Inhibition of Myosin ATPase Activity by Halogenated Pseudilins: A Structure–Activity Study

Abstract

Myosin activity is crucial for many biological functions. Strong links have been established between changes in the activity of specific myosin isoforms and diseases such as cancer, cardiovascular failure, and disorders of sensory organs and the central nervous system. The modulation of specific myosin isoforms therefore holds a strong therapeutic potential. In recent work, we identified members of the marine alkaloid family of pseudilins as potent inhibitors of myosin-dependent processes. Here, we report the crystal structure of the complex between the Dictyostelium myosin 2 motor domain and 2,4-dichloro-6-(3,4,5-tribromo-1H-pyrrole-2-yl)phenol (3). Detailed comparison with previously solved structures of the myosin 2 complex with bound pentabromopseudilin (2a) or pentachloropseudilin (4a) provides insights into the molecular basis of the allosteric communication between the catalytic and the allosteric sites. Moreover, we describe the inhibitory potency for a congeneric series of halogenated pseudilins. Insight into their mode of action is gained by applying a combination of experimental and computational approaches.

Small-Molecule Inhibitors of Myosin Proteins

“…The PCIP-binding pocket is located near actin-binding residues at the tip of the 50-kDa cleft in the myosin motor domain, approximately 16 Å from the nucleotide binding site and 7.5 Å from the hydrophobic pocket bound by blebbistatin. PCIP binding to this allosteric pocket reduces ATPase activity via a conserved communication pathway between the allosteric and nucleotide binding sites that results in local structural changes in the myosin, reduction of coupling between the actin and nucleotide binding sites, and global changes in myosin dynamics ( Figure 1) [54,55]. Complementary computational studies indicate that the specific preference of PCIP for the binding pocket in class I myosins may stem from the presence of a relatively higher number of polar/charged residues within the binding site of this myosin class [55].…”

“…In particular, PBP most potently inhibits myosin V due to its affinity for the increased hydrophobicity of the binding pocket in this class of myosins [55]. PBP has been shown to inhibit the ATPase activity of this myosin by increasing its affinity for ADP, decreasing ATP binding and hydrolysis rates, and reducing coupling between the actin-and nucleotide-binding sites in the motor domain (Figure 1).…”

“…PCIP binding to this allosteric pocket reduces ATPase activity via a conserved communication pathway between the allosteric and nucleotide binding sites that results in local structural changes in the myosin, reduction of coupling between the actin and nucleotide binding sites, and global changes in myosin dynamics (Figure 1) [54,55]. Complementary computational studies indicate that the specific preference of PCIP for the binding pocket in class I myosins may stem from the presence of a relatively higher number of polar/charged residues within the binding site of this myosin class [55].It is important to note that although the potent inhibition of myosin I proteins by PCIP has been characterized in greatest detail, the drug also inhibits the ATPase activity of myosin Vb and nonmuscle myosin II at higher concentrations (IC 50 >90 μM) [54]. This lack of specificity at higher concentrations must be taken into account when designing in vivo experiments using PCIP.…”

“…PBP has been shown to inhibit the ATPase activity of this myosin by increasing its affinity for ADP, decreasing ATP binding and hydrolysis rates, and reducing coupling between the actin-and nucleotide-binding sites in the motor domain (Figure 1). These changes occur via a conserved communication pathway between the allosteric and nucleotide-binding sites [55,58].The potential for the targeted use of PBP as a myosin Va inhibitor in vivo is limited by the fact that this molecule has a wide range of effects on other myosins and myosin-dependent processes. At higher concentrations than those required for myosin Va inhibition, PBP inhibits the ATPase activity of myosin Vb (IC 50 = ~20 μM), non-muscle myosin II (IC 50 = 25 μM) and myosin Ie (IC 50 = ~50 μM) [58].…”

Azidoblebbistatin, a photoreactive myosin inhibitor

“…As BDM has turned out to have a broad effect on many other proteins (7) and the inhibitory effect of BTS is limited to fast skeletal muscle myosin, until now blebbistatin has been the only potent tool for specific blocking of myosin II-dependent processes in various species and cell types. Recent data have shown that halogenated pseudilins also have nonspecific inhibitory effects on myosin II isoforms (8)(9)(10).…”