New mechanistic insights into targeted protein degradation using Raman-active theranostic hydrophobic tags

Hydrophobic tagging (HyT) has emerged as a useful tool within the field of targeted protein degradation for the targeting and removal of a protein of interest (POI) from the cell. Development of new hydrophobic tags with increased efficiency not only allows increased POI degradation but also provides an opportunity to further study the mechanisms of HyT, which are not fully understood. Herein, we describe the development of new theranostic hydrophobic tags that can be used to induce protein degradation and simultaneously enable drug imaging using stimulated Raman scattering (SRS) microscopy. By coupling our dual-purpose theranostic hydrophobic tags to the PARP inhibiting, anti-cancer therapeutic olaparib, we explore the degradation efficiency, mode of action and potency that these theranostics confer in ovarian cancer cells. Lead compound 2c effectively degrades intracellular PARP-1 (DC₅₀ = 0.618 μM, D_max = 55 %) and has more potent antiproliferative activity than the parent compound olaparib (IC₅₀ = 62 nM vs 11.52 μM for olaparib), whilst maintaining on-target PARP inhibitory activity. Using mass spectrometry-based proteomic analysis, we demonstrate activation of the unfolded protein response, ER-stress and cell cycle arrest after incubation with 2c. Using SRS imaging, via the inherent Raman activity of the theranostic alkyne tag, we demonstrate involvement of the ER and autophagy after treatment with our drug conjugates. These results provide new insight into the mechanisms involved in HyT-induced protein degradation.