The biochemical mode of action of bistramides

Bistramide A is a highly potent antiproliferative marine natural product from Lissoclinum histratum. It is capable of severing actin filaments. This study provides comprehensive characterization of the biochemical mode of action of bistramide A and identifies the structural requirements of bistramide-based compounds that are responsible for severing actin filaments and inhibiting growth of cancer cells in vitro and in vivo. We studied herein the X-ray structure of bistramide A bound to monomeric actin at a resolution of 1.35 A. The most notable aspect of the bistramide A-actin structure is an extensive hydrogen-bonding network established upon a deep penetration of the central segment of bistramide A into the actin-binding cleft between subdomains 1 and 3. The structure presents the first insight into the observed ability of bistramide A to modulate G-actin polymerization. The structural information combined with our ability to chemically modify the bistramide framework provided the basis for rational design and assembly of a series of new synthetic analogues. We used TIRF microscopy to directly observe actin filament severing by this series of small molecules, which established that the combination of the spiroketal and the amide subunits was sufficient to enable rapid actin filament disassembly in vitro. In addition, we demonstrated that the enone subunit of bistramide A is responsible for covalent modification of the protein in vitro and in A549 cells, resulting in further increase in the cytotoxicity of the natural product. Our results demonstrate that bistramide A elicits its potent antiproliferative activity by a dual mechanism of action, which entails both severing of actin filaments and covalent sequestration of monomeric actin in the cell.;Formins are a family of proteins involved in the regulation of cytokinesis. We developed a high-throughput assay of formin-mediated polymerization of actin. In search for inhibitors of this process, we screened a representative chemical library and identified a number of small molecules that effectively blocked formin-mediated actin polymerization. This represents the first step en route to the development of selective and potent inhibitors of formin-actin interaction.