ULK1 (IC50 = 1.6 nM); ULK2 (IC50 = 30 nM)

BafA1-induced LC3B-II accumulation in U2OS cells is inhibited by ULK-101 (0-5 μM) in a concentration-dependent manner[1].

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•ULK-101 has improved potency and selectivity when compared with SBI-0206965.[1]
•ULK-101 inhibits both the nucleation of autophagic vesicles and turnover.[1]
•ULK-101 sensitizes KRAS-driven lung cancer cells to nutrient restriction.[1]
•ULK-101 is a valuable molecular tool to study the function of ULK1 and autophagy.[1]

Kinase Assays and IC50 Calculations [1]
IC50 data for ULK1 and ULK2 was generated using 10-point IC50Profiler assays with half-log dilutions from top concentrations of 10 μM (4 replicates) and 1 μM (4 replicates), giving 8 data points for most concentrations in the curve. For selectivity profiling, KinaseProfiler assays with wild-type human kinase panels were performed in duplicate using 500 nM SBI-0206965, 40 nM ULK-101, or 15 nM ULK-100. For each kinase reaction, the Km concentration of ATP was used. The percent activity remaining and percent inhibition were calculated from negative control wells. For selectivity profiling, relative inhibition was calculated by dividing the percent inhibition of each kinase by the percent inhibition of ULK1. GraphPad Prism 7 was used for IC50 determinations by fitting curves with variable slope (four-parameter) non-linear regression models using top and bottom constraints of 100% and 0%, respectively.

Immunoblotting [1]
Cells were lysed in ice-cold lysis buffer [10 mM KPO4, 1 mM EDTA, 10 mM MgCl2, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO4, 5 mM NaF, 2 mM DTT, and complete protease inhibitors] and proteins resolved by SDS-PAGE. Hand-poured 10% acrylamide gels were used for Figure 1 and pre-cast BOLT 4-12% Bis-Tris Plus gels were used for all other blots. Proteins were transferred to nitrocellulose membranes (or PVDF membranes for LC3 blots) and probed with primary antibodies overnight at 4°C followed by secondary antibodies for 1 hour at room temperature. Proteins were detected by enhanced chemiluminescence (Figure 1C), or imaged and quantified on an Odyssey Classic or Odyssey Clx imager (for all other blots).

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Fluorescent Microscopy [1]
A monoclonal U2OS-EGFP-DFCP1 cell line was generated by transducing cells with retrovirus expressing this plasmid and selecting a low-expressing monoclone. Cells were seeded at 20,000 per chamber of a 4-chamber 35 mm no. 1.5 glass-bottom dish for 24 hours. Media was replenished and cells treated with 5 μM ULK101 (or DMSO control) and 100 nM AZD8055 (or DMSO control). Images were captured in the FITC channel every 30 minutes using a Nikon Ti Eclipse microscope enclosed with a cage incubator and maintained at 37°C with humidified 5% CO2. For quantification, images were deconvolved, smoothed, top-hat transformed (“detect peaks” function), and thresholded (by intensity) using NIS Elements to obtain the number of DFCP1-positive objects per cell. For Figure 4C, U2OS cells stably expressing ptfLC3B were seeded for approximately 48 hours on no. 1.5 coverglass discs in a 24-well dish. Cells were treated with 5 μM ULK-101 (or DMSO control) for 3 hours. 1.5 hours later, media was supplemented with 100 nM BafA1 (or a volume-equivalent of DMSO). Cells were then fixed with 4% formaldehyde and nuclei stained with Hoechst-33342. Cells were imaged in the FITC (green) and DAPI (blue) channels. For quantification, images were deconvolved, top-hat transformed (“detect peaks” function), and thresholded (by intensity) using NIS Elements to obtain the number of GFP-LC3-positive objects per cell

