Diverse chemical scaffolds support direct inhibition of the membrane-bound O-acyltransferase porcupine

Abstract

Secreted Wnt proteins constitute one of the largest families of intercellular signaling molecules in vertebrates with essential roles in embryonic development and adult tissue homeostasis. The functional redundancy of Wnt genes and the many forms of cellular responses they elicit, including some utilizing the transcriptional co-activator β-catenin, has limited the ability of classical genetic strategies to uncover their roles in vivo. We had previously identified a chemical compound class termed Inhibitor of Wnt Production (or IWP) that targets Porcupine (Porcn), an acyltransferase catalyzing the addition of fatty acid adducts onto Wnt proteins. Here we demonstrate that diverse chemical structures are able to inhibit Porcn by targeting its putative active site. When deployed in concert with small molecules that modulate the activity of Tankyrase enzymes and glycogen synthase kinase 3 β (GSK3β), additional transducers of Wnt/β-catenin signaling, the IWP compounds reveal an essential role for Wnt protein fatty acylation in eliciting β-catenin-dependent and -independent forms of Wnt signaling during zebrafish development. This collection of small molecules facilitates rapid dissection of Wnt gene function in vivo by limiting the influence of redundant Wnt gene functions on phenotypic outcomes and enables temporal manipulation of Wnt-mediated signaling in vertebrates.

FIGURE 1.

The IWP compounds directly attack Porcn. A, synthesis of a fluorescently labeled Porcn inhibitor. The IWP2 molecule was modified as shown with a linker and Cy3 adduct to generate an IWP-Cy3 fluorescently labeled probe. B, Porcn mutant with an altered putative active site residue (Porcn H335D) does not engage IWP-Cy3. Wild type Porcn or Porcn H335D sequence fused to Gaussia luciferase DNA (to stabilize the Porcn H335D protein) was transfected into COS1 cells. IWP binding to Porcn proteins was assessed by treating transfected cells with IWP-Cy3 and scoring the number of Cy3-positive cells in each experiment. Percentages shown are relative to control (wild type Porcn-GL-transfected cells). Competition with unlabeled IWP2 serves as a specificity control for IWP-Cy3 binding. C, Porcn fusion proteins exhibit comparable activity to unmodified Porcn protein. The ability of various Porcn proteins to counter the effects of IWP2 on Wnt/β-catenin pathway response was monitored using the SuperTopFlash (STF) reporter in HEK293 cells. Both wild-type Porcn and mutant proteins harboring a putative active site alteration (histidine 335) exhibited comparable activity in this assay regardless of whether GL or Myc epitope were appended to the Porcn C terminus. D, expression levels of wild-type and H335D Porcn are comparable. Western blot analysis of wt and H335D Porcn in COS1 cells in the presence or absence of IWP2. Kif3A serves as a loading control. E, Porcn and Porcn-H335D predominantly exhibit intracellular subcellular localization. Porcn-and Porcn-H335D-GL proteins exhibit a reticular expression pattern consistent with previous assignment of Porcn localization to the endoplasmic reticulum.

FIGURE 2.

The IWP compounds specifically inhibit Porcn acyltransferase activity. A, IWP2 inhibits Wnt fatty acylation. Cells transfected with an expression construct expressing either a fusion molecule consisting of Wnt3A and the Fc region of human IgG (Wnt3A-Fc) or IgG-Fc (Fc) alone are treated with C16 ω-alkynyl fatty acid (alkynyl-PA). Purified alkynyl-PA-labeled fusion protein bound to protein A-Sepharose is treated with biotin-azide reagent which enables protein detection using streptavidin-HRP. RNAi-mediated knock-down or overexpression of Porcn respectively results in loss or increase in Wnt3A-Fc protein labeling with alkynyl-PA. IWP2 is able to block the labeling of Wnt3A. B, IWP2 does not inhibit Hh fatty acylation. The same click chemistry strategy is used to monitor fatty acylation of Hh protein. C, IWP-Cy3 specifically binds to Porcn. COS-1 cells overexpressing Porcn or other members of the MBOAT family with recognized protein substrates (HHAT and GOAT) were treated with IWP-Cy3 and then gated for Cy3 staining. The number of IWP-Cy3-associated cells was scored as before.

FIGURE 3.

