Clinical evaluation of pathognomonic salivary protease fingerprinting for oral disease diagnosis

Dental decay (Caries) and periodontal disease are prevalent diseases globally, with significant clinical need for improved diagnosis. As mediators of dental disease-specific extracellular matrix degradation, proteinases are promising analytes. We hypothesized that dysregulation of active proteases is functionally associated with oral disease status, and may be used diagnostically. To address this, we examined a total of 52 patients with varying oral disease states, including healthy controls. Whole mouth saliva samples and dentine caries biopsies were collected and subjected to analysis. Overall proteolytic and substrate specific activities were assessed using five multiplexed, fluorogenic peptides. Peptide cleavage was further described by inhibitors targeting matrix metalloproteases (MMPs) and cysteine, serine, calpain proteases (CSC). Proteolytic fingerprints, supported by supervised machine-learning analysis, were delineated by total proteolytic activity (PepE) and substrate preference combined with inhibition profiles. Caries and periodontal disease showed increased enzymatic activities, elevated MMP/CSC levels with common (PepA) and divergent substrate cleavage patterns (PepE), suggesting differential MMP/CSC contributions in disease states. For dentine caries classification, a sensitivity of 84.6% and specificity of 90.0% was attained. Thus, a combined analysis of protease derived individual and arrayed substrate cleavage rates in conjunction with inhibitor profiles may represent a sensitive and specific tool for oral disease detection