Activity-based probes for the proteomic profiling of metalloproteases

Abstract

Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activitybased profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.

Towards third generation matrix metalloproteinase inhibitors for cancer therapy

“…Since TIMPs do not cause the musculoskeletal side effects that occurred with some MMPIs, then it is possible that neither MMPs nor ADAMs are the cause of these adverse reactions. This further supports incorporating weaker Zn 2 þ ion binding groups into the third generation MMPIs to reduce off target binding that was seen in activity based probe studies (Saghatelian et al, 2004). An alternative view though is that more potent and selective zinc-binding groups might improve MMPI specificity.…”

“…However, strong Zn 2 þ -chelating moieties disproportionately drive binding and so overwhelm the contribution from the rest of the compound, reducing other opportunities for improved specificity. Indeed, hydroxamate activity-based MMP probes related to marimastat bound many off-target metalloproteinases that were not MMPs (Saghatelian et al, 2004). This may be related to the lack of selectivity of hydroxamic acid for zinc over other divalent transition metals, including the ability to bind metal ions in several oxidation states such as iron (III).…”

Dual role of proapoptotic BAD in insulin secretion and beta cell survival

“…These findings prompted us to evaluate whether the BAD BH3 domain targets glucokinase directly. To test this mechanism, we derivatized BAD SAHBs to contain both FITC and a photoactivatable benzophenone moiety for covalent capture of protein targets 29 (Fig. 5f).…”

Application of activity-based probes to the study of enzymes involved in cancer progression

“…This warhead is often derived from the electrophilic group of an irreversible enzyme inhibitor, such as an epoxide or fluorophosphonate. For enzymes that do not covalently bind their substrates, such as metalloproteases, ABPs have been developed that contain a chelator moiety (for noncovalent binding to the conserved metal atom in the active site) and a photoinducible chemical crosslinker (for covalent binding to the enzyme active site upon ultraviolet irradiation) [17]. The linker region of an ABP can influence the specificity of the probe.…”

“…However, ABPs for these enzymes have been constructed that contain a zincchelating moiety and a photocrosslinking group that allows for covalent labeling of the target enzymes. A library of metalloprotease probes was used to profile the activities of metalloproteases in both breast carcinoma and melanoma cell lines [17,22]. Neprilysin, alanyl aminopeptidase, and ADAM10 activities were found to be elevated in invasive cells.…”