Project Summary Immunoglobulin light chain amyloidosis (AL) is a degenerative disease that putatively arises from light chain (LC) misfolding and aggregation. Full-length (FL) LCs are secreted from a clonally expanded plasma cell population in AL. Thus, AL patients suffer from both a plasma cell cancer and a LC misfolding and aggregation-associated proteinopathy that appears to compromise organ function, leading to progressive organ deterioration. Currently, AL is treated by repurposed multiple myeloma drugs that kill the clonal plasma cells secreting FL LCs. Mechanistically distinct therapeutic approaches are needed, particularly for patients with cardiac involvement who cannot tolerate the currently available chemotherapy regimens. In this proposal, we seek to produce lead candidates that ultimately would enable a clinical trial on a FL LC kinetic stabilizer targeting the proteinopathy component of AL. In Specific Aim 1, we hypothesize that the LC kinetic stabilizers to be synthesized in our hit-to-lead medicinal chemistry efforts can be dissected into four substructures—the “anchor substructure”, the “aromatic core”, the “linker module” and the “terminal aromatic component”. This hypothesis is based on 11 (FL LC)2•kinetic stabilizer crystal structures solved to date as well as the structure- activity relationship data resulting from the synthesis of over 300 FL LC2 kinetic stabilizers during the past 18 months. We will continue to use structure-based design principles that we learned developing the drug tafamidis, in addition to computational tools, especially the Schrödinger LiveDesign software, to replace the metabolically unstable and potentially toxic coumarin aromatic core and the diethyl aniline anchor substructure in our LC kinetic stabilizers, while introducing functionality to reduce albumin binding. The LiveDesign software utilizes a weighted combination of the predicted LogP values and docking scores to accurately predict albumin binding. In Specific Aim 2, we will develop FL LC2 plasma binding selectivity assays to validate our computational efforts to diminish albumin binding. First, we will add fluorescently labeled FL amyloidogenic LCs to pooled healthy donor plasma, along with proteinase K and a candidate kinetic stabilizer, and follow proteinase K endoproteolysis linked to FL LC2 conformational excursions chromatographically over time. If the kinetic stabilizer candidate selectively binds the amyloidogenic FL LC2 over all the other plasma proteins, including albumin, FL LC2 proteinase K endoproteolysis will be prevented–this assay is largely developed. Next, a subunit exchange assay will be developed to quantify pharmacologic FL LC2 kinetic stabilization in human plasma. This assay also quantifies kinetic stabilizer binding to albumin. A fluorescently labeled Cys214Ser FL LC2 variant facilitates subunit exchange experiments that afford the KD of kinetic stabilizer binding to the FL LC2 variants added to healthy plasma, as wel...