Synthesis of C-ring-modified blebbistatin derivatives and evaluation of their myosin II ATPase inhibitory potency.

(S)-Blebbistatin is a micromolar myosin II ATPase inhibitor that is extensively used in research. In search of analogs with improved potency, we have synthesized for the first time C-ring modified analogs. We introduced hydroxymethyl or allyloxymethyl functionalities in search of additional favorable interactions and a more optimal filling of the binding pocket. Unfortunately, the resulting compounds did not significantly inhibit the ATPase activity of rabbit skeletal-muscle myosin II. This and earlier reports suggest that rational design of potent myosin II inhibitors based on the architecture of the blebbistatin binding pocket is an ineffective strategy.

Mertk drives myosin II redistribution during retinal pigment epithelial phagocytosis.

PURPOSE: Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis. METHODS: Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells. RESULTS: Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect. CONCLUSIONS: Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.

Inhibition of skeletal muscle S1-myosin ATPase by peroxynitrite.

Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity, reaching 50% inhibition with 46.7 +/- 8.3 microM SIN-1 for 8.7 microM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca(2+)-dependent and K(+)/EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys(707) and Cys(697)), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5'-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys(707) labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP.V(1) to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

Thymol: a classical small-molecule compound that has a dual effect (potentiating and inhibitory) on myosin.

The effect of thymol on the ATPase activity of myosin subfragment-1 (S1) and on the contractile properties of skinned skeletal muscle fibers was studied. At concentrations of 1.5-2 mM, thymol activated the S1 ATPase substantially and the actin-activated S1 ATPase modestly. At the same concentrations, the isometric force of skinned skeletal muscle fibers was modestly suppressed (11% at 2 mM). However, the kinetic parameters of contraction were suppressed more: the velocity of shortening and the rate of force redevelopment after shortening were suppressed by 43% and 31% at 2 mM, respectively. Thus, among other small-molecule inhibitors, thymol is unique in that it has opposite effects on the enzymatic activity and kinetic parameters of contraction. Thymol may serve as a potent tool for studying the mechanism of coupling between the ATPase reaction and contraction in muscle.

Synthesis of biotinylated xestoquinone that retains inhibitory activity against Ca2+ ATPase of skeletal muscle myosin.

Xestoquinone isolated from a marine sponge binds to skeletal muscle myosin and inhibits its Ca(2+) ATPase activity. In this study, we first examined xestoquinone and its analogues to assess the relationships between structure and myosin Ca(2+) ATPase inhibitory activity. On the basis of the resultant data, we then designed a biotinylated xestoquinone analogue. Xestoquinone and its analogues were derived from extracts of the marine sponge Xestospongia sapra. Four xestoquinone analogues with a quinone structure significantly inhibited Ca(2+) ATPase activity. In contrast, four xestoquinone analogues in which the quinone structure was converted to a quinol dimethyl ether did not inhibit Ca(2+) ATPase activity. This suggests that the quinone moiety is essential for inhibitory activity. Then, we synthesized a biotinylated xestoquinone in which a biotin tag was introduced to a site far from the quinone moiety, and this molecule exhibited stronger inhibitory activity than that of xestoquinone. This biotinylated xestoquinone could be useful as a probe in studies of the xestoquinone-myosin binding mode.

A small-molecule inhibitor of skeletal muscle myosin II.

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.