CaMK II ULK1 (IC50 = 26.6 nM)

XST-14 has been shown to inhibit various enzymes, including ULK1 (IC50 = 13.6 nM), MAP2K1/MEK1 (IC50 = 721.8 nM), MAPK14/p38 alpha (IC50 = 283.9 nM), TGFBR2 (IC50 = 809.3 nM), ACVR1/ALK2 (IC50 = 183.8 nM), ULK2 (IC50 = 70.9 nM), and CAMK2A (IC50 = 66.3 nM). Cell proliferative activity is reduced by XST-14 (20-80 μM) for a 24-hour period[1]. In HepG2 and human primary HCC cells, XST-14 (5 μM) causes apoptosis for a duration of 24 hours[1]. CHO cells producing GFP-LC3 steadily thanks to LC3-II[1]. XST-14 (5 μM) suppresses the phosphorylation of PIK3C3 at Ser249 and BECN1 at Ser15 after 12 hours[1].

In nude mice, XST-14 (15, 30 mg/kg/day; IP) exhibits anti-HCC efficacies, leading to reduced tumor weights and inhibited HCC cell proliferation in tumors[1]. T1/2 values for mg/kg for IV and 10 mg/kg for IP are 2.69 and 2.31 hours, respectively[1].

ULK1 kinase assays [1]
ULK1 kinase, its substrate, ATP and XST-14 were diluted in kinase buffer (40 mM Tris, pH 7.5, 20 mM MgCl2, 0.1 mg mL−1 BSA, 50 μM DTT). Then, 2 μL of XST-14, 4 μL of ULK1 kinase enzyme, and 4 μL of myelin basic protein (MBP) (0.1 μg μL−1)/ATP (10 μM) were added and incubated at room temperature for 60 min, and 10 μL of ADP-Glo™ reagent was added. The plates were incubated at room temperature for 40 min, and then 20 μL of kinase detection reagent was added. After incubation at room temperature for 30 min, the luminescence was recorded. The ADP formed from the kinase reactions was measured using an ADP-Glo Kinase Assay Kit following the manufacturer’s instructions as previously described. The IC50 values were calculated using a nonlinear regression with normalized dose-response fitting using Prism software.

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Surface plasmon resonance analysis [1]
Recombinant His-tagged wild-type ULK1, as well as K46A, Y94A and D165A kinase domain (KD) truncation mutants, were expressed in 293F cells, purified, and eluted from a Ni column. The binding kinetics between XST-14 and ULK1 or the ULK1 mutants were measured by a surface plasmon resonance assay using a Biacore T200 instrument. The dissociation constant (KD) was calculated according to BIAevaluation software.

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ULK kinase selectivity panels [1]
Kinase panel testing was performed to determine the potency of XST-14 toward 403 different kinases using SelectScreen™ Kinase Profiling Services. XST-14 was evaluated at a concentration of 5 μM in kinase-specific assay conditions, and the data analyzes are described on the company’s website (services/services/custom-services/screening-and-profiling-services/selectscreen-profiling-service/selectscreen-kinase-profiling-service.html). All tests were run in duplicate.

Apoptosis Analysis[1]
Cell Types: HepG2 and human primary cells
Tested Concentrations: 5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced apoptosis in HepG2 and human primary HCC cells. Cell Autophagy Assay[1]
Cell Types: CHO, HepG2 cells stably expressing GFP -LC3
Tested Concentrations: 5 μM
Incubation Duration: 12 hrs (hours)
Experimental Results: Strongly inhibited the conversion of LC3-I to LC3-II in CHO cells. Dramatically diminished the number of GFP-LC3 puncta in HepG2 cells. diminished autophagosome formation and blocked autophagosome/lysosome fusion in HepG2 cells.

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Western Blot Analysis[1]
Cell Types: HepG2 cells
Tested Concentrations: 5 μM
Incubation Duration: 12 hrs (hours)
Experimental Results: Inhibited the Ser249 phosphorylation of PIK3C3 and Ser15 phosphorylation of BECN1.

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Autophagy flux assay [1]
CHO cells were infected with the mCherry-GFP-LC3 adenovirus. After 12 h, the cells were starved in Earle’s balanced salt solution for 4 h and treated with or without XST-14 (5 μM) for 12 h. Then, the autophagic flux rate was detected by live cell imaging microscopy.

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EdU assays [1]
HCC cells (5 × 10~4 cells per well) were plated onto 12-well plates. After 24 h, the cells were treated with 5 μM XST-14, 5 μM sorafenib, or a combination of XST-14 (5 μM) and sorafenib (5 μM). After incubation for 24 h, the cell culture medium in each well was replaced with 1 mL of EdU medium (50 μM) and incubated for 2 h. The cells were harvested by trypsinization, and cell proliferation activities were determined using a Cell-Light™ EdU Apollo® 488 In Vitro Flow Cytometry Kit (20T).

Animal/Disease Models: Nude mice bearing HepG2 tumor xenografts[1]
Doses: 15, 30 mg/kg
Route of Administration: IP; daily; for 4 consecutive weeks
Experimental Results: Displayed anti-HCC efficacies, resulting in diminished tumor weights and suppressed tumor growth of HCC cells in nude mice.

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Animal/Disease Models: SD (Sprague-Dawley) rat[1]
Doses: 2 mg/kg for IV; 10 mg/kg for IP (pharmacokinetic/PK Analysis)
Route of Administration: IV or IP
Experimental Results: Had a T1/2 of 2.31 hrs (hours), a CL of 26.28 mL/min·kg, and and an AUC of 1269 hr·ng/mL for IV. Had a T1/2 of 2.69 hrs (hours), a Cmax of 2033 ng/mL, and an AUC of 5979 hr •ng/mL for IP.

