Protease Signatures in Health and Disease: Toward Smart Diagnostic Platforms

Abstract Proteases, a vast and functionally diverse class of enzymes, are fundamental regulators of many important biological processes. Their activity is tightly controlled in cells; aberrant protease activity is associated with numerous pathologies including cancer, infectious diseases, neurodegeneration, and metabolic disorders. Protease dysregulation is not necessarily a mere consequence of disease but often a critical driver of pathogenesis, making protease activity a powerful functional biomarker. Importantly, protease activity offers a dynamic readout of biological function, integrating upstream regulatory signals with local environmental conditions. This Perspective explores the field of protease-based diagnostics, charting its evolution from fundamental research to emerging clinical applications. We first establish the rationale for using proteases as functional biomarkers across a spectrum of diseases, with particular focus on oncology, infectious diseases, neurodegeneration, and diabetes mellitus. Current and emerging technologies for detecting protease activity are then critically evaluated, from traditional substrate-based assays to sophisticated mass spectrometry platforms and innovative biosensing strategies involving smart probes, nanotechnology, and synthetic biology; we distinguish approaches that are clinically established from those validated in human samples, demonstrated preclinically, or still at the proof-of-concept stage. We then discuss the translational potential and inherent challenges of the approaches. Finally, we examine how multiplexed, activity-based profiling could generate dynamic “protease signatures” that, when combined with appropriate statistical and artificial intelligence methods, may support early disease detection, longitudinal monitoring, patient stratification, and personalized therapeutic strategies. Despite significant challenges related to biological complexity, analytical specificity, and clinical translation, protease diagnostics have the potential to provide disease information that complements established biomarkers and imaging modalities and may contribute to precision medicine.