LGK974 (NVP-LGK974) is an effective and selective PORCN inhibitor and inhibits Wnt signaling (IC50: 0.4 nM) in TM3 cells. It has been used in trials studying the treatment of Metastatic Colorectal Cancer and Squamous Cell Carcinoma, Head And Neck.

In both the MMTV-WNT1 mouse model of breast carcinoma and the human head and neck squamous cell carcinoma model (hn30), LGK-974 (3 mg/kg) inhibits the Wnt signaling pathway, leading to tumor regression without affecting mouse body weight. Additionally, LGK-974 (5 mg/kg, twice daily, orally) also suppresses the growth of RNF43-mutant pancreatic tumors (HPAF-II and Capan-2).

LGK-974 inhibits a range of tested Wnts, with IC50 values between 0.05 to 2.4 nM. In the PORCN radioligand binding assay, LGK-974 effectively displaces [3H]GNF-1331 with an IC50 of 1 nM and exhibits minimal cytotoxicity at 20 μM. It specifically inhibits the growth of RNF43 mutant cell lines HPAF-II, PaTu 8988S, and Capan-2.

Radioligand binding assay: using the aforementioned membrane preps, filtration binding assays are performed. To reduce nonspecific binding, 96-well filtration plates are precoated as suggested by the manufacturer with 0.1% BSA and then washed four times with 0.1% BSA. Membrane preps (50 μg total protein) are incubated in polypropylene 96-well plates with 6.6 nM 3H-GNF-1331 in the presence or absence of a testing compound in binding buffer (50 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA) plus EDTA-free protease inhibitor mixture in a final volume of 150 μL for 3 h at room temperature. Binding reaction mixtures are then transferred to the precoated 96-well filtration plates, filtered, and washed using a 96-pin FilterMate Harvester. Radioactive signals are obtained using a Microplate Scintillation Counter TopCount. Curve fitting is performed using Prism[1].

Cells are plated in growth medium in a 96-well plate at a density of 6,000–12,000 cells per well and treated with DMSO or 1 μM LGK974. After 3 d, the cells are treated with fresh growth medium containing 20 μM EdU, which is included in the Click-iT EdU Alexa Fluor 488 HCS assay kit, and the plate was incubated for 2 h at 37 °C in a humidi?ed atmosphere containing 5% CO2. Cells are ?xed with ?nal 4% (mass/vol) paraformaldehyde for 30 min, washed with PBS, permeabilized, and stained with 50 μg/mL Hoechst in PBS for 30 min. After wash, the cells are proceeded to EdU detection according to the instruction of Click-iT EdU assay kit. Triplet wells are performed for each condition. (Only for Reference)