Future advances in the in vivo
Future advances in the in vivo molecular imaging of enzyme activity will greatly benefit from the progress towards the quantitative detection of enzymes. Quantitative imaging requires knowledge of both the location and concentration of a specific enzyme, which is dependent on the simultaneous measurement of both the product and substrate concentrations at the same location. However, the dynamic in vivo environment presents significant barriers to quantification. Traditional imaging studies are normally dependent on a single imaging modality, which may lead to an inaccurate or even false quantitative results. Therefore, it is necessary to develop new activatable imaging probes with a combination of two or three imaging modalities to provide an additional imaging signal for the quantitative measurement of the probe concentration, independent of enzyme activity . Indeed, the use of a continuous PET signal to correct the MMP activity measured through optical imaging using an MMP-activatable bimodal probe was recently demonstrated, suggesting a high potential for the quantitative measurement of enzyme levels in vivo . Considering the great progress that has been made, we strongly believe that activatable imaging probes will find more applications in clinics, including (i) earlier and more accurate diagnosis of diseases, (ii) imaging-guided surgery, and (iii) real-time assessment of therapeutic efficacy.
Conflict of interest
Acknowledgments This work was supported by the National Natural Science Foundation of China (21505070, 21632008) and Natural Foundation of Jiangsu Province (BK20150567).
Introduction The post-translational modifier ubiquitin influences virtually all aspects of eukaryotic biology (Hershko and Ciechanover, 1998). The single-chain ubiquitin-activating enzyme E1 (UBA1) adenylates the C terminus of ubiquitin (Ub), followed by Ub transfer onto the UBA1 catalytic cysteine (Haas et al., 1983). Thioesterified Ub is subsequently relayed to a ubiquitin-conjugating enzyme (E2) and, with the aid of ubiquitin ligases (E3), onto target Terazosin (Hershko and Ciechanover, 1998). Besides Ub, there are 16 additional ubiquitin-like proteins (Ubls) including SUMO and NEDD8, which are activated by cognate E1s (Schulman and Harper, 2009, Cappadocia and Lima, 2017). SUMO and NEDD8 E1s are heterodimers (SAE1/UBA2 and APPBP1/UBA3) with UBA2 and UBA3, respectively, representing the catalytic subunit (Lee and Schindelin, 2008, Schulman and Harper, 2009, Streich and Haas, 2010). Ever since the approval by the US Food and Drug Administration of the proteasome inhibitor bortezomib for the treatment of multiple myeloma and mantle cell lymphoma, extensive research has been carried out to target the components of the ubiquitin proteasome system for cancer therapy. UBA1, 2, and 3 were established as promising oncological drug targets (da Silva et al., 2013). Furthermore, targeting UBA1 may overcome clinical resistance to bortezomib and be more effective in treating solid tumors (An and Statsyuk, 2015a, Xu et al., 2010). Wilkinson et al. (1990) demonstrated that a non-hydrolyzable analog of the ubiquitin adenylate could potently inhibit the ubi-quitin-activating enzyme. Later, Ub-AMSN and SUMO-AMSN probes, which mimic the ubiquitin and SUMO adenylates, respectively, were shown to selectively inhibit Uba1 and the SUMO E1 (Lu et al., 2010); however, due to their large size these semisynthetic protein inhibitors could not be used for therapeutic purposes. Adenosyl sulfamates were described as cell membrane-permeable E1 inhibitors with the NEDD8 E1-selective inhibitor MLN4924 as first-in-class representative (Soucy et al., 2009). These compounds form N-acylsulfamates with Ub/Ubls (Nawrocki et al., 2012) in the presence of ATP and the E1 (Brownell et al., 2010, Chen et al., 2011) via a mechanism whereby the sulfamate NH2 group attacks the E1∼Ub/Ubl thioester to produce a covalent Ub/Ubl-inhibitor adduct. The resulting adduct mimics the Ub/Ubl adenylate and binds tightly (with picomolar affinity) to the E1, thus causing its inhibition (Chen et al., 2011). Although adenosyl sulfamates specifically inhibit E1 enzymes, their specificities differ vastly as reflected in low-nanomolar to high-micromolar half-maximal inhibitory concentration (IC50) values reported for these compounds toward various E1 enzymes (Figure S1). Recently, the adenosyl sulfamates MLN7243 (Traore et al., 2014), a UBA1-selective inhibitor, and ABPA3 (An and Statsyuk, 2015a), a dual inhibitor of the Ub and NEDD8 E1s, were reported. Despite the structure of MLN4924 bound to NEDD8 E1, knowledge of how chemically diverse adenosyl sulfamates occupy the ATP-binding pocket of UBA1 and display variable specificities toward different E1 enzymes has been missing.