Design, Synthesis, and Docking Studies of New Torin2 Analogs as Potential ATR/mTOR Kinase Inhibitors †

Targeting DNA damage and response (DDR) pathway has become an attractive approach in cancer therapy. The key mediators involved in this pathway are ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia-mutated, Rad3-related kinase (ATR). These kinases induce cell cycle arrest in response to chemo- and radio-therapy and facilitate DNA repair via their major downstream targets. Targeting ATP-binding site of these kinases is currently under study. Torin2 is a second generation ATP competitive mTOR kinase inhibitor (EC50 = 250 pmol/L) with better pharmacokinetic profile. Torin2 also exhibits potent biochemical and cellular activity against ATM (EC50 = 28 nmol/L) and ATR (EC50 = 35 nmol/L) kinases. In this study, eight new Torin2 analogs were designed and synthesized through multistep synthesis. All the synthesized compounds were characterized by NMR and mass analysis. The newly synthesized analogs were evaluated for their anti-cancer activity via CellTiter-Glo® assay. Additionally, compounds 13 and 14 also showed significant inhibition for ATR and mTOR substrates, i.e., p-Chk1 Ser 317 and p70 S6K Thr 389, respectively. Compounds 13 and 14 displayed promising anti-cancer activity with HCT-116 cell lines in the preliminary study. Further, a comparative model of ATR kinase was generated using the SWISS-MODEL server and validated using PROCHECK, ProSA analysis. Synthesized compounds were docked into the ATP-binding site to understand the binding modes and for the rational design of new inhibitors.


Figure S37: LC-Mass of compound 9a
Cell based studies

Cell viability assay
Human HCT-116 cells were grown in DMEM/10% FBS/1% Pen-Strep medium at 37 0 C in a humidified incubator with 5% CO2. For the cell viability assay, cells were plated into 96-well plates at a count of 2000 cells per well in 198 μl medium, incubated for 24 hours and then treated with increasing concentrations of compound, respectively. After 72 hours of compound treatment, cell viability was determined using CellTiter-Glo® (Promega). Luminescence was measured by Envision Hybrid and modular multimode reader. All data were calculated by GraphPad Prism 6 software to get GI50 of each compound.

Immunoblot assay
HCT-116 cells were seeded in 6-well plates at a count of 0.5 x 10 6 cells per well and incubated overnight in a humidified CO2 incubator maintained at 37 0 C. For ATR assay, cells were exposed to 50 mJ/cm 2 of UV radiation energy (using UVP cross linker) after an hour of pre-treatment with appropriate compounds.
Culture media was saved before UV treatment and added back to the cells after UV treatment. After another 1-hour incubation, cells were rinsed with ice-cold PBS and lysed in ice-cold cell extraction buffer.
The soluble fractions of cell lysate were isolated by centrifugation at 13000rpm for 10 minutes at 4 0 C.
Following that, concentration of the protein was normalized by Bradford assay. Cell lysates were then subjected to SDS-PAGE and immunoblotting.

Discussion
The anticancer activity of all the synthesized compounds was performed against HCT-116 cell line at 1 µM concentration (Figure 7). Compound 13 and 14 showed strong inhibition similar to compound 11 (Torin2).
Two novel potent compound inhibitors i.e., 13 and 14 were selected from the initial screening based cell viability assay and used to sensitize colon cancer cell line. We performed another cell viability assay across a dose range of both the compounds in colon cancer cell line. A dose range between 0nM and 1000nM was chosen. With an incubation time of up to 72 hours, compound 14 was more toxic than 13 with a GI50 of 57 nM ( Figure S38). Compound 13 also inhibited viability of colon cancer cells with a GI50 of 138 nM ( Figure   S38). We confirmed that both 13 and 14 helped in sensitization of HCT-116 cells.
Based on the above mentioned results, to confirm if the compounds inhibit ATR and mTOR signaling in colon cancer cell line treated with radiation, an immunoblot assay was performed. Radiation was used as a part of combinatorial treatment as it is commonly used in colon cancer treatment. We assessed phosphorylation of Chk1 (p-Chk1 S317 ) and p70 S6 kinase (p70 S6K T389 ) by immunoblotting of treated HCT-116 cells. p-Chk1 S317 and p70 S6K T389 are downstream targets of ATR and mTOR, respectively. It was observed that 250 nM of 14 treatment inhibited phosphorylation of Chk1 (Ser 317) after treatment with UV radiation ( Figure S40). Interestingly, phosphorylation of p70 S6 kinase (Thr 389) was observed to be inhibited by both compounds at 50 nM for 14 and 250 nM for 13 ( Figure S40 and S41). Importantly, we confirmed that compound 14 inhibited the phosphorylation of ATR and mTOR substrates under these conditions. Whereas compound 13 inhibited the phosphorylation of mTOR substrate but not the ATR kinase substrate under UV treatment.