Structural and biophysical characterization of RAD52 as novel pharmacological target for synthetic lethality

RAD52 is a protein involved in various DNA reparation mechanisms. In the last few years, RAD52 has been proposed as a novel pharmacological target for cancer synthetic lethality strategies. Hence, this work has the purpose to investigate RAD52 protein, with biophysical and structural tools to shed light on proteins features and mechanistic details that are, up to now poorly described, and to design novel strategies for its inhibition. My PhD work had two goals: the structural and functional characterization of RAD52 and the identification of novel RAD52 inhibitors. For the first part, RAD52 was characterized both for its DNA interaction and oligomerization state together with its propensity to form high molecular weight superstructures. Moreover, using EM and Cryo-EM techniques, additional RAD52 structural hallmarks were obtained, valuable both for understanding protein mechanism of action and for drug discovery purpose. The second part of my PhD project focused on the design and characterization of novel RAD52 inhibitors to be potentially used in combination therapies with PARPi to achieve cancer cells synthetic lethality, avoiding resistance occurrence and side effects. With this aim we selected and characterized promising RAD52 inhibitors through three different approaches: 19F NMR fragment-based screening; virtual screening campaign; aptamers computational design. Selected hits (fragments, molecules and aptamers) were investigated for their binding to RAD52 and for their mechanism of inhibition. Collected data highlighted the identification of hits worthy to be developed into more potent and selective RAD52 inhibitors. Finally, a side project carried out during my PhD is reported. GSK-3β protein, an already validated pharmacological target was investigated using biophysical and structural biology tools. Here, an innovative and adaptable drug discovery screening pipeline able to directly identify selective compounds with binding affinities not higher than a reference binder was developed.