SUMMARYThe pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two novel classes of small molecules that disrupt Wnt pathway responses - whereas one class inhibits the activity of Porcupine (Porcn), a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, suppressors of Wnt/β-catenin pathway activity. With these small molecules we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/β-catenin pathway response in vivo, and establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the liquid-ordered phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Increasing evidence suggests that cholesterol and sphingolipid-rich lipid microdomains or rafts exist in eukaryotic cell membranes and have important functions there (1-3). These rafts are likely to be important in the structure and function of caveolae, plasma membrane invaginations that are implicated in signal transduction (4, 5), endocytosis (6), transcytosis across endothelial cells (7,8), and cholesterol trafficking (9 -11). However, rafts are not restricted to caveolae (2, 3, 12) and recent evidence suggests that they act in signal transduction in cells that lack distinct caveolae, such as T lymphocytes (13-16) and basophils (17)(18)(19). Rafts have also been implicated in protein and lipid sorting in the secretory and endocytic pathways (1, 20 -22).Cholesterol and sphingolipid-rich detergent-resistant membranes (DRMs) 1 can be isolated from mammalian cells (23). DRM lipids are in a state similar to the liquid-ordered (l o ) phase (3, 24 -26). The l o phase, which requires cholesterol to form, is favored by lipids like sphingolipids, whose long saturated acyl chains give them a high degree of order and a high acyl-chain melting temperature (3). Acyl chain order explains the detergent-insolubility of DRMs (3). We hypothesize that DRMs are an in vitro correlate of rafts in intact membranes. It is important to note that detergent insolubility can underestimate the association of proteins and lipids with the l o phase; some proteins and lipids that are in rafts can be solubilized (25). Nevertheless, DRM association provides a powerful tool for identifying molecules that are likely to have a high affinity for rafts. DRMs isolated from cells contain a number of proteins (27-29) which are undoubtedly crucial for the function of the domains in vivo. For this reason, it is important to determine how proteins associate with DRMs. Three DRM targeting signals have been defined. First, glycosylphosphatidylinositol (GPI)-anchored proteins are targeted to DRMs through acyl chain interactions (23)(24)(25)30). An N-terminal Met-Gly-Cys motif that is present in some Src family kinases and heterotrimeric G protein ␣ subunits, in which Gly is myristoylated and Cys is palmitoylated, can also serve as a DRM targeting signal (31,32). Third, dual palmitoylated Cys residues are required for raft association of the T cell adaptor protein LAT (15) and the neu...

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.A key challenge for medicine is to develop new drugs against pathogens that are resistant to current antimicrobial agents (1,2). A promising strategy is to identify agents that inhibit microbial virulence without inhibiting growth, because these present less selective pressure for the generation of resistance (3-5). Many bacterial pathogens recognize the host environment by sensing and responding to the host adrenergic signaling molecules epinephrine and norepinephrine (NE) in order to promote the expression of virulence factors (6,7). These pathogens appear to use the same membrane-embedded sensor histidine kinase, QseC (7), to recognize both host-derived adrenergic signals and the bacterial aromatic signal autoinducer-3 (AI-3) to activate their virulence genes (5,6). Upon sensing any of these signaling molecules, QseC autophosphorylates and subsequently phosphorylates a transcription factor, QseB (Fig. 1A) (7), which initiates a relay to a complex regulatory cascade and leads to the transcription of key virulence genes (Fig. 1B) (5-8).QseC homologs are present in at least 25 important human and plant pathogens (table S1), and qseC mutants of enterohemorrhagic Escherichia coli (EHEC) (Fig. 1C and fig. S1) (7), Salmonella typhimurium ( Fig. 2A) (8), and Francisella tularensis (9) are attenuated in infected †To whom correspondence should be addressed.