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Mouse models for tumor growth [1]
To generate mouse models for tumor growth, 2 × 106 HepG2 cells per mouse in 100 μL of PBS solution were injected subcutaneously into the right flanks of 5- to 6-week-old male nude mice. Tumor volumes were determined every 3 d using the formula TV = W2 × L × 0.5. For sorafenib treatment alone in vivo, the tumors were allowed to grow for 7 d (designated as day 0), and sorafenib treatment was then administered. At that time, the tumor volume reached approximately 40 mm3. The mice received vehicle (PBS, 100 μl, per os [P.O.]) or sorafenib (30 mg/kg, P.O., every other day [q.o.d.]) for 4 consecutive weeks. For XST-14 treatment or sorafenib and XST-14 combination treatment in vivo, when the tumor volume reached approximately 100 mm3 (designated as day 0), the treatments were administered. The mice received vehicle (PBS, 100 μL, intraperitoneal [IP]), sorafenib (30 mg/kg, P.O., daily), XST-14low (15 mg/kg, IP, daily), XST-14high (30 mg/kg, IP, daily), XST-14low and sorafenib (15 mg/kg, IP, daily for XST-14low; 30 mg/kg, P.O., daily for sorafenib), or XST-14high and sorafenib (30 mg/kg, IP, daily for XST-14high; 30 mg/kg, P.O., daily for sorafenib) for 4 consecutive weeks. After 4 weeks, all mice were sacrificed, and necropsies were performed. The tumors were excised and analyzed by western blot analysis for protein expression.

[1]. The role of the key autophagy kinase ULK1 in hepatocellular carcinoma and its validation as a treatment target . Autophagy. 2020 Oct;16(10):1823-1837.

Although macroautophagy/autophagy is involved in hepatocellular carcinoma (HCC) initiation and development and has been identified as a mechanism of HCC therapy resistance, the role of ULK1 (unc-51 like autophagy activating kinase 1) in HCC remains unclear. Here, we report that both knockdown and knockout of ULK1 inhibited human HCC cell proliferation and invasion, and Ulk1 deletion abrogated tumor growth in a xenograft mouse model. Furthermore, ULK1 ablation in combination with sorafenib significantly inhibited HCC progression compared with sorafenib treatment alone or vehicle control. To identify candidate ULK1 inhibitors, we used a structure-based virtual docking approach to screen 3428 compounds. Among these compounds, XST-14 showed the highest affinity for the ULK1 protein and specifically blocked ULK1 kinase activity. Moreover, the Lys46, Tyr94 and Asp165 amino acid residues of ULK1 were required for its binding to XST-14 according to molecular docking and mutagenesis experiments. Functional assays revealed that XST-14 blocked autophagy and subsequently induced apoptosis and inhibited growth in HCC cells. More importantly, XST-14 acted synergistically with sorafenib to attenuate HCC progression by inhibiting sorafenib-induced autophagy activation both in vitro and in vivo. In addition, XST-14 was well tolerated and exhibited favorable drug metabolism and pharmacokinetic properties and low toxicity in mice. In summary, our study determined that ULK1 may represent a new therapeutic target for HCC and that targeting ULK1 in combination with sorafenib treatment may serve as a promising interventional strategy for treating HCC.[1]

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In our study, we screened XST-14, a highly selective ULK1 kinase inhibitor, to block autophagy and induce apoptosis in HCC cells. XST-14 significantly synergized with sorafenib to suppress HCC progression both in vitro and in vivo. Interestingly, 100 μM XST-14 treatment induced apoptosis in 60–70% of HepG2 cells and in only 20–30% of normal cells (Figure S12). The differential roles of XST-14 in HCC and normal cells are still unclear in this study, which requires further investigation in the next study.
In this study, XST-14 had good pharmacokinetic properties and appeared to be tolerable in mice. In addition, the effects of the XST-14 metabolite (XST-14Metab) on autophagy and ULK1 activities were further evaluated. We found that XST-14Metab has similar autophagy and ULK1 inhibitory properties as XST-14 in vitro. XST-14Metab also suppressed tumor cell proliferation in vitro (Figure S5B–E). Hence, the antitumor effects of XST-14 in animal studies may be produced by XST-14 itself and/or its metabolite due to the short half-life of XST-14. Further efforts will be needed to explore the use of XST-14Metab in animal models of HCC. In summary, this study confirmed that ULK1 could be a therapeutic target for HCC. XST-14, a novel ULK1 inhibitor, exerts anti-HCC effects and is a good candidate for future investigation. Moreover, ULK1 inhibitor coadministration with sorafenib may become a promising interventional strategy for HCC and other types of cancer treatment.[1]

C16H21NO4

291.34

291.147

C, 65.96; H, 7.27; N, 4.81; O, 21.97

2607143-50-8

155291452

White to off-white solid powder

3.8

1

4

6

21

358

0

CC(C)OC1C2C=C(NC=2C=C(OC(C)C)C=1)C(OC)=O

ZNFMBFABCSHXPM-UHFFFAOYSA-N

InChI=1S/C16H21NO4/c1-9(2)20-11-6-13-12(15(7-11)21-10(3)4)8-14(17-13)16(18)19-5/h6-10,17H,1-5H3

methyl 4,6-di(propan-2-yloxy)-1H-indole-2-carboxylate

XST-14; 2607143-50-8; Methyl 4,6-diisopropoxy-1H-indole-2-carboxylate; methyl 4,6-di(propan-2-yloxy)-1H-indole-2-carboxylate;

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 : 250 mg/mL (858.10 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.14 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 20.8 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 2: ≥ 2.08 mg/mL (7.14 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 20.8 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.)