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ATG12 Immunofluorescence[1]
U2OS cells were treated with 100 nM AZD8055 or a volume-equivalent of DMSO with or without 5 μM ULK-101 (or DMSO control) for 2.5 hours. Cells were fixed with 4% formaldehyde, permeabilized with 0.2% triton-X100 in 1xDPBS, blocked with 3% bovine serum albumin (BSA) and 5% goat serum in 1xDPBS, and stained with anti-ATG12 antibodies (diluted 1:100 in blocking buffer) overnight at 4°C. Cells were then stained with AF488-conjugated secondary antibodies (at 1:1000) for 1 hour at room temperature, nuclei counterstained with Hoechst-33342, and coverglass inverted onto microslides with gel mount. Cells were imaged with a 60x oil objective in the FITC (green) and DAPI (blue) channels on a Nikon Ti Eclipse microscope. 30-50 cells per condition were imaged and representative images shown in Figure 3C.

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Clonogenic Survival Assays[1]
Cells (U2OS or NSCLC) were seeded on tissue culture treated 96-well plates at 1,000 cells per well in RPMI-1640 media supplemented with 10% FBS. Twenty four hours later, media was aspirated, wells rinsed with 1x DPBS, and replaced with full media (FM) or Optistarve (OS) with a concentration gradient of ULK-101 (final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.1 μM, 1.6 μM, 0.8 μM, 0.4 μM, or 0 μM). Two days later, media (and ULK-101) was aspirated, wells were rinsed with 1x DPBS, and all wells replaced with FM. Five days later, relative ATP levels were measured using a luminescent CellTiter-Glo assay following manufacturer’s instructions

[1]. A Potent and Selective ULK1 Inhibitor Suppresses Autophagy and Sensitizes Cancer Cells to Nutrient Stress. iScience. 2018 Oct 26;8:74-84.

Cancer cells generate nutrients and energy through a cellular cycle called autophagy in response to stress, thereby promoting cell survival and tumor progression. Therefore, inhibiting autophagy has become a potential cancer treatment strategy. Inhibitors targeting ULK1 (an important regulator of early autophagy) have demonstrated the concept of inhibiting autophagy by targeting this kinase; however, these inhibitors have limitations in terms of potency, selectivity, and cellular activity. This study reports two small-molecule ULK1 inhibitors, ULK-100 and ULK-101, and demonstrates their superior potency and selectivity compared to a well-known published inhibitor. Furthermore, we found that ULK-101 can inhibit autophagy and autophagy flux induced by different stimuli. Finally, we used ULK-101 to demonstrate that ULK1 inhibition enhances the sensitivity of KRAS-mutant lung cancer cells to nutritional stress. ULK-101 is a powerful molecular tool for investigating the role of autophagy in cancer cells and evaluating the therapeutic potential of autophagy inhibition. [1]

