Discussion

Ryan Mordue, Michelle Newman, Alison Levy, Paul Wright, Puneet Khurana, Simon Osborne, Catherine Kettleborough, Barbara Saxty and Ahmad Kamal
1 MRC Technology, Centre for Therapeutics Discovery, 1-3 Burtonhole Lane, Mill Hill, London, NW7 1AD, United Kingdom.

Autophagy is a primary catabolic pathway involving lysosomal-dependant degradation and recycling of cellular components to maintain cell health and survival. ULK1 (Unc-51-Like Autophagy Activating Kinase 1 or Atg1) is a serine/threonine kinase shown to be crucial for the initiation of autophagy and targeting this kinase activity may be a tractable option for developing compounds that block autophagy. As well as being a homeostatic process, autophagy has been proposed as a potential cancer cell survival mechanism in response to hypoxia, nutrient deprivation and chemotherapy that may in part drive resistance to anti-cancer treatments. Therapeutic intervention that targets ULK1 kinase activity and block key steps in autophagy in combination with existing chemotherapeutic strategies could be important in overcoming resistance in cancer.
A focussed screen (approximately 20,000 compounds), identified a panel of ULK1 inhibitors with moderate kinase selectivity profiles. One candidate molecule (MRT1) was used as a chemical probe to pharmacologically validate the role of ULK1 in autophagy as well as the cell-based assays employed. A range of assays employing a number of cancer cell lines (A549, DLD-1, MCF-7, Panc-1, PC-3, HCT-116 and SkBR3) were used to assess the effect of MRT1 on phenotypic readouts of autophagy (LC3 turnover) and cell viability as well as comprehensive profiling of a number of cellular ULK substrates as potential pharmacodynamic markers of target engagement in cells (e.g. Atg13 and Beclin-1).
MRT1 blocked LC3 turnover in A549 as shown by a reduction in total LC3 accumulation (mean IC50, 0.25 ± 0.04 M), lipdated LC3 (LC3-II) and autophagosome and autolysosome formation. Modulation of a number of potential ULK-dependent biomarkers was also assessed in response to autophagy and in combination with MRT1. Autophagy consistently reduced the basal phosphorylation of ULK1 (S757) in response to mTOR inhibition or amino acid starvation, but this marker was not further modulated by compound treatment. We confirmed that recombinant ULK1 and ULK2 phosphorylated previously reported substrates, Beclin-1, Atg13 and Atg4b which was inhibited by MRT1, and we are currently monitoring the phosphorylation status of these substrates in A549 cells.
Further optimisation of compound potency and kinase selectivity through exploratory SAR is ongoing and may result in improved tool compounds for probing and validating the role of ULK1 kinase activity in autophagy through complimentary studies in additional cancer cell lines and investigation of alternative ULK biomarkers. These compounds may help in defining the role of ULK1 in disease settings such as cancer and validate its potential as a novel therapeutic target in chemotherapy resistance.