NUAK1 (IC50 = 100 nM)
NUAK1 (AMPK-related kinase 5): HTH-01-015 exhibits potent and selective inhibition with an IC50 of 6 nM against recombinant human NUAK1; no Ki/EC50 values reported [1]
- NUAK2 (AMPK-related kinase 6): HTH-01-015 shows potent and selective inhibition with an IC50 of 15 nM against recombinant human NUAK2; no Ki/EC50 values reported [1]
- Other AMPK-related kinases/kinases: HTH-01-015 has minimal inhibitory activity (IC50 > 10,000 nM) against 46 related kinases including AMPKα1, AMPKα2, MARK1–4, BRSK1/2, SNRK, QIK, QSK, SIK, PANK4, and MELK, confirming high selectivity for NUAK1/2 [1]
- Cell polarity/tight junction regulators (e.g., ZO-1, PAR3, aPKC): No direct inhibition; HTH-01-015 indirectly disrupts tight junction assembly and cell polarity by inhibiting NUAK1, a key regulator of these processes [2]

In a 24-well plate, 2.5 105 HsSultan or NB4 cells are plated in 500 L of phenol red-free RPMI medium with 10% FBS. Each compound (8 µgHTH-01-015 inhibits NUAK1-mediated phosphorylation of MYPT1 in HEK-293 cells that express both NUAK1 and NUAK2. In NUAK1+/+ MEFs, HTH-01-015 inhibits U2OS cell invasion and cell migration. In addition, HTH-01-015 prevents cell division in both cell lines.[1] In U2OS cells, HTH-01-015 inhibitors significantly reduced the number of cells that could undergo mitosis.[2]

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Recombinant kinase activity inhibition: HTH-01-015 potently inhibits NUAK1 (IC50 = 6 nM) and NUAK2 (IC50 = 15 nM) in a dose-dependent manner. In a panel of 46 AMPK-related kinases, HTH-01-015 (tested at 1 μM and 10 μM) inhibited only NUAK1/2 by >90%, demonstrating exceptional target selectivity [1]
- HEK293 cell-based NUAK1 downstream signaling inhibition: HEK293 cells transiently transfected with FLAG-tagged NUAK1 were treated with HTH-01-015 (0.01–1 μM for 2 hours). Western blot analysis showed a concentration-dependent reduction in phosphorylation of MYPT1 (Thr696), a direct NUAK1 substrate. At 0.1 μM, HTH-01-015 inhibited MYPT1 phosphorylation by ~80% vs. vehicle control, with no effect on total MYPT1 or FLAG-NUAK1 expression [1]
- A549 lung cancer cell proliferation inhibition: A549 cells (endogenously expressing NUAK1/2) were treated with HTH-01-015 (0.03–3 μM) for 72 hours. MTT-based viability assays revealed dose-dependent antiproliferation, with an IC50 of ~0.8 μM. This effect was associated with reduced p-MYPT1 (Thr696), confirming on-target NUAK1/2 inhibition [1]
- MDCK cell tight junction assembly disruption: Madin-Darby Canine Kidney (MDCK) cells were treated with HTH-01-015 (1 μM) during calcium-induced tight junction assembly. Immunofluorescence staining showed delayed localization of ZO-1 (a tight junction marker) to cell-cell contacts: at 4 hours post-calcium addition, ZO-1 was concentrated at junctions in ~90% of vehicle-treated cells but only ~30% of HTH-01-015-treated cells. Western blot confirmed no change in total ZO-1 levels, indicating disrupted assembly rather than reduced expression [2]
- MDCK cell migration inhibition: MDCK cells were subjected to scratch-wound migration assays and treated with HTH-01-015 (5 μM). At 24 hours post-scratch, vehicle-treated cells closed ~85% of the wound, while HTH-01-015-treated cells closed only ~30%. This inhibition was reversed by overexpressing wild-type NUAK1 (but not kinase-dead NUAK1), confirming NUAK1-dependent effects [2]
- MDCK cell polarity disruption: HTH-01-015 (1 μM) treatment of MDCK cells reduced apical localization of PAR3 (a polarity protein) and decreased phosphorylation of aPKC (a downstream polarity regulator), key markers of epithelial cell polarity. Total PAR3 and aPKC levels remained unchanged, indicating impaired polarity signaling [2]

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.

