A myosin V inhibitor based on privileged chemical scaffolds.

Small molecules that perturb the function of their targets on fast time scales can be powerful probes of dynamic cellular processes, such as intracellular transport. A number of inhibitors for motor proteins, ATPases that drive the movement of cellular cargo, have been reported. These chemical inhibitors (with micromolar potency) have served as valuable tools for the dissection of complex cellular mechanisms and have even provided an impetus for the development of chemotherapeutics that target motor proteins. However, chemical inhibitors are available for only approximately 6% of the motor proteins (there are over 100 in humans) involved in a variety of biological processes, including development, hearing, intracellular signaling, and muscle function. Myosins are motor proteins that move along the actin cytoskeleton (Figure 1). Since their initial characterization as proteins that drive muscle contraction, 18 different classes of myosins have been characterized, and it is now known that myosins are involved in almost every aspect of biological motion. However, specific small-molecule probes are available only for class II myosins. Therefore, we have set the development of chemical probes for members of the other myosin classes as our long-term goal. The analysis of myosin structures reveals that although these enzymes bind ATP, their structures are similar to those of GTPases. Good inhibitors for GTPases have been generally difficult to obtain. We reasoned that the high nucleotide affinity (low nanomolar), and not the structure of the nucleotide-binding pocket itself, is a key factor limiting the identification of GTPase inhibitors. This factor has also been noted in the context of inhibitor development for kinases that have unusually high ATP affinity. Myosins have aKM value for ATP that is typically in themicromolar range, [8] which raises the possibility that inhibitors for these enzymes may be more readily accessible. To test this hypothesis, we focused on class V myosins. These motor proteins are essential for survival in eukaryotes, and mutations that impair activity give rise to pigmentation and neurological defects in mice and humans. The micromechanics and mechanochemistry of myosin V have been the focus of intense research. However, the precise cellular functions of myosin V remain poorly characterized, particularly in vertebrates, and a small-molecule inhibitor would be a valuable tool. We have shown that small molecules based on “privileged” chemical scaffolds, which map to the region of chemical space occupied by known bioactive compounds, can yield diverse cellular phenotypes. These results, along with other studies, suggest that privileged-scaffold-based compounds may provide efficient starting points for the development of inhibitors of different target proteins. We noted that such scaffolds include pyrimidines 1, oxindoles 2, pyrrolopyrimidines 3, and pyrazolopyrimidines 4 (Figure 1). We also noted that such scaffolds are common to many known kinase inhibitors. The specificity of kinase inhibitors is typically examined in vitro against a large panel of known kinases. However, the ability of these inhibitors to target motor proteins has not been examined systematically. To determine whether kinase inhibitors could inhibit myosin V, Figure 1. Illustration of myosin V walking on actin filaments and structures of compounds based on “privileged” scaffolds (bold) as potential inhibitors (X=NH or CH2; R 1–3 are various aliphatic or aromatic groups; ADP=adenosine diphosphate, ATP=adenosine triphosphate).