PROJECT SUMMARY Targeting the ubiquitin-proteasome system has greatly contributed to the treatment of hematological cancers over the past fifteen years. Despite the success of these proteasome-targeted drugs (such as bortezomib, Btz) that inhibit the proteasome’s catalytic function, eventual drug resistance and off-target toxicities demand for alternative therapies to treat patients that become resistant to current treatments or for refractory cases. To overcome the current challenges, many groups have begun targeting the regulatory subunits of the proteasome. Rpn-13, a ubiquitin receptor on the proteasome’s regulatory particle, has been one of the most promising targets as it is not an essential proteasome subunit in healthy cells. Our central hypothesis is that inhibition of the proteasome’s regulatory function, specifically the Rpn-13 subunit, can overcome Btz-resistant mechanisms, working as an alternative therapy for multiple myeloma (MM) patients. With current inhibitors RA190 and KDT-11, Rpn-13 inhibition is observed to be toxic to MM in in vitro and in vivo applications. Since KDT-11 has been shown to be more selective than RA190, the long-term goal of this research project is to develop Rpn-13 inhibitors with improved potency and physical properties that bind to the same surface as KDT- 11 and maintain KDT-11’s selectively. The objective for this application is to utilize KDT-11 as a chemical probe to characterize and to explore its mechanism of action with Rpn-13 and in MM cell lines. To complete this objective, we propose the following specific aims: (1) To determine the binding site of the peptoid probe KDT-11 on Rpn-13, and (2) to evaluate KDT-11’s effect on Rpn-13 and apoptotic pathways in MM and Btz-resistant MM. Peptoids, such as KDT-11, have been considered potential therapeutic agents but previously reported peptoid probes have not been further investigated or optimized either due to low-affinity or non-selectivity. This proposal is innovative, in our opinion, because we plan to transition a selective peptoid probe with modest affinity to a more lead-like scaffold to develop more potent and pharmacologically-amenable Rpn-13 inhibitors. This contribution is expected to be significant because the discovery of KDT-11’s binding site and the evaluation of its mechanism of action in Btz-resistant MM would allow for optimization of the only reported reversible selective inhibitor for Rpn-13, which would lead to a greater understanding for alternative targets for MM treatment.