The development of highly potent inhibitors for porcupine

Abstract

Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It catalyzes the palmitoylation of Wnt proteins, a process required for their secretion and activity. We recently disclosed a class of small molecules (IWPs) as the first reported Porcn inhibitors. We now describe the structure-activity relationship studies and the identification of subnanomolar inhibitors. We also report herein the effects of IWPs on Wnt-dependent developmental processes, including zebrafish posterior axis formation and kidney tubule formation.

Figure 1

The structures and activities of IWPs identified from a high-throughput screen in cells exhibiting cell-autonomous Wnt signaling.

Figure 2

The phthalazinone/pyrimidinone and the benzothiazole moieties of IWPs are important for their binding to Porcn.

Figure 3

Effects of the arylamide groups on the Porcn-inhibiting activity.

Figure 4

IWP-L6 (27) blocks the phosphorylation of the cytoplasmic Wnt pathway effector Dvl2.

Figure 5

Stability of IWP-L6 (27) in plasma.

Figure 6

Inhibition of the regeneration of tailfin of juvenile zebrafish.

Figure 7

IWP-L6 (27) inhibits posterior axis formation. Zebrafish embryos harboring an mCherry-based reporter of Wnt/β-catenin signaling were treated with increasing concentrations of IWP-L6 (27) in the aquarium water (top). Fluorescence intensity of animals was quantified as before. Activity of the IWP compounds IWP-L6 (27), 30, 32, and 35 was measured using the posterior axis formation assay (bottom).

Figure 8

IWP-L6 (27) inhibits Wnt mediated branching morphogenesis in cultured embryonic kidneys. Hoxb7Cre;RosaTomato kidneys were dissected from E11.5 embryos and cultured at the air/media interface (top).^, Media was replaced every 24 hours. Images were captured every 24 hours using a Zeiss Lumar V12 fluorescent stereoscope. Magnitude: 80× at 0 h and 24 h; 60× at 48h. The fold increase in the number of ureteric bud tips between time=0 and 48 hours for each treatment was quantified (bottom). Statistics were performed using Student T-test.