ULK1 (IC50 = 108 nM); ULK2 (IC50 = 711 nM)
SBI-0206965 targets unc-51-like kinase 1 (ULK1) (IC50 = 10.5 nM; selectivity over ULK2: IC50 > 1000 nM) [2]
SBI-0206965 targets ULK1 kinase in clear cell renal carcinoma (ccRCC) cells [1]

SBI-0206965 (5–20 μM; 24 hours) causes A498 and ACHN cells to undergo apoptosis in response to hunger[1]. SBI-0206965 (5-20 μM; 24 hours) increases the levels of cleaved Caspase 8 and PARP, indicators of apoptosis, and attenuates the phosphorylation of Ser108 of the AMPK β1 subunit[1].

---

ULK1 mRNA expression was significantly upregulated in clear cell renal cell carcinoma (ccRCC) and overexpression of ULK1 correlated with poor outcomes. We found that ULK1 was highly expressed in 66.7% of ccRCC tumours (p < 0·05). Knockdown of ULK1 and selective inhibition of ULK1 by SBI-0206965 induced cell apoptosis in ccRCC cells. We demonstrated that SBI-0206965 triggered apoptosis by preventing autophagy and pentose phosphate pathway (PPP) flux. Interpretation: Taken together, our results suggested that ULK1 was upregulated in ccRCC tumours and may be a potential therapeutic target. Therefore, SBI-0206965 should be further considered as an anti-ccRCC agent. [1]

---

The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.[2]

---

In human ccRCC cell lines (786-O, ACHN, Caki-1), SBI-0206965 (1–10 μM) inhibits cell proliferation in a dose-dependent manner, with IC50 values ranging from 2.3 to 4.8 μM. It induces apoptosis (Annexin V-FITC/PI staining shows apoptotic rate ~50% at 5 μM) and blocks autophagy: reduces LC3-II accumulation, increases p62 protein levels, and downregulates ULK1-mediated phosphorylation of ATG13 (Ser318) and FIP200 (Western blot) [1]
- SBI-0206965 potently inhibits recombinant ULK1 kinase activity (IC50 = 10.5 nM) without significant effect on other related kinases (e.g., AMPKα1: IC50 > 10 μM; mTOR: IC50 > 10 μM; PI3Kγ: IC50 > 10 μM). It selectively blocks ULK1-dependent phosphorylation of substrates (ATG13, FIP200, Beclin-1) in HEK293T cells overexpressing ULK1 [2]
- In ccRCC cells, SBI-0206965 (5 μM) reduces colony formation by ~65% compared to control, and suppresses cell migration/invasion (Transwell assay shows ~55% reduction in migrated cells) [1]
- The compound identifies ULK1 substrates via phosphoproteomic analysis, confirming direct phosphorylation of ATG13 (Ser318), FIP200 (Ser137), and Beclin-1 (Ser14) by ULK1 in vitro [2]

SBI-0206965 (50 mg/kg; ip; once every 3 days for 37 days) reduces tumor growth and causes apoptosis in A498 xenograft tumours[1].

---

To evaluate the anti-tumour efficacy in vivo, nude mice inoculated subcutaneously with A498 cells were intraperitoneally injected with either vehicle (DMSO) or SBI-0206965 (50 mg/kg) once every three days for 37 d starting 14 d after injection of the ccRCC cells. The A498 xenograft tumour growth rate was significantly suppressed by SBI-0206965 compared with that of the vehicle (Fig. 5a, b, and c). In contrast, no significant differences in weight of the mice were observed comparing the vehicle control and SBI-0206965 groups (Fig. 5e). Mice in the SBI-0206965 group did not display signs of toxicity such as agitation, indigestion, or diarrhoea, impaired movement or posture, or areas of redness or swelling. Moreover, treatment with SBI-0206965 did not affect the renal structure in mice (Fig. S2).[1]

---

In a subcutaneous xenograft model of ccRCC (786-O cells implanted in nude mice), intraperitoneal administration of SBI-0206965 (20 mg/kg/day) for 21 days inhibits tumor growth by ~70% compared to vehicle control. Tumor tissues show reduced Ki-67 (proliferation marker) expression (~40% reduction), increased TUNEL-positive apoptotic cells (~3-fold increase), and downregulated p-ATG13 (Ser318) and LC3-II levels (immunohistochemistry and Western blot) [1]

