Guidelines for the Design of Custom Affinity-Based Probes for Metalloproteases.

Although metalloproteases (MPs) have been studied for decades, their precise roles in various pathological processes remain to be fully elucidated. These enzymes often function within complex networks, and their activation states can vary over time and depending on the pathological context. In this dynamic landscape, there is a critical need for robust analytical tools capable of accurately identifying which MPs are present in their active forms and how their activation patterns evolve. Among the available technologies for profiling active MPs, affinity-based probes (AfBPs) have emerged as powerful chemical tools. These probes enable both the tracking and unambiguous identification of active MPs in complex biological systems. Here we attempt to provide guidelines for the design of AfBPs that could show selectivity for specific MP subclasses. We present the different alternatives for each structural element of a custom AfBP for MPs and the parameters that need to be considered in order to generate an effective AfBP for a target enzyme.