Optimization and Synthesis of Ubiquitin-Specific Protease 7 Inhibitors for Alzheimer’s Disease

The ubiquitin proteasomal system (UPS) is one of the primary biological mediators of abnormal protein degradation. Defects in this system are linked to neurodegeneration. Ubiquitin-specific protease 7 (USP7) is a deubiquitinatinase (DUB) within the UPS that is linked to neuroinflammation and tau aggregation, both of which are biomarkers of Alzheimer’s disease (AD). Herein, we synthesized a series of USP7 inhibitors (compounds 5–17) to explore the structure-activity relationships (SAR) of literature compounds 2–4. An unpublished USP7 NanoBRET cellular target engagement assay was utilized to quantify target engagement of the compounds in intact HEK293 cells. This study seeks to identify how positional modifications and molecular substitutions on literature compounds 2–4 impact USP7 potency and to compare the performance of analogues in the published enzyme activity assay and our NanoBRET assay. Modifying the position bearing a methoxy group on the phenyl ring of compound 4 from the para- to the meta- position and substituting the methoxy group on the phenyl ring with a para-fluoro resulted in similar USP7 potency when compared to the literature compounds in permeabilized cells. However, substitution of sulfur containing groups on phenyl ring in compound 3 at the para- and meta- positions depleted inhibition. Literature compounds 2 and 3 which featured para-substituted groups such as a secondary alkyl amine and methoxy on the phenyl ring were most potent in the published enzymatic activity assay, however, 2 and 3 did not demonstrate high in-cell affinity for USP7 in the NanoBRET assay. This discrepancy provides insight on a potential shortcoming of published USP7 inhibitors: cell penetrance.  On the other hand, compound 4 exhibited potency in the NanoBRET assay in intact and permeabilized cells.  As a result, a second series of compounds (9-17) were developed to expand SAR on 4.  Extensions of the alkyl group on the piperidine nitrogen (9), homologation of the piperidine (11) and extensions of the alkyl chain extending from the amide (12 and 14) maintained USP7 potency.  However, compounds containing a tetrohydropyran (10), pyrrolidine, or pyrazole (15, 16, and 17) were not active in the NanoBRET assay.  Evaluation of these compounds has expanded our knowledge of USP7 inhibition in enzymatic activity versus USP7 in-cell engagement and provide valuable direction for future research, including analogues to improve potency.