Molecular Weight (MW)

468.55

Formula

C26H28N8O

CAS No.

1613724-42-7

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 58 mg/mL (123.8 mM)

Water: <1 mg/mL

Ethanol: 30 mg/mL (64.0 mM)

SMILES Code

CC1=C(N(C)C(C2=CC(C=CC=C3)=C3C=C2N4C)=O)C4=NC(NC5=CN(N=C5)C6CCNCC6)=N1

Synonyms

HTH-01015; HTH01015; HTH 01015; HTH-01-015; HTH01-015; HTH 01-015

In Vitro

In vitro activity: In HEK-293 cells express NUAK1 as well as NUAK2, HTH-01-015 suppresses NUAK1-mediated MYPT1 phosphorylation. HTH-01-015 suppresses cell migration In NUAK1+/+ MEFs, and inhibit U2OS cell invasion. Moreover, HTH-01-015 inhibits cell proliferation in both cell lines. HTH-01-015 inhibitors markedly restricted cells from entering into mitosis in U2OS cells.

Kinase Assay: 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 Assay: Cell proliferation assays are carried out colorimetrically in 96-well plates using the CellTiter 96(R) 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.

In Vivo

Short-term treatment of normal Sprague Dawley rats with A-769662 decreases liver malonyl CoA levels and the respiratory exchange ratio, VCO2/VO2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreases hepatic expression of PEPCK, G6Pase, and FAS, lowers plasma glucose by 40%, reduced body weight gain and significantly decreases both plasma and liver triglyceride levels.

Animal model

Formulation & Dosage

References

Biochem J. 2014 Jan 1;457(1):215-25; Biochem J. 2014 Jul 15;461(2):233-45.