PROJECT SUMMARY Human African Trypanosomiases (HAT) is a disease of poverty caused by various sub-species of the Trypanosoma brucei protozoan parasite. Transmitted by the bite of the tse-tse fly, Stage 1 disease is characterized by trypanosomes that disseminate through the blood and lymphatic systems, and then cross the blood-brain barrier into the CNS to cause Stage 2 disease. Infiltration of the CNS is associated with a frightening array of progressively deteriorating and eventually lethal psychological and physiological disorders. Drug therapy relies on antiquated and often toxic drugs that must be administered under medical supervision and for which resistance is established or a constant threat. New drugs are needed. The proteasome is an evolutionarily- conserved, multi-subunit protein complex in the cell that regulates normal protein turnover and degradation of misfolded proteins. Inhibition of the T. brucei proteasome (Tb20S) with small molecule inhibitors kills trypanosomes. However, there is little information regarding the proteolytic cleavage preferences or the inhibition profile of the Tb20S target. Uncovering this information would support the development of proteasome inhibitors with improved specificity and potency that could then form the basis for a new treatment of HAT. Accordingly, in Aim 1 we will comprehensively profile the cleavage specificity of Tb20S using a technology called multiplex substrate profiling by mass spectrometry (MSP-MS). The data arising will inform (i) the synthesis of optimized peptidyl substrates with which the activity of Tb20S can be measured and (ii) a medicinal chemistry effort (Aim 3) to synthesize new Tb20S inhibitors with improved specificity. In Aim 2, we will screen Tb20S with various proteasome inhibitor libraries containing diverse scaffolds and reactive groups that have been developed in-house and by collaborators. The data arising will inform (i) the broader chemical space associated with inhibition of Tb20S and (ii), again, the medicinal chemistry program planned for Aim 3. For Aim 3, we will screen both Trypanosoma brucei and mammalian cell lines with the top performing Tb20S inhibitors to understand the differential between parasite-killing and cytotoxicity. This Aim also involves a focused medicinal chemistry campaign that evaluates all of the proteasome inhibition and cell screening data to synthesize Tb20S inhibitors with improved specificity using the scaffold of the marine natural product known as carmaphycin B. Upon completion of the proposed R21 studies, we will have identified a series of Tb20S inhibitors ready for further development as therapeutics for HAT.