Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIB

Ramamurthy, Bhagavathi; Yengo, Christopher M.; Straight, Aaron F.; Mitchison, Timothy J.; Sweeney, H. Lee

doi:10.1021/bi0490284

Cited by 90 publications

(91 citation statements)

References 18 publications

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“…1B). These results are consistent with blebbistatin binding to the myosin-ADP-Pi complex, inhibiting the rate of release of P i and trapping myosin in an F-actin weak-binding state, as has been proposed from more detailed studies of skeletal muscle myosin II (25) and nonmuscle myosin IIB (26).…”

Section: Resultssupporting

confidence: 88%

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Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

Shu

Liu

Korn

2005

Proc. Natl. Acad. Sci. U.S.A.

101

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Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatininactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II.phagocytosis ͉ macropinocytosis ͉ cell streaming ͉ development B lebbistatin (1), a cell-permeable inhibitor of myosin II ATPase activity, promises to be a very useful tool for identifying and studying myosin II-dependent processes in cells. As for any pharmacologic agent, however, extended use of blebbistatin will depend on the specificity and generality of its inhibition of class-II myosins. Thus far, blebbistatin's effect has been tested in vitro (1, 2) on only a few representatives of four of the 18 myosin classes that, together, contain Ͼ200 myosins. Blebbistatin inhibits myosin II from striated muscle, vertebrate nonmuscle cells, and Dictyostelium by Ϸ95%, with an IC 50 of 0.5-5 M. Smooth muscle and Acanthamoeba myosin II are only incompletely inhibited at blebbistatin concentrations as high as 160 M, with IC 50 values of Ϸ80 M, and blebbistatin does not inhibit either rat myosin Ib, Acanthamoeba myosin IC, mouse myosin V, or bovine myosin X. It is not practical to test the blebbistatin sensitivity of all of the myosins or to determine whether blebbistatin might inhibit enzymes other than myosins, whether some functions of myosin II might not require enzymatic activity (and, therefore, not be inhibited by blebbistatin), or whether enzymatically inactive myosin II might directly or indirectly affect any non-myosin II-dependent cell function in all cells.The social amoeba Dictyostelium discoideum provides a useful system for assessing some of these variables. Dictyos...

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Section: Resultssupporting

confidence: 88%

“…1A). As was reported for skeletal muscle myosin II (25) and nonmuscle myosin IIB (26), blebbistatin also inhibits the basal (non-actin-activated) MgATPase of Dictyostelium myosin II (Fig. 1 A), with a slightly lower IC 50 .…”

Section: Resultssupporting

confidence: 81%

Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

Shu

Liu

Korn

2005

Proc. Natl. Acad. Sci. U.S.A.

101

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“…During the myosin ATPase cycle, this cleft characteristically closes as a result of structural rearrangements in the nucleotide-binding site mediating the process of Pi release and subsequent ADP release [28]. Blebbistatin binding stabilizes the closed ADP/Pi bound myosin II intermediate state, thus preventing the release of Pi and the associated myosin power stroke (i.e., the force generating step) ( Figure 1) [27,29,30]. Blebbistatin is highly suited for studies of the cellular functions of cytoplasmic myosin II, as it blocks myosin II activity in an actin-detached state, thus, preventing artifacts from the formation of strongly bound nonfunctional actomyosin complexes [29].…”

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confidence: 99%

Small-Molecule Inhibitors of Myosin Proteins

et al. 2012

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Advances in screening and computational methods have enhanced recent efforts to discover/ design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design.The identification and characterization of pharmacological compounds that inhibit the functional activity of one or more specific proteins or processes has been the subject of much scientific investigation. On a basic science level, these membrane-permeable compounds provide the scientific community with a tool for the targeted and functional inhibition of a given protein in the cell; a potent means of evaluating the intracellular functions of that protein [1,2]. From a biomedical standpoint, the characterization of these small-molecule inhibitors affords an opportunity for the development of novel disease treatments centering on the repression of an offensive molecule or the reversal of its downstream effects [3][4][5].At present, several complementary methods for obtaining suitable small-molecule inhibitors of specific proteins exist. Traditional methods in inhibitor discovery involve the systematic testing of a series of chemically synthesized or naturally occurring compounds. Advances in robotics and data processing have made it possible to use high-throughput screens to test libraries of thousands or even millions of potential drugs for their ability to inhibit the function of a specific protein in a targeted biochemical or cellular assay [6][7][8]. These inhibitor discovery processes are complemented by more precise methods in small-molecule inhibitor design. Structure-based methods rely on the use of x-ray crystallographic or NMRbased structures of a protein of interest to design small molecules likely to bind and inhibit protein function [9,10]. Computer-aided inhibitor design uses computational methods to optimize potential inhibitors identified by screening or structure-based methods, to virtually © 2013 Folma Buss * Author for correspondence: Tel.: +44 1223 763348, Fax: +44 1223 762640, fb207@cam.ac.uk. Financial & competing interests disclosureThe authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance w...

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“…However, a series of functional, kinetic and structural investigations have shown that (S)-(-)-blebbistatin (1) achieves selectivity by binding in a highly hydrophobic cleft in the myosin motor domain. [5][6][7][8] Surprisingly, this is one of the most conserved components of all myosin motor domains where a few small sequence and structural differences lead to the ability of 1 to discriminate between myosin isoforms.…”

Section: Introductionmentioning

confidence: 99%

The small molecule tool (S)-(−)-blebbistatin: novel insights of relevance to myosin inhibitor design

et al. 2008

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The small molecule blebbistatin is now a front line tool in the study of myosin function. Chemical modification of the tricyclic core of blebbistatin could deliver the next generation of myosin inhibitors and to help address this we report here on the impact of structural changes in the methyl-substituted aromatic ring of blebbistatin on its biological activity. Chemical methods for the preparation of isomeric methyl-containing analogues are reported and a series of co-crystal structures are used to rationalise the observed variations in their biological activity. These studies further support the view that the previously identified binding mode of blebbistatin to Dictyostelium discoideum myosin II is of relevance to its mode of action. A discussion of the role that these observations have on planning the synthesis of focused libraries of blebbistatin analogues is also provided including an assessment of possibilities by computational methods. These studies are ultimately directed at the development of novel myosin inhibitors with improved affinity and different selectivity profiles from blebbistatin itself.

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Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIB

Cited by 90 publications

References 18 publications

Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

Small-Molecule Inhibitors of Myosin Proteins

The small molecule tool (S)-(−)-blebbistatin: novel insights of relevance to myosin inhibitor design

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