1). The mechanical energy needed to power cell contractility is driven by the ATPase activity of myosin. ATP binding causes myosin to detach from actin. ATP is hydrolyzed to ADP and inorganic phosphate (P_i) by myosin ATPase, inducing a conformational change in the myosin head that moves it into a high-energy, "cocked" position where ADP and P_i remain bound to the myosin head. Next, the myosin head binds to a new position on the actin filament, re-forming the actin cross-bridge. During the power stroke, ADP and P_i are released, and the myosin head pivots and bends, moving myosin along the actin filament and returning it to its low-energy conformation. Then, ATP can bind to myosin again, repeating the cycle. 

Figure 1. The sliding filament model of muscle contraction. The energy from ATP hydrolysis drives a conformational change in the myosin head, which moves myosin along the actin filament with each round of ATP hydrolysis. Contractile assemblies are also present in non-muscle cells, where a similar mechanism occurs.

Contractile assemblies are also present in non-muscle cells, where a similar mechanism of actin-myosin interactions occurs to mediate cytoskeletal rearrangements. The main difference in contractile assemblies between muscle and non-muscle cells are the accessory proteins that regulate myosin activity. In skeletal and cardiac muscle cells, myosin activity is regulated by troponin and tropomyosin. Troponin is activated by calcium, inducing a conformational change in troponin that moves tropomyosin away from myosin binding sites on actin filaments. In non-muscle cells and smooth muscle cells, myosin activity is regulated via phosphorylation by myosin light chain kinase (MLCK or MYLK). MLCK is a calcium/calmodulin-dependent protein kinase that phosphorylates the regulatory MLC, promoting actin-myosin interactions and cell contractility.

Actin and myosin dynamics are critical for normal cell processes like muscle contraction or cytoskeletal rearrangements that mediate cell division, signaling, adhesion, and migration. Neuronal plasticity, which is required for memory formation, learning, and recovery from CNS injury, is also dependent on actin and myosin. However, these same processes parallel morphological and signaling changes that drive cancer progression and metastasis. Hence, the identification of modulators of actin and myosin dynamics holds promise for the treatment of many diseases.

Actin Polymerization

Actin dynamics can be controlled with actin depolymerizing agents and actin polymerization inhibitors. Actin polymerization inhibitors are invaluable in cell biology experiments requiring inhibition of processes related to cytoskeletal rearrangement like endocytosis or intracellular trafficking. Natural products like cytochalasins and latrunculins bind monomeric actin to inhibit actin polymerization. Another strategy to inhibit actin polymerization is to target the actin-related protein 2/3 (Arp2/3) complex, which regulates the initiation of actin polymerization, with agents such as CK-636, CK-869, and CK-666. Because actin is key to many normal cell functions, broad pharmacological inhibition of actin polymerization is not a viable therapeutic strategy. Finding new approaches for targeted drug delivery, like lipid-based nanoparticles, may one day overcome this challenge.

Myosin ATPase

Myosin ATPase modulators have uses ranging from basic cell biology research to preclinical applications. The most well-known myosin ATPase inhibitor is (–)-blebbistatin, a selective inhibitor of non-muscle myosin II ATPases. It is cell-permeable and commonly used to investigate the role of myosin in in vitro and in vivo studies. Blebbistatin has been used to inhibit cancer cell migration in vitro and to promote neuronal axon growth, indicating that non-muscle myosin II ATPases inhibitors have potential application in the treatment of cancer and neurodegenerative disorders. Blebbistatin derivatives like para-amino-blebbistatin and (S)-4'-nitro-blebbistatin, which are offered exclusively by Cayman, have improved stability, reduced phototoxicity, and enhanced water solubility. Blebbistatins and other myosin ATPase inhibitors like 2,3-butanedione-2-monoxime and N-benzyl-p-toluenesulfonamide (BTS) have been used in experiments using isolated myocytes or muscle preparations to suppress contractions that would otherwise confound the interpretation of experimental results.

Myosin ATPases have roles in several pathologies, including cardiovascular disease, cancer, and neuronal disorders. Muscle myosin is the most downstream effector of muscle contraction. Hence, targeting myosin activity with ATPase or MLCK modulators permits direct modulation of the contractile machinery and avoids interference with upstream effectors like calcium, which has many physiological effects. Modulators of cardiac myosin II ATPases are actively being investigated for the treatment of cardiovascular diseases. Mavacamten and aficamten are cardiac myosin ATPase inhibitors that reduce cardiac contractility and have been used in FDA-approved formulations for the treatment of symptomatic obstructive hypertrophic cardiomyopathy. Omecamtiv mecarbil and danicamtiv, formerly known as MYK-491, are cardiac myosin ATPase activators. They increase cardiomyocyte contractility and are in clinical trials for the treatment of heart failure.   

MLCK

Increased expression or activity of MLCK has been observed in cancer, cardiac diseases like atherosclerosis, hypertension, and heart failure, and respiratory conditions like asthma. Wortmannin, a fungal metabolite commonly used in basic research applications to inhibit PI3K, also inhibits MLCK. ML-7 and its congener, ML-9, have been investigated for use in hypertension and atherosclerosis. While MLCK inhibitors have potential therapeutic utility, very few are commercially available. Hence, there is a need to identify new MLCK inhibitors. Cayman's Medicinal and Computational Chemistry Division can help identify new inhibitors through their screening, structure-based drug design, and hit-to-lead services.

Tools to Study Actin-Myosin Dynamics

Finding new ways to disrupt actin-myosin dynamics holds promise for the treatment of countless diseases. Cayman supports these therapeutic opportunities by providing dedicated toolsets to study microtubule dynamics and cytoskeleton and motor proteins like actin and myosin. We also offer fluorogenic dyes that can be used to visualize actin filaments in viable or fixed cells as well as assay kits and fluorogenic probes to study cellular processes like phagocytosis and the endomembrane system and vesicular trafficking, respectively.

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Suggested Reading

Bond, L.M., Tumbarello, D.A., Kendrick-Jones, J., et al. Small-molecule inhibitors of myosin proteins. Future Med. Chem. 5(1), 41-52 (2012).