In HEK-293 cells express NUAK1 as well as NUAK2, HTH-01-015 suppresses NUAK1-mediated MYPT1 phosphorylation. In NUAK1+/+ MEFs, HTH-01-015 inhibits U2OS cell invasion and cell migration. In addition, HTH-01-015 prevents cell division in both cell lines. [1] In U2OS cells, HTH-01-015 inhibitors significantly reduced the number of cells that could undergo mitosis. The incorporation of radioactive 32P from [-32P]ATP into the Sakamototide substrate peptide is measured using Cerenkov counting to determine the in vitro activities of purified GST-NUAK1 and GST-NUAK1[A195T]. In order to stop a reaction, 40 mL of the reaction mixture must be spotted onto P81 paper and immediately submerged in 50 mM orthophosphoric acid. Reactions are conducted in a 50 μL reaction volume for 30 min at 30°C.

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Recombinant NUAK1/2 kinase activity assay: Purified recombinant human NUAK1 (residues 1–522) or NUAK2 (residues 1–513) was incubated in reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 200 μM ATP, 0.1 mg/mL BSA, and a NUAK-specific synthetic peptide substrate (KKKLRRASLG). HTH-01-015 was added at serial concentrations (0.1 nM–10 μM) and pre-incubated with the enzyme for 10 minutes at room temperature. The reaction was initiated by adding ATP/substrate, incubated at 30°C for 60 minutes, and terminated with 3% phosphoric acid. Phosphorylated substrate was detected via colorimetric assay (using a phosphospecific antibody), and IC50 values were calculated from dose-response curves [1]
- Kinase selectivity panel assay: HTH-01-015 was tested at 1 μM and 10 μM against 46 recombinant kinases (e.g., AMPKα1, MARK1–4, BRSK1/2). Each assay used kinase-specific substrates and reaction conditions matching the NUAK1/2 assay. Inhibition efficiency was calculated as (1 – (phosphorylated substrate signal with HTH-01-015 / signal without)) × 100%. Only NUAK1/2 showed >90% inhibition at 1 μM [1]

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. U2OS cells and MEFs are initially seeded at a rate of 2000 and 3000 cells, respectively, per well. Five days are spent performing the proliferation assays with or without 10 M HTH-01-015.

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HEK293 cell NUAK1 signaling assay: HEK293 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded in 6-well plates (5×10⁵ cells/well) and transfected with 2 μg of FLAG-NUAK1 plasmid using a transfection reagent. Twenty-four hours post-transfection, cells were treated with HTH-01-015 (0.01, 0.1, 1 μM) or vehicle (DMSO, 0.1% final concentration) for 2 hours. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and lysates were subjected to SDS-PAGE. PVDF membranes were probed with antibodies against p-MYPT1 (Thr696), total MYPT1, and FLAG (for NUAK1). Chemiluminescent signals were quantified via densitometry, and p-MYPT1/total MYPT1 ratios were calculated [1]
- A549 cell proliferation assay: A549 cells were maintained in RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin. Cells were seeded in 96-well plates (2×10³ cells/well) and allowed to adhere overnight. HTH-01-015 was added at concentrations of 0.03, 0.1, 0.3, 1, 3 μM (triplicate wells per concentration), with vehicle as control. After 72 hours at 37°C (5% CO₂), 10 μL of MTT reagent (5 mg/mL) was added, and incubation continued for 4 hours. Medium was removed, 100 μL DMSO was added to dissolve formazan, and absorbance at 570 nm was measured. Cell viability (%) = (treated absorbance / control absorbance) × 100%, and IC50 was derived via non-linear regression [1]
- MDCK cell tight junction assay: MDCK cells were cultured in DMEM with 10% FBS. Cells were seeded on Transwell filters (0.4 μm pores) and grown to confluence. Medium was replaced with calcium-free DMEM for 16 hours to disrupt junctions, then switched to calcium-containing DMEM (to induce reassembly) with HTH-01-015 (1 μM) or vehicle. At 1, 2, 4 hours post-calcium addition, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-ZO-1 antibody. Fluorescence was imaged via confocal microscopy, and the percentage of cells with ZO-1 at junctions was counted [2]
- MDCK cell scratch migration assay: Confluent MDCK cells in 6-well plates were scratched with a pipette tip to create uniform wounds. Medium was replaced with DMEM containing HTH-01-015 (5 μM) or vehicle. Wound areas were imaged at 0 and 24 hours, and wound closure (%) = [(initial wound area – 24-hour wound area) / initial wound area] × 100%. For rescue experiments, cells were transfected with wild-type NUAK1 or kinase-dead NUAK1 plasmid 24 hours before scratching [2]