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Autophagy is a conserved recycling process that has become a key effector of oncogenes and tumor suppressor genes and a potent regulator of cancer cell fate (Liu and Ryan, 2012; Rosenfeldt and Ryan, 2009). Although autophagy is accomplished by the synergistic action of more than 30 proteins, only a few enzymes have clear drug targeting potential. ULK1 is one of them and has attracted much attention as a small molecule target due to its crucial early role in the autophagy pathway. This article introduces a highly potent and selective ULK1 inhibitor, ULK-101, and demonstrates its ability to inhibit autophagy in human cells. [1]
ULK-101 is one of at least six ULK1 inhibitors reported since 2015. The Shokat laboratory has developed a series of compounds that target ULK1, and although these compounds have limited selectivity and potency in cells, they have provided valuable insights into the structure of ULK1 (Lazarus et al., 2015; Lazarus and Shokat, 2015). By mining drug data to identify compounds active against ULK1, two other noteworthy inhibitors were discovered, similar to our approach. SBI-0206965, developed from a FAK inhibitor, has been shown to reduce phosphorylation levels of Beclin 1 Ser15 in cells (Egan et al., 2015). This compound has been reported to be selective, primarily based on large-scale competitive binding experiments; however, our direct comparison using in vitro kinase activity assays revealed that ULK-101 exhibited significantly higher selectivity than SBI-0206965. MRT68921, derived from a TBK1 inhibitor, effectively inhibits ULK1 in vitro and strongly suppresses autophagy; 1 μM of MRT68921 blocked BafA1-induced LC3-II accumulation in nutrient-deprived mouse embryonic fibroblasts (Petherick et al., 2015). Although the authors screened 80 other kinases to test the inhibitory activity of MRT68921, it was difficult to compare the selectivity profiles of MRT68921 and ULK-101. ULK-101 screened 327 kinases. Notably, MRT68921 cross-reacts with AMPK, which may pose a therapeutic risk given the extensive tumor-suppressive function of the AMPK signaling pathway. Interestingly, although in vitro experiments showed that ULK-100 also inhibited AMPK, ULK-101 did not inhibit AMPK (Table S1). Finally, a study using computer screening and structure-activity relationship analysis identified some effective indazole ULK1 inhibitors, but their selectivity and activity in cells remain to be determined (Wood et al., 2017). [1]
A major unresolved question in the field of autophagy is under what genetic and environmental contexts autophagy promotes tumor growth and becomes a therapeutic target. Here, we use ULK-101 to demonstrate that nutritionally stressed cells may be particularly sensitive to ULK1 inhibition. Similarly, SBI-0206965 was also found to increase the death of nutrient-deprived cells or cells whose mTORC1 was inhibited by chemical methods (Egan et al., 2015). These findings are consistent with other studies showing that autophagy inhibition is particularly effective in nutrient-deprived cells (Eng et al., 2016; Guo et al., 2016). In summary, this suggests that nutrient depletion caused by rapid tumor growth may make cells uniquely vulnerable to autophagy inhibition. Finally, although we found that several lung cancer cell lines carrying oncogenic KRAS were sensitive to ULK-101, further research is needed to fully determine the genetic background of the effectiveness of targeting ULK1 and autophagy. [1]
Research limitations: There is growing interest in developing novel therapies that can modulate fundamental mechanisms of human diseases, including autophagy. Despite encouraging progress, only a few compounds targeting autophagy have been developed outside of basic research. Therefore, our goal is to advance these autophagy inhibitors into the preclinical development stage. ULK-100 and ULK-101 showed promising results in in vitro studies, but these compounds require further in vivo validation before entering preclinical trials. Furthermore, the therapeutic mechanism targeting ULK1 may not be effective in all genetic or environmental contexts, thus further research is needed to determine under what conditions this strategy is most effective.

C22H16F4N4OS

460.45

460.098

C, 57.39; H, 3.50; F, 16.50; N, 12.17; O, 3.47; S, 6.96

2443816-45-1

137628686

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.684

3.38

1

8

5

32

685

1

C1CC1[C@@H](C(F)(F)F)NC(=O)C2=CC(=CS2)C3=C4N=CC(=CN4N=C3)C5=CC=C(C=C5)F

PFZRXJIYAFANHP-IBGZPJMESA-N

InChI=1S/C22H16F4N4OS/c23-16-5-3-12(4-6-16)15-8-27-20-17(9-28-30(20)10-15)14-7-18(32-11-14)21(31)29-19(13-1-2-13)22(24,25)26/h3-11,13,19H,1-2H2,(H,29,31)/t19-/m0/s1

N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]-4-[6-(4-fluorophenyl)pyrazolo[1,5-a]pyrimidin-3-yl]thiophene-2-carboxamide

ULK-101; 2443816-45-1; (S)-N-(1-cyclopropyl-2,2,2-trifluoroethyl)-4-(6-(4-fluorophenyl)pyrazolo[1,5-a]pyrimidin-3-yl)thiophene-2-carboxamide; N-[(1S)-1-Cyclopropyl-2,2,2-trifluoroethyl]-4-[6-(4-fluorophenyl)pyrazolo[1,5-a]pyrimidin-3-yl]thiophene-2-carboxamide; ULK101; CHEMBL4744680; SCHEMBL25395801; EX-A4693;

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: 83.33 mg/mL (180.98 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)