Mechanistic studies of a novel inhibitor of the ubiquitin-proteasome system

Abstract: The ubiquitin-proteasome system (UPS) not only maintains cellular proteostasis, but plays an essential role in multiple vital cellular processes, including survival and growth. Proteasomal inhibitors, such as bortezomib, have been shown to have effective anti-cancer properties, verifying the UPS as a viable drug target in cancer treatment. Yet, these compounds have encountered problems regarding toxicity, and inevitably development of resistance. The search for alternative targets within the UPS has revealed the 19S regulatory particle-associated deubiquitinases USP14 and UCHL5. Their inhibition blocks the deubiquitinating activity necessary for protein degradation by the proteasome. This has been shown to have cytotoxic effects in a range of cancer cells lines, as well as inhibiting tumor growth in several in vivo models. The small molecule inhibitor b-AP15 and its optimized lead VLX1570 were first discovered and characterized by the Linder research group at Karolinska Institute. Though thought to be highly promiscuous due to its , - unsaturated ketone motif, b-AP15 was demonstrated to selectively bind and inhibit the proteasomal deubiquitinases USP14 and UCHL5, with preferential binding to USP14. Inhibition of USP14 by b-AP15 results in a strong proteotoxic stress characterized by elevated levels of poly-ubiquitin, activation of the ER stress response, and oxidative stress, followed by apoptosis. We show here that the mechanism of b-AP15-induced apoptosis is characteristic of proteasome inhibition, but significantly differs from the effects of catalytic proteasome inhibitors. The results of b-AP15 treatment manifest as: severe proteotoxicity, mitochondrial damage without mitophagy induction, and lack of cytoprotective aggresome formation. Available evidence supports that the cellular response to b-AP15 is primarily dependent on USP14. Additionally, we use a drug screen of compounds that share a reactive unsaturated ketone motif with b-AP15, to show their potential pharmacological applications, relative selectivity for USP14, and ability to inhibit the UPS. This thesis describes in detail the proteotoxic effects induced by b-AP15 and its derivative VLX1570, and shows that despite its potential reactivity, b-AP15 selectively targets USP14. Similarly reactive compounds are shown to also display selectivity for the 19S deubiquitinases, indicating a potential for phamacological application in cancer therapy.