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.

In response to stress, cancer cells generate nutrients and energy through a cellular recycling process called autophagy, which can promote survival and tumor progression. Accordingly, autophagy inhibition has emerged as a potential cancer treatment strategy. Inhibitors targeting ULK1, an essential and early autophagy regulator, have provided proof of concept for targeting this kinase to inhibit autophagy; however, these are limited individually in their potency, selectivity, or cellular activity. In this study, we report two small molecule ULK1 inhibitors, ULK-100 and ULK-101, and establish superior potency and selectivity over a noteworthy published inhibitor. Moreover, we show that ULK-101 suppresses autophagy induction and autophagic flux in response to different stimuli. Finally, we use ULK-101 to demonstrate that ULK1 inhibition sensitizes KRAS mutant lung cancer cells to nutrient stress. ULK-101 represents a powerful molecular tool to study the role of autophagy in cancer cells and to evaluate the therapeutic potential of autophagy inhibition.[1]

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Autophagy is a conserved recycling process that has emerged as a critical effector of both oncogenes and tumor suppressors and a potent regulator of cancer cell fate (Liu and Ryan, 2012, Rosenfeldt and Ryan, 2009). Although autophagy is carried out by the coordinated activity of more than 30 proteins, just a few are enzymes with clear drug-targeting potential. Among these is ULK1, which has garnered interest as a small molecule target given its essential and early role in the pathway. Here, we have presented ULK-101 as a potent and selective ULK1 inhibitor and demonstrated its ability to suppress autophagy in human cells.[1]

ULK-101 joins at least six other ULK1 inhibitors reported since 2015. The Shokat laboratory has developed a series of ULK1-targeted compounds that have provided valuable insights into the structure of ULK1, despite limited selectivity and potency in cells (Lazarus et al., 2015, Lazarus and Shokat, 2015). Two other notable inhibitors were found by mining pharmaceutical data for compounds with activity against ULK1, analogous to our approach. SBI-0206965 was developed from a FAK inhibitor and shown to reduce Beclin 1 Ser15 phosphorylation in cells (Egan et al., 2015). This compound was reported as selective, based primarily on a large-scale competition binding assay; however, our direct comparison using in vitro kinase assays found ULK-101 to be considerably more selective than SBI-0206965. MRT68921, derived from a TBK1 inhibitor, inhibited ULK1 potently in vitro and strongly suppressed autophagy in cells, with 1 μM shown to block BafA1-induced LC3-II accumulation in nutrient-starved murine embryonic fibroblasts (Petherick et al., 2015). Although the authors screened 80 other kinases for inhibition by MRT68921, it is difficult to compare the selectivity profiles of MRT68921 and ULK-101. ULK-101 was screened against 327 kinases. Of interest, MRT68921 cross-reacts with AMPK, which may represent a therapeutic liability given the broad tumor suppressive functions of AMPK signaling. Interestingly, whereas AMPK was also inhibited by ULK-100 in vitro, it was spared by ULK-101 (Table S1). Finally, a study employing in silico screening and structure-activity relationship analyses identified potent indazole-derived ULK1 inhibitors, although their selectivity and activity in cells remains to be determined (Wood et al., 2017).[1]

A major unresolved issue in the autophagy field concerns the genetic and environmental contexts in which autophagy promotes tumor growth and represents a therapeutic target. Here, we have used ULK-101 to show that nutrient-stressed cells may be particularly susceptible to ULK1 inhibition. SBI-0206965 was similarly found to increase cell death in nutrient-starved cells or in those with chemical mTORC1 inhibition (Egan et al., 2015). These findings are consistent with other studies in which autophagy inhibition was particularly effective in cells deprived of nutrients (Eng et al., 2016, Guo et al., 2016). Together, this suggests that nutrient depletion caused by rapid tumor growth may create a unique vulnerability to autophagy inhibition. Finally, although we found that several lung cancer cell lines with oncogenic KRAS were sensitive to ULK-101, future work is required to fully define the genetic backgrounds in which targeting ULK1 and autophagy will be effective.[1]

Limitations of the Study Interest is mounting in developing novel therapeutics that can modulate the fundamental mechanisms of human disease, including autophagy. Despite encouraging research progress, only a limited number of compounds that target autophagy are developed beyond basic research. Accordingly, we aim to move these autophagy inhibitors through preclinical development. ULK-100 and ULK-101 have performed well in vitro, but these compounds require further validation in vivo to proceed with preclinical testing. In addition, ULK1 targeting as a therapeutic mechanism may not be effective in all genetic or environmental contexts, and further research is needed to identify when this strategy would be 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.)