PROJECT SUMMARY/ABSTRACT Charcot neuroarthropathy (CN) is a progressive, denervation-induced degeneration of weight bearing joints. Any patient with loss of afferent proprioceptive fibers is susceptible to this degeneration. CN presents as a discrete local inflammatory reaction resulting in the sterile destruction of joint bone in the foot and ankle. The 5-year mortality in patients is as high as 28% with greater than 67% of these patients developing severe ulcerative complications that may lead to amputation. The CN inflammatory process results in localized osteopenia in a focal area of the foot or ankle causing instability and collapse when bearing weight. It is a self-limiting process which appears to be triggered by a trauma or repetitive microtrauma. Once activated, CN is characterized by resorptive osteoclastic activity leading to a progressive bone lesion. CN is often misdiagnosed as cellulitis or osteomyelitis, delaying diagnosis and increasing bone destruction. Osteolysis of bone contributes significantly to the morbidity and mortality of patients with CN and early assessment is critical to preserve function and to reduce the risk of major amputation. There are currently no prognostic tests available for early detection of CN. Cathepsin K (CatK) is a cysteine protease produced by osteoclasts that is overexpressed in many resorptive bone diseases, including CN. We have designed a novel molecular imaging probe, OFS (Osteoadsorptive Fluorogenic Substrate), to detect CatK on bone surfaces. This probe incorporates a bisphosphonate moiety to allow it to target bone with very high tissue selectivity, together with a fluorophore linked to a Förster resonance energy transfer (FRET) quenching dye by a peptide that is specifically cleaved by CatK, thereby generating a strong fluorescent signal in the presence of the enzyme. OFS thus offers a unique method to detect abnormal osteoclast-mediated bone resorption with high sensitivity. We propose to develop this highly innovative molecular probe for early detection of osteolysis and surveillance of CN by mapping OFS fluorescence in intraoperative bone samples from CN patients, using healthy bone as a control and radiography as the reference diagnostic imaging technology. If successful, the proposed research would provide the foundation for a powerful new diagnostic and prognostic tool to improve outcomes for CN patients, among whom disadvantaged minorities are disproportionately represented. The project, led by Dr. Laura Shin, DPM, PhD (expert on limb salvage, bone reconstruction related to CN, cell culture and confocal microscopy) and Dr. Charles McKenna, PhD (expert on imaging bisphosphonates), will be performed in the PIs’ laboratories at the University of Southern California’s Keck School of Medicine (Shin) and Drug Discovery Center (McKenna).