The human proteasome plays an essential role in both protein homeostasis and in the regulation of multiple cellular processes from signal transduction to transcription. It has also become a proven target for developing drugs for treating multiple diseases including multiple myeloma and lymphoma. Several inhibitors of the proteasome, including bortezomib, carfilzomib and ixazomib, have been used in the clinic, responsible for prolonging lives of multiple myeloma patients. However, the existing proteasome-targeted drugs suffer from severe toxicity and rapid emergence of drug resistance, which also limits their potential in treating other types of diseases. We have developed a rapamycin-inspired macrocycle library known as rapafucins by fusing the FKBP-binding domain (FKBD) of rapamycin with a combinatorial peptide library. A screen of the rapafucin library in multiple myeloma cell line NCI-H929 led to the identification of a potent inhibitor, Rapaprotin that induces apoptosis in NCI-H929 cells. Rapaprotin exhibited selective toxicity to cancer cells over normal cells. It also has a unique mechanism of action, requiring activation by an intracellular protease through cleavage of the macrocycle into a linear form, Rapaprotin-L, which inhibits all three types of protease activities of the proteasome. Moreover, Rapaprotin is synergistic with bortezomib and is capable of resensitizing bortezomib-resistant cancer cells to the drug. In this application, we will investigate the mechanism of activation of Rapaprotin by the cellular protease, validating Rapaprotin-L as the active species for its cellular activity. We will obtain a high-resolution cryo-EM structure of the complex between proteasome and Rapaprotin-L. We will optimize the potency and pharmacokinetic property of Rapaprotin through design and synthesis of new analogs. The optimized analogs of Rapaprotin will be assessed for their efficacy in animal models of multiple myeloma and other diseases. The newly developed Rapaprotin analogs will serve as useful chemical probes to facilitate the study of the function and pharmacology of the proteasome and promising leads for developing a new class of anticancer and immunosuppressive drugs with lower adverse effects.