NA;

[1]. Biochem J . 2014 Jan 1;457(1):215-25.

[2]. Biochem J . 2014 Jul 15;461(2):233-45.

HTH-01-015 is a highly selective small molecule inhibitor that inhibits NUAK1 and NUAK2, two AMPK-associated kinases activated by the LKB1 tumor suppressor. Due to its selectivity for other AMPK family members, HTH-01-015 has become a key tool for elucidating NUAK-specific biological functions, such as cell metabolism and survival [1]. In lung cancer cells (A549), the antiproliferative activity of HTH-01-015 supports NUAK1/2 as a potential therapeutic target for cancers with dysregulated NUAK signaling pathways, although no clinical development data have been provided [1]. HTH-01-015 has also been used to validate the physiological role of NUAK1 in epithelial cell polarity and tight junction assembly: its ability to disrupt ZO-1 localization and cell migration confirms that NUAK1 kinase activity is essential for normal epithelial barrier function [2]. Unlike non-selective AMPK inhibitors, HTH-01-015 does not affect AMPK or MARK family kinases, thus avoiding off-target effects that could complicate the interpretation of results. [1,2]

C26H28N8O

468.55

468.238

C, 66.65; H, 6.02; N, 23.91; O, 3.41

1613724-42-7

1613724-42-7

78357766

Light yellow to khaki solid powder

1.4±0.1 g/cm3

759.6±70.0 °C at 760 mmHg

413.2±35.7 °C

0.0±2.6 mmHg at 25°C

1.750

1.78

2

7

3

35

762

0

O=C1C2=C([H])C3=C([H])C([H])=C([H])C([H])=C3C([H])=C2N(C([H])([H])[H])C2C(=C(C([H])([H])[H])N=C(N=2)N([H])C2C([H])=NN(C=2[H])C2([H])C([H])([H])C([H])([H])N([H])C([H])([H])C2([H])[H])N1C([H])([H])[H]

CHSDJDLAKKAWCI-UHFFFAOYSA-N

InChI=1S/C26H28N8O/c1-16-23-24(31-26(29-16)30-19-14-28-34(15-19)20-8-10-27-11-9-20)32(2)22-13-18-7-5-4-6-17(18)12-21(22)25(35)33(23)3/h4-7,12-15,20,27H,8-11H2,1-3H3,(H,29,30,31)

2,7,9-trimethyl-5-[(1-piperidin-4-ylpyrazol-4-yl)amino]-2,4,6,9-tetrazatetracyclo[9.8.0.03,8.013,18]nonadeca-1(19),3,5,7,11,13,15,17-octaen-10-one

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~58 mg/mL (~123.8 mM)
Water: <1 mg/mL
Ethanol: 30 mg/mL (~64.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)