Insulin and glucagon play a central role in the management of diabetes mellitus (DM). This MPI application combines key clinical objectives of molecular endocrinology with frontiers of structural biology to design analogs of insulin and glucagon with enhanced pharmacologic properties. Our overarching goal is to reduce the burden of T1D through an innovative program of “device-driven protein engineering.” Animal studies will be performed in STZ rats (Subcontract to CWRU) and non-diabetic dogs (Subcontract to Vanderbilt University). This long-standing R01 program, now in its 30th year, began as a basic investigation of structure-activity relationships (SARs) in insulin with application to insulinopathies. The general flowering of this field internationally now affords translational opportunities to enhance T1D technologies while at the same time enriching basic insights into foundational principles of hormone SARs. Protein design rests upon two premises: Hypothesis 1: That optimization of the molecular properties of insulin and glucagon (i.e., non- incremental advances in pharmacologic parameters) can enable transformative delivery devices; and Hypothesis 2: That such translational research can provide broad scientific guidance for protein engineering in other diseases and therapeutic spaces. Our preliminary results suggest that two-chain insulin analogs can be made sufficiently stable and rapid acting to make feasible prefilled, disposable insulin pumps—and indeed that such pumps may be miniaturized to the size of a postage stamp (Aim 1). Data are also provided that for the first time suggest that insulin can be made more potent in vivo on a per nanomole basis and without increased mitogenicity (Aim 2). Such modifications may find particular utility in basal insulin analogs and reduce the high cost of these formulations (Aim 3). Our preliminary studies toward ultra-stable glucagon analogs (Aim 4) promise to make practical a one-week bihormonal pump as part of a closed-loop algorithm-based system. We emphasize these technologies because they may markedly enhance the long-term health of T1D patients, especially in the adolescent age group. Aims 1-3 exploit general principles of protein design and protein stability, including both natural amino-acid substitutions and innovative unnatural amino-acid substitutions amenable to biosynthetic manufacture. Aim 4 generalizes these principles to focus on the exquisite challenge posed by the instability of glucagon. Protein design will be based both on classical insulin crystal structures and on recent advances in the structural dissection of the insulin receptor (IR) and its mode of hormone binding. Aims 1-3 thus exploit the ongoing cryo- EM “resolution revolution” via Subcontract to M.C. Lawrence. An interdisciplinary Approach is proposed by a unique team with integrated MPI Management Plan. Translational significance is highlighted by the JDRF and recent American Diabetes Association President L. Philip...