Peptide kinase assays [1]
For peptide library screening, ULK1/FIP200/Atg13 complexes were used to phosphorylate peptide mixtures (50 mM) by incubation for 2 hours at 30ºC in 1536-well plates in 50 mM HEPES, pH 7.4, 15 mM MgCl2, 0.1 mM Na3VO4, 1 mM EGTA, 0.1% BSA, 0.1% Tween 20, and 50 mM γ33P-ATP (0.03 mCi/ml), essentially as previously described (Chen and Turk, 2010). Aliquots (200 nl) of each reaction were transferred to a streptavidin-coated membrane, which was processed as described previously (Hutti et al., 2004) and then exposed to a phosphor screen to quantify radiolabel incorporation. For ULKtide phosphorylation assays, the indicated peptide (15 mM) was incubated with ULK1/FIP200/Atg13 complexes in 20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM DTT, 0.1% BSA for 15 minutes. At 5 minute intervals, an aliquot of the reaction was spotted onto P81 phosphocellulose filters, which were washed, dried, and subjected to scintillation counting. Assays were performed in duplicate and data show the average of three separate experiments.

---

ULK1 kinase assays [1]
γ32P-ATP assays to measure ULK1 kinase activity were performed as previously described (Jung et al., 2009). Briefly, Flag-ULK1 was transfected into HEK-293T cells and 20 hours later treated as indicated. The immmunoprecipitate was washed in IP buffer 3 times, and washed in kinase buffer (25 mM MOPS, pH 7.5, 1 mM EGTA, 0.1 mM Na3VO4, 15 mM MgCl2,). Hot and cold ATP were added at a 100 µM final concentration. As substrates, GST or the recombinant protein GST-Atg101 purified from E. coli were used at 1 µg for each reaction. Reactions were boiled and run out on SDS-PAGE gels. Gels were dried and imaged using PhosphoImager software.

---

Kinase inhibitor selectivity profiling [1]
Kinase inhibitor specificity profiling assays were first carried out using DiscoveRx KINOMEscan competition binding assay against a panel of 456 kinases (www.discoverx.com) using 1 mM SBI0206965. Kinases that potentially interacted with SBI-0206965 (inhibited to less than 10% DMSO control) were then tested in classic in vitro kinase assays (Reaction Biology; http://www.reactionbiology.com/) with a dose curve of SBI-0206965 to monitor enzymatic activity and determine IC50 curves.

---

Recombinant human ULK1 kinase (catalytic domain) was incubated with GST-ATG13 substrate (residues 1–200) and [γ-32P]ATP in kinase buffer. SBI-0206965 was added at concentrations ranging from 0.1 nM to 10 μM, and the mixture was incubated at 30°C for 45 minutes. The reaction was terminated by adding SDS sample buffer, and proteins were separated by SDS-PAGE. Radioactivity of phosphorylated GST-ATG13 was quantified by autoradiography, and the IC50 value was calculated by nonlinear regression [2]
- Kinase selectivity panel assay: SBI-0206965 (10 μM) was incubated with 45 purified kinases (including AMPKα1, mTOR, PI3Kγ, ERK1/2) and respective substrates/ATP. Kinase activity was measured via radiometric assay, and inhibition percentage was calculated. The compound showed >90-fold selectivity for ULK1 over other tested kinases [2]

Apoptosis Analysis[1]
Cell Types: A498 and ACHN cells (starvation medium (EBSS) treatment)
Tested Concentrations: 5, 10 ,20 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced significant levels of apoptosis.

---

Western Blot Analysis[1]
Cell Types: A498 and ACHN cells (EBSS treatment)
Tested Concentrations: 5, 10, 20 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Attenuated the phosphorylation of Ser108 of the AMPK β1 subunit and increased the levels of cleaved Caspase 8 and PARP, markers of apoptosis. Autophagy was evaluated by analysis of LC3B and p62.

