Objective

Autophagy, also referred to as “self-eating”, plays a pivotal role in the degradation of defective cellular components and under nutrient replete conditions maintains cellular homeostasis and integrity. This largely non selective mechanism involves the formation of double membrane structures, called autophagosomes, into which lysosomes release enzymes responsible for cargo degradation, with recyclable material being re-released into the cytosol. Our current understanding of the role of autophagy in cancer is complicated by the fact that it can be both pro-survival and pro-death. However, it is becoming increasingly clear that established tumours challenged by chemotherapeutic drugs can take advantage of the autophagic pathway to evade cell death and, furthermore, that suppression of this pathway can re-sensitise cells to therapeutic drugs. There are currently no clinically available drugs that specifically interact with regulatory molecules in the autophagy pathway, the autophagy related (Atg) proteins, and in light of this we have designed a high-throughput screening assay to identify drugs that inhibit autophagy. We selected two crucial autophagy proteins, Atg5 and Atg16, that are centrally involved in autophagosome formation and tagged them with two subunits, LgBiT and SmBiT, derived from the NanoLuc® Luciferase enzyme (NanoLuc® Binary Technology; Promega). The interaction of Atg5 and Atg16 during autophagosome formation allows the LgBiT and SmBiT subunits to interact generating a bioluminescent signal. Induction of autophagy increases the bioluminescent signal approximately 4-fold. We have also validated the technique by testing compounds known to bind to Atg5 and block interaction with Atg16¹. Further screening of compound libraries will allow us to refine and improve structure activity relationships and to identify possible lead compounds that can be used to re-sensitise chemoresistant cancers.