Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC50 values ranging from 0.5 to 5 microM. Interestingly, smooth muscle, which is highly homologous to vertebrate nonmuscle myosin, is only poorly inhibited (IC50=80 microM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X. In cells, blebbistatin has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. ? We conducted a detailed investigation of blebbistatins mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a product complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.? We have recently evaluated the effectiveness of several blebbistatin derivatives towards rabbit skeletal muscle myosin and Dictyostelium myosin. These studies were combined with a crystallographic study in which some of the inhibitors were co-crystallized with Dictyostelium myosin. From these studies we have learned which of the positions on the blebbistatin rings can be modified which will serve as references for future derivatives.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL004234-05
Application #
7734994
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2008
Total Cost
$187,757
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Lucas-Lopez, Cristina; Allingham, John S; Lebl, Tomas et al. (2008) The small molecule tool (S)-(-)-blebbistatin: novel insights of relevance to myosin inhibitor design. Org Biomol Chem 6:2076-84
Sakamoto, Takeshi; Limouze, John; Combs, Christian A et al. (2005) Blebbistatin, a myosin II inhibitor, is photoinactivated by blue light. Biochemistry 44:584-8
Limouze, John; Straight, Aaron F; Mitchison, Timothy et al. (2004) Specificity of blebbistatin, an inhibitor of myosin II. J Muscle Res Cell Motil 25:337-41
Kovacs, Mihaly; Toth, Judit; Hetenyi, Csaba et al. (2004) Mechanism of blebbistatin inhibition of myosin II. J Biol Chem 279:35557-63
Straight, Aaron F; Cheung, Amy; Limouze, John et al. (2003) Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299:1743-7