Identification of drug target engagement of repurposing candidates for polycystic kidney disease by thermal proteome profiling

Autosomal dominant polycystic kidney disease (ADPKD) is a chronic, progressive hereditary disease characterized primarily by fluid-filled cysts in the kidneys and caused by mutations in the polycystin-1 or -2 gene. The treatment is currently limited to a single approved drug worldwide. In the search for additional drugs, drug repurposing provides an alternative. With this approach, clinically validated drugs with established pharmacokinetic and safety profiles are used for new indications. Here, 2D-thermal proteome profiling (2DTPP) was used to validate known and identify novel targets of ADPKD repurposing candidates. The repurposing candidates birinapant, bardoxolone methyl and salicylic acid were selected based on their previously demonstrated attenuation of cyst growth in vitro, in clinical trials and in preclinical studies, respectively. To mimic in vivo conditions, whole cell experiments were performed under physiological oxygen tension in the renal proximal tubular epithelial cell line RPTEC/TERT1. Our results confirmed the expected modes of action (MoA) for each of these three repurposing candidates. We also identified shared effects on mRNA translation, monocarboxylate transport, and mitochondrial nucleotide transport and energy metabolism. In addition, we discovered focal adhesion kinase, an activator of the proliferative PI3K/Akt/mTOR pathway, as a novel potential target that may contribute to the anti-proliferative effect of salicylic acid. Furthermore, we observed thermal destabilization of the glucosidase GANAB, an ADPKD-causing gene involved in polycystin maturation and trafficking, in response to salicylic acid treatment, highlighting the broad range of mechanisms that salicylic acid exerts on cyst growth. Finally, we corrected the observed dose-dependent changes in thermal stability with measured unbound drug concentrations at the target site, defined as the intracellular drug bioavailability. Overall, our findings highlight the utility of 2DTPP for evaluation of drug target engagement of repurposing candidates, enabling complex MoA validation and target discovery. Salicylic acid emerges as a particularly promising repurposing candidate for ADPKD due to its anti-proliferative effects and favorable safety profile.