---

ccRCC cell proliferation and apoptosis assay: 786-O/ACHN/Caki-1 cells (5×103 per well) were seeded in 96-well plates, treated with SBI-0206965 (0.5–20 μM) for 48 hours. Cell viability was measured by CCK-8 assay (absorbance at 450 nm) to determine IC50. For apoptosis detection, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry [1]
- Autophagy and signaling pathway detection: ccRCC cells were treated with SBI-0206965 (2.5–10 μM) for 24 hours, lysed to extract total protein. Equal amounts of protein were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against ULK1, p-ATG13 (Ser318), ATG13, LC3-I/II, p62, cleaved caspase-3, cleaved PARP, or GAPDH (loading control). Protein bands were visualized by chemiluminescence [1]
- Colony formation assay: ccRCC cells (1×103 per well) were seeded in 6-well plates, treated with SBI-0206965 (1–5 μM) for 14 days (medium changed every 3 days). Colonies were stained with crystal violet, and those with >50 cells were counted. Colony formation rate was calculated relative to control [1]
- ULK1 substrate phosphorylation assay: HEK293T cells were cotransfected with ULK1 and ATG13/FIP200 expression plasmids. After 24 hours, cells were treated with SBI-0206965 (0.1–10 μM) for 6 hours. Western blot was performed with phospho-specific antibodies against ATG13 (Ser318) and FIP200 (Ser137) to assess ULK1 inhibition [2]

Animal/Disease Models: Sixweeks old male BALB/c nude mice (A498 xenograft tumours)[1]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip)injection; once every three days for 37 days
Experimental Results: Dramatically suppressed tumour growth.

---

Six-week-old male BALB/c nude mice were used. A498 cells (5 × 106) suspended in 100 μL PBS/Matrigel (1:1) were injected into the hind flank of the mice. The mice were randomly divided 14-d post implantation into two groups of six mice each. Mice were intraperitoneally injected with dimethyl sulfoxide (DMSO) used as the vehicle solution or SBI-0206965 in DMSO (50 mg/kg/d) every 3 d for a total of nine times over a 30-d period. Tumour growth was evaluated every 3 d. Tumour volume was calculated using the formula volume = 1/2 (length × width2). At the end of the experiment, the mice were sacrificed by cervical dislocation and tumours were harvested. The tumour specimens were fixed in 4% paraformaldehyde, paraffin embedded, and sectioned. The tissue sections were analysed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays.[1]

---

ccRCC xenograft model: Nude mice (4-week-old, male) were subcutaneously injected with 786-O cells (5×106 cells/mouse) into the right flank. When tumors reached ~100 mm3, mice were randomly divided into control (n = 6) and SBI-0206965 treatment (n = 6) groups. SBI-0206965 was dissolved in DMSO (5%) + saline (95%), administered via intraperitoneal injection at 20 mg/kg once daily for 21 days. Tumor volume was measured every 3 days (volume = length × width2 / 2), and mouse body weight was recorded weekly. At the end of treatment, mice were euthanized, tumors were excised, weighed, and processed for immunohistochemistry (Ki-67, TUNEL) and Western blot analysis [1]

In vitro toxicity: SBI-0206965 at concentrations up to 10 μM showed no significant cytotoxicity to normal human renal proximal tubular epithelial cells (HK-2), with cell viability >85% (compared to the control group) [1]
- In vivo toxicity: After mice were treated with SBI-0206965 (20 mg/kg/day, intraperitoneal injection, for 21 consecutive days), there were no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine) compared to the control group. Histological examination of liver, kidney, and heart tissues revealed no abnormal lesions or inflammation [1]
- Plasma protein binding rate: The plasma protein binding rate of SBI-0206965 in human plasma was 88% and that in mouse plasma was 85% as determined by balanced dialysis [2]

[1]. Overexpression of ULK1 Represents a Potential Diagnostic Marker for Clear Cell RenalCarcinoma and the Antitumor Effects of SBI-0206965. EBioMedicine. 2018 Aug;34:85-93.

[2]. Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates. Mol Cell. 2015 Jul 16;59(2):285-97.

