Project Summary. Cyclophilins and FKBPs are members of a large class of Peptidyl-Proline Isomerases (PPIases) and play a central role in modern medicine with clinical applications in immunosuppression, infectious disease, and chemotherapy. Although decades of effort have revealed the remarkable structural features of immunophilin enzymes, many members of this family, including Cyp40, have yet to meet the critical level of therapeutic examination. Recently our team described a system that uses beads to rapidly enhance the discovery of probes that target specific domains within a protein. In this program our team develops this method, as a technique to discover molecular probes to study the neurological roles of the immunophilins. The extracellular amyloid plaques and the intracellular neurofibrillary tangles (tau) that define the neuropathology of Alzheimer's disease are both regulated by the large immunophilins. Cyclophilin 40 (Cyp40), FK506-binding proteins FKBP51 and FKBP52 play important, but antagonistic roles. Cyp40 disaggregates α-synuclein and overexpression rescues tau-induced cognitive deficits and unravels neurotoxic plaques. Both Cyp40 and FKBP51 promote the stabilisation of microtubules while FKBP52 has been shown to cause memory impairments in a tau transgenic mouse model and to promote tau pathogenesis. A likely mechanism involves the opposing roles played by Cyp40 and FKPB52 in regulating microtubule depolymerisation. FKBP52 has been shown to bind and inhibit tubulin formation via direct binding by its TPR domain. As the amino acid identity between these three large immunophilin TPR domains is less than 26%, it will be possible to identify small molecule binders that distinguish between the TPR domains of Cyp40, FKBP51 and FKBP52. Modulating the effects of the large immunophilins will provide a new form of therapy to treat neurodegenerative diseases caused by plaque formation including Alzheimer's disease. Here, our team adapts an optimized workflow that uses beads, called Confocal Nanoscanning (CONA) as a tool to identify and validate fluorescent natural products that bind with high or medium affinity to Cyp40, FKBP51 or FKBP52. The resulting materials are then used to establish methods for cellular imaging as well as establish a high-throughput screen for each of these large immunophilins.