The IWP compounds target both β-catenin-dependent and -independent Wnt pathway responses. A, IWP2 inhibits the secretion of Wnt1 protein in embryonic kidneys. Urogenital systems from E11.5 mice expressing Wnt1-GFP were removed, bisected, and treated with either DMSO or IWP2 in vitro for 24 h. A cross section of a ureteric bud was analyzed for Wnt1-GFP expression (top panels). The effect of IWP2 on branching morphogenesis in the kidney was assayed using Wnt1-GFP protein to outline the ureteric buds (bottom panels). IWP2 markedly inhibited branching morphogenesis. B, similar experiment as in A except cellular boundaries were revealed by E-cadherin staining. White arrows indicate representative Wnt1-GFP cell surface staining. C, IWP inhibits the accumulation of Wnt3A on the cell surface in cultured cells. COS1 cells transiently expressing Wnt3A-myc were treated for 48 h in the presence or absence of IWP prior to cell surface biotinylation using a cell-impermeable labeling agent. Amounts of cell surface Wnt3A-myc protein was then determined by comparing total and streptavidin agarose-precipitated Wnt3A protein by Western blot analysis. D, IWP2 inhibits the secretion of Wnt proteins regardless of their ability to induce transcriptional responses in HEK293 cells. The secretion of several Wnt-GL fusion proteins introduced by DNA transfection into HEK293 cells was tested for sensitivity to IWP2 (top). In parallel, the ability of the same Wnt molecules lacking GL to activate Wnt/β-catenin pathway response as measured using the STF reporter was determined (bottom). Data are mean ± S.E. from three measurements. E, evidence that IWP2 inhibits the production of a non-canonical Wnt (Wnt5A). Antagonism of Wnt/β-catenin signaling by expression of Wnt5A in the presence or absence of IWP compound was determined in HEK293 cells transfected with the STF reporter and treated with Wnt3A-containing conditioned medium. STF activity was normalized to the activity of a co-transfected control reporter. Data are mean ± S.E. from three measurements. p values for change from control response are indicated. F, IWP inhibits Wnt-dependent activation of Jnk. Mouse L fibroblasts transfected with Wnt7B DNA induce IWP-sensitive phosphorylation of Jnk, a target of multiple β-catenin-independent Wnt pathways.

FIGURE 4.

Diverse chemical scaffolds support Porcn inhibition by targeting the putative active site. A, Dvl2 phosphorylation status in HeLa cells reflects Porcn activity. IWP2 inhibits Dvl2 phosphorylation in HeLa cells indicating cell-autonomous Wnt-mediated signaling in these cells as previously described. B, identification of additional Porcn inhibitors. The IWP compound collection of Wnt/β-catenin pathway inhibitors was tested for their ability to inhibit Dvl2 phosphorylation in HeLa cells. The ratio of phosphorylated to unphosphorylated Dvl protein in cells treated with each IWP compound was determined by densitometric analysis of Western blot results as shown in A. Compounds inhibiting 90% or more of Dvl phosphorylation are labeled. C, shared chemical scaffolds yielding the most active IWP molecules. Compounds are clustered based on their similarity to IWP2 or shared chemical structures. IC50 against Wnt/β-catenin pathway response as measured by STF is provided for at least one representative compound from each class. D, novel IWP compounds disrupt Wnt protein acylation. Wnt3A-Fc protein from cells treated with alkynyl-PA in the presence of indicated IWP compound or DMSO was subjected to an alkyne cycloaddition reaction to label fatty acylated Wnt3A with biotin. Biotinylated protein separated on SDS-PAGE was visualized with streptavidin HRP. E, novel Porcn inhibitors likely bind directly to Porcn. The ability of indicated IWP compounds to compete for IWP-Cy3 binding to Porcn was determined as before.

FIGURE 5.

Concerted deployment of IWP and IWR compounds distinguishes β-catenin-dependent and -independent responses in vivo. A, identification of an IWP compound with in vivo activity in zebrafish. IWP12 inhibits the expression of an EGFP fluorescent protein reporter driven by synthetic TCF-binding elements in a transgenic line (Tg(7xTCF-Xla.Siam:GFP)ia4]. A ∼10-fold excess of IWP12 is equivalent in activity to IWR1 compound. Fluorescence intensity was quantified (below) in an area that covers most of the posterior region (box). Data are mean ± S.E. from three animals. B, IWR and IWP compounds inhibit Wnt signaling in zebrafish primary embryonic fibroblasts. Embryonic fibroblasts isolated from 6 hpf Tg(7xTCF-Xla.Siam:GFP)ia4 embryos were cultured in the presence or absence of indicated compound. GFP expression was visualized 20 h later. C, IWP compounds inhibit tailfin regeneration, a Wnt-dependent process. Tailfins of zebrafish larvae at 3 days post fertilization were resected and the larvae subsequently reared in medium containing DMSO, IWR1 (10 μm), or IWP12 (50 μm) for an additional 4 days. D, IWP12 inhibits embryonic convergent extension by targeting β-catenin-independent Wnt signaling. Zebrafish embryos were treated with GSK3β inhibitor (a Wnt/β-catenin pathway activator), IWP12 compound, or both starting 4 hpf followed by whole mount in situ analysis at 24 hpf with probes and the respective developmental structures they label indicated: hgg1 (ctsl1b) [prechordal plate (pcp)], ntl [prospective notochord (n) and germ ring blastopore margin], and dlx3b [anterior edge of the neural plate (np)]. Changes in the distance between the neural plates and prechordal plates, as well as the notochord were quantified based upon the severity of the phenotype as represented. Number of animals examined under each condition is indicated above each plot. E, inactivation of GSK3β rescues Wnt/β-catenin pathway activity in animals treated with IWP12. F, engrailed expression in the midbrain-hindbrain boundary (MHB) is suppressed by chemical inhibition of Porcn. Zebrafish embryos (4 hpf) treated with IWP12 for 20 h were subjected to in situ analysis with a probe for eng1a. Number of animals examined in each condition is indicated within each plot.