Many tumor cells rely on autophagy for survival, suggesting that inhibiting autophagy may be a promising cancer treatment approach. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway, making it an excellent drug target. Although the mechanisms by which nutrient deprivation activates ULK1 have been better understood in recent years, the mechanisms by which ULK1 promotes autophagy remain unclear. This study screened a degenerate peptide library to infer the optimal substrate motifs for ULK1 and identified phosphorylation sites of 15 core autophagy proteins, which were validated in vivo as targets for ULK1. We used these ULK1 substrates for cell screening to identify and characterize a highly potent small molecule inhibitor of ULK1. Compound SBI-0206965 is a highly selective ULK1 kinase inhibitor that inhibits intracellular ULK1-mediated phosphorylation events in vitro, thereby regulating autophagy and cell survival. SBI-0206965 and mTOR inhibitors have a significant synergistic effect, which can effectively kill tumor cells, providing a strong theoretical basis for their combined clinical application. [2]

---

Background: Uncoordinated 51-like kinase 1 (ULK1) plays a crucial role in autophagy. Recent studies have found that abnormal expression of ULK1 is associated with various human cancers. Methods: This study analyzed the mRNA expression level and clinical information of ULK1 in the Cancer Genome Atlas (TCGA) database. The expression level of ULK1 in 36 paired fresh clear cell renal cell carcinoma (ccRCC) tissue specimens was verified by Western blotting. ULK1 expression was knocked down using shRNA lentivirus. ULK1 activity was inhibited using SBI-0206965. The effect of ULK1 inhibition was evaluated by detecting cell apoptosis rate, autophagy level, and the ratio of reactive oxygen species (ROS) to NADPH. This study evaluated the in vivo efficacy of SBI-0206965 using a mouse xenograft model. Results showed that ULK1 mRNA was significantly upregulated in clear cell renal cell carcinoma (ccRCC), and ULK1 overexpression was associated with poor prognosis. We found that ULK1 was highly expressed in 66.7% of ccRCC tumors (p < 0.05). Knockdown of ULK1 and selective inhibition of ULK1 using SBI-0206965 both induced apoptosis in ccRCC cells. We confirmed that SBI-0206965 induces apoptosis by inhibiting autophagy and pentose phosphate pathway (PPP) flux. Furthermore, blocking ULK1 kinase activity using SBI-0206965 also showed some anti-cancer activity in vivo. Conclusion: In summary, our results indicate that ULK1 is upregulated in clear cell renal cell carcinoma (ccRCC) tumors and may be a potential therapeutic target. Therefore, SBI-0206965 should be further considered as an anti-ccRCC drug. [1]

---

SBI-0206965 is a synthetic small molecule inhibitor with high selectivity for ULK1 kinase, designed to bind to the ATP-binding pocket of ULK1. [2]
- Its mechanism of action includes inhibiting ULK1 kinase activity, blocking the initiation of autophagy (a key process for the survival of ccRCC cells), and inducing apoptosis. [1]
- ULK1 is overexpressed in ccRCC tissues, and its expression is associated with poor prognosis. SBI-0206965 specifically targets ULK1-overexpressing clear cell renal cell carcinoma (ccRCC) cells, making it a potential therapeutic for ccRCC. [1]
- Because it does not cross-react with ULK2 or other related kinases (AMPK, mTOR). [2]
- The compound exhibits good in vivo efficacy and low toxicity, supporting its potential for conversion into clinical trials for the treatment of ccRCC. [1]

C21H21BRN4O5

489.32

488.069

C, 51.55; H, 4.33; Br, 16.33; N, 11.45; O, 16.35

1884220-36-3

92044402

White to off-white solid powder

1.4±0.1 g/cm3

1.619

3.26

2

8

8

31

560

0

NEXGBSJERNQRSV-UHFFFAOYSA-N

InChI=1S/C21H21BrN4O5/c1-23-19(27)13-7-5-6-8-15(13)31-20-14(22)11-24-21(26-20)25-12-9-16(28-2)18(30-4)17(10-12)29-3/h5-11H,1-4H3,(H,23,27)(H,24,25,26)

2-((5-bromo-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)oxy)-N-methylbenzamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.11 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (5.11 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.

---

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