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Development of Epoxyketone‐based Immunoproteasome‐selective Inhibitors with Enhanced Brain Distribution In Vivo

The proteasomes play an essential role in the maintenance of cellular protein homeostasis. As an inducible form of the proteasomes, the immunoproteasome (IP) has been implicated in various pathological conditions including neurodegenerative diseases. Different from the constitutive proteasome (CP), the IP harbors a distinct set of three catalytic subunits (β1i, β2i, β5i) replacing the respective counterparts (β1, β2, β5 in the CP). Several IP‐selective inhibitors have been so far reported including YU‐102 (a peptide epoxyketone). Recently, we obtained initial evidence for the protective effect of YU‐102 in the mouse models of neurodegenerative diseases. Being a substrate for P‐glycoprotein (Pgp), YU‐102 likely has poor penetration across the blood brain barrier (BBB). In order to achieve the IP inhibition in the brain parenchyma, we prepared a focused library of YU‐102 analogs and evaluated their interactions with Pgp and penetration across BBB. In the current study, a focused library of YU‐102 analogs were prepared by modifying the functional groups mainly at P1, P2 and N‐termial positions and we evaluated their potential to inhibit the IP behind the intact BBB via step‐wise screening assays. Initially, the analogs were evaluated for their potencies to inhibit the IP in cell lysates. The compounds with promising inhibitory potencies (comparable to or greater than YU‐102) were further tested using DLD‐1 cells overexpressing Pgp. The screening assays identified compound #16 as a lead candidate with its inhibitory potency similar to YU‐102 in cell lysates and with minimal interactions with Pgp (based on no enhancement by the Pgp inhibitor, reversin 121). For in vivo evaluation, mice received a single intraperitoneal injection (10 mg/kg) of compound #16 or YU‐102. At 2 or 4 h post‐dosing, the brain, whole blood and major organs were collected and analyzed for the remaining IP activity (activity assessed by kinetics assays using fluorogenic peptide substrates). The results indicated that the systemically administered YU‐102 effectively inhibited the IP in the major organs, but not in the brain. Compound #16 inhibited the IP activity in the major organs to the extent less than YU‐102. However, compound #16 was more effective in inhibiting the IP activity in the brain (IP inhibition by ~20% at 4 h post‐dosing). Together, these results suggest that compound #16 can have an enhanced brain distribution and IP target engagement in the brain parenchyma, potentially achieving therapeutic effects with lower systemic doses than YU‐102. We are currently investigating the pharmacokinetic profiles (drug exposure) and dose‐dependent nature of compound #16 in inhibiting the IP in the brain in order to proceed to the efficacy testing against neurodegenerative diseases.