br Structure of USP Schematic representation of the
Structure of USP7 Schematic representation of the USP7 domain architecture is shown in Fig. 3B. USP7 is a 135 kDa protein that consists of seven domains, including the N-terminal TRAF-like (Tumor necrosis factor Receptor–Associated Factor) domain, followed by the catalytic core domain and the five C-terminal ubiquitin-like domains, UBL1-5, (Fig. 3B) [50,59]. The TRAF-like and the UBL domains recognize various USP7 substrates [4,7,33,, , ,74,75], and the regulatory C-terminus of the protein is thought to be crucial for enhancement of the USP7 catalytic activity [5,59,64]. The domains of this dynamic DUB are connected by linkers that allow for flexibility of interdomain arrangement (Fig. 3B and Supplementary Movie 1). Such flexibility is likely important for regulation of the enzyme’s activity [59,63,64,76].
Inhibition of USP7 Manipulating stability of proteins that are mutated, overexpressed or downregulated in human malignancies represents a promising therapeutic strategy for cancer treatment. Between the two protein degradation pathways, lysosomal proteolysis and UPS, the latter is highly selective and, therefore, its inhibition provides a strategy for the development of highly specific targeted therapies . E3 ligases and DUBs are of special interest since they determine the selectivity of UPS. USP7 is a promising pharmaceutical target because of (i) its role in cellular pathways involving regulators of DNA damage, oncogenes and tumor suppressors and (ii) growing evidence of its aberrant pgp inhibitors in various cancer cell lines. Despite several USP7 inhibitors have been reported in the literature [94,95], the lack of co-crystal structures of USP7 with small-molecule compounds until recently has been a limiting factor in the development of potent and selective USP7 inhibitors. In the past year, several groups reported structures of USP7 in complexes with small molecule inhibitors (Fig. 6, Supplementary Table 1) [83,85,, , ], which triggered a rapid structure-based design of a number of potent and highly specific analogues [, , , , ]. Solution NMR and mass spectrometry studies of interactions between the USP7 catalytic domain and P22077  and P50429  inhibitors uncovered the molecular mechanism of action of these thiophene-based compounds (Fig. 6, green, and Supplementary Table 1). Both inhibitors bind to the active site of USP7 and covalently and irreversibly modify the catalytic cysteine C223 with remarkable specificity . Co-crystal structures of several pyrimidinone based USP7 inhibitors were reported by several independent groups [, , , ] and shown to bind the same narrow and long grove of the catalytic site normally occupied by the C-terminal tail of ubiquitin (Fig. 6, purple). One of the inhibitors, FT827, carries vinylsulfonamide moiety, which reaches the catalytic triad and covalently modifies the catalytic cysteine. All other reported USP7 inhibitors are non-covalent. Another class of allosteric inhibitors, GNE-6640, GNE-6776  and USP7 inhibitor 2 , were shown to bind 12 Å away from the catalytic cysteine and impede ubiquitin binding  (Fig. 6, blue). Using a different approach, Zhang et al. reported ubiquitin variants that selectively interact with USP7 blocking its interaction with ubiquitin . All these USP7 inhibitors exhibit cytotoxic activity in several cancer cell lines making them promising leads for further development and optimization that through rational structure-based design.
Concluding remarks and perspectives
Introduction Prodrugs are the inactive derivatives of drug molecules, which can be used to improve drugs' stability, water-solubility, selectivity, etc. , , , , , , , , . The key step for performing prodrug therapy is that the reversible prodrugs can be effectively activated when and where they are needed. Prodrug activation by enzymes is a common method for converting prodrugs into effective drugs , , , , , , . In order to realize selective prodrug activation and supplement the lack of endogenous enzymes for matching different types of prodrugs, introduction of the exogenous natural enzymes to target cells is needed. This usually depends on gene transfection or antibody-enzyme delivery , , , . However, this process is complicated and great skills are often required. As a result, the prodrug therapy based on natural enzymes needs to be further improved, especially with the development of enzyme mimics.