HTH-01-015 is a selective NUAK1 inhibitor (IC50=100 nM).

NUAK1:100 nM

在HEK-293细胞中表达NUAK1和NUAK2时，HTH-01-015能够抑制NUAK1介导的MYPT1磷酸化。此外，HTH-01-015能够抑制NUAK1+/+ MEFs的细胞迁移，以及限制U2OS细胞的侵袭性。HTH-01-015还能够抑制这两种细胞系的细胞增殖。[1]在U2OS细胞中，HTH-01-015抑制剂显著阻止细胞进入有丝分裂阶段。[2]

Kinase activity assays: In vitro activities of purified GST–NUAK1 and GST–NUAK1[A195T] are measured using Cerenkov counting of incorporation of radioactive 32P from [γ-32P]ATP into Sakamototide substrate peptide. Reactions are carried out in a 50 μL reaction volume for 30 min at 30°C and reactions are terminated by spotting 40 μL of the reaction mix on to P81 paper and immediately immersing in 50 mM orthophosphoric acid. Samples are washed three times in 50 mM orthophosphoric acid followed by a single acetone rinse and air drying. The kinase-mediated incorporation of [γ-32P]ATP into Sakamototide is quantified by Cerenkov counting. One unit of activity is defined as that which catalyses the incorporation of 1 nmol of [32P]phosphate into the substrate over 1 h.

Cell proliferation assays are carried out colorimetrically in 96-well plates using the CellTiter 96? AQueous Non-Radioactive Cell Proliferation Assay kit following the manufacturer's protocol. Initially, 2000 cells per well are seeded for U2OS cells and 3000 cells per well are seeded for MEFs. The proliferation assays are carried out over 5 days in the presence or absence of 10 μM HTH-01-015.(Only for Reference)