Cell morphology and motility are governed largely by complex signaling networks that ultimately engage the actin cytoskeleton. Understanding how individual circuits contribute to the process of forming cellular structures would be aided greatly by the availability of specific chemical inhibitors. We have used a novel chemical screen in Xenopus cell-free extracts to identify compounds that inhibit signaling pathways regulating actin polymerization. Here we report the results of a high-throughput screen for compounds that inhibit phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin assembly and the identification of the first compound, a cyclic peptide, known to block actin assembly by inhibiting an upstream signaling component. We identify the target of this compound as N-WASP, a protein that has been investigated for its role as a node interconnecting various actin signaling networks. We show that this compound prevents activation of the Arp2/3 complex by N-WASP by allosterically stabilizing the autoinhibited conformation of N-WASP.

The yeast transcriptional repressor Sir2p silences gene expression from the telomeric, rDNA, and silent mating-type loci and may play a role in higher order processes such as aging. Sir2p is the founding member of a large family of NAD-dependent deacetylase enzymes, named the sirtuins. These proteins are conserved from prokaryotes to eukaryotes, but most remain uncharacterized, including all seven human sirtuins. A reverse chemical genetic approach would be useful in identifying the biological function of sirtuins in a wide variety of experimental systems, but no cell-permeable small molecule inhibitors of sirtuins have been reported previously. Herein we describe a high throughput, phenotypic screen in cells that led to the discovery of a class of sirtuin inhibitors. All three compounds inhibited yeast Sir2p transcriptional silencing activity in vivo, and yeast Sir2p and human SIRT2 deacetylase activity in vitro. Such specific results demonstrate the utility and robustness of this screening methodology. Structure-activity relationship analysis of the compounds identified a key hydroxy-napthaldehyde moiety that is necessary and sufficient for inhibitory activity. Preliminary studies using one of these compounds suggest that inhibition of sirtuins interferes with body axis formation in Arabidopsis.

To study the role of the BH3 domain in mediating pro-apoptotic and anti-apoptotic activities of Bcl-2 family members, we identified a series of novel small molecules (BH3Is) that inhibit the binding of the Bak BH3 peptide to Bcl-xL. NMR analyses revealed that BH3Is target the BH3-binding pocket of Bcl-xL. Inhibitors specifically block the BH3-domain-mediated heterodimerization between Bcl-2 family members in vitro and in vivo and induce apoptosis. Our results indicate that BH3-dependent heterodimerization is the key function of anti-apoptotic Bcl-2 family members and is required for the maintenance of cellular homeostasis.