HTH-01-015

Catalog No.S7318 Batch:S731801

Technical Data

Formula	C₂₆H₂₈N₈O
Molecular Weight	468.55	CAS No.	1613724-42-7
Solubility (25°C)*	In vitro	DMSO	58 mg/mL (123.78 mM)
		Ethanol	30 mg/mL (64.02 mM)
		Water	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

HTH-01-015 is a potent and selective NUAK1 inhibitor with IC50 of 100 nM, >100-fold selectivity over NUAK2.

Targets

NUAK1 ^[1]

100 nM

In vitro

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. ^[1] HTH-01-015 inhibitors markedly restricted cells from entering into mitosis in U2OS cells. ^[2]

Protocol (from reference)

Kinase Assay:^{^[1]}	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 Assay:^{^[1]}	Cell lines U2OS cells and MEFs Concentrations 10 μM Incubation Time 5 days Method 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.

Kinase Assay:^{^[1]}

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 Assay:^{^[1]}

Cell lines
U2OS cells and MEFs
Concentrations
10 μM
Incubation Time
5 days
Method
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.

References

Customer Product Validation

: Data from [Data independently produced by , , Brain Behav Immun, 2017, doi: 10.1016/j.bbi.2017.11.003]

Selleck's HTH-01-015 has been cited by 3 publications

Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription. [ Mol Cell, 2020, 77(6):1322-1339]	PubMed: 32006464
Development of MAP4 Kinase Inhibitors as Motor Neuron-Protecting Agents. [ Cell Chem Biol, 2019, 10.1016/j.chembiol.2019.10.005]	PubMed: 31676236
Functional genomics identifies specific vulnerabilities in PTEN-deficient breast cancer [ Breast Cancer Res, 2018, 20(1):22]	PubMed: 29566768

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SHIPPING AND STORAGE
Selleck products are transported at room temperature. If you receive the product at room temperature, please rest assured, the Selleck Quality Inspection Department has conducted experiments to verify that the normal temperature placement of one month will not affect the biological activity of powder products. After collecting, please store the product according to the requirements described in the datasheet. Most Selleck products are stable under the recommended conditions.

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