Project Summary / Abstract Chronic neuroinflammation occurs in early stages of Alzheimer’s disease (AD). Patients with mild cognitive impairment (MCI) or a diagnosis of AD express lower levels of programmed death-1 (PD-1) on CD4+ T cells. There is a positive correlation between PD-1 blockade and the remission of AD. In mice, inhibition of PD-1 by monoclonal antibodies evokes an interferon γ-dependent systemic immune response, leading to the recruitment of monocyte-derived macrophages to the brain, clearance of cerebral amyloid-β plaques, and improved cognitive performance. Epidemiological studies suggested an inverse association between cancer diagnosis early in life and subsequently developing AD or dementia, and vice versa. FDA-approved PD-1 monoclonal antibody drugs has been proved to be a validated drug target in humans and transformed immuno-oncology. Small-molecule PD-1 drugs have the potential to offer increased efficacy, safety, and global access. For the treatment of MCI or AD in elderly patients, the argument could be made that orally bioavailable, small-molecule drugs would almost be required. Identifying small molecules that bind to PD-1 and disrupt the interactions of PD-1 with its endogenous ligands (PD-L1 and PD-L2) has proven to be very difficult. Indeed, there are no small-molecule anti- PD-1 drugs that have entered clinical trials. In our published work (Tang and Kim, 2019), we have identified a prominent pocket on the ligand- binding surface of human PD-1 that appears to be an attractive small-molecule drug target. The pocket forms when PD-1 is bound to one of its endogenous ligands, PD-L2. The structure of an apo- PD-1 variant shows that the CC′ loop of PD-1 adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. In the parent award, we are investigating the sperm surface fertilization receptor IZUMO1 in sperm- egg recognition, adhesion, and fusion. We are pursuing a strategy coupling deep mutational scanning with yeast surface display to uncover the structural and mutational landscapes essential for IZUMO1’s homo-oligomerization, as well as for IZUMO1’s interactions with its egg ligand, JUNO. In this supplement, we propose to use a similar yeast surface display approach to investigate the conformational dynamics of T cell surface receptor PD-1. Our central hypothesis is that conformational changes in the CC′ and FG loops and formation of pockets in the ligand-binding interface of PD-1 are thermodynamically coupled. We propose to establish strategies to conformationally stabilize the PD-1 pocket in the absence of PD-L2. If successful, this pilot project would set the stage for the discovery of small-molecule drugs that target the pocket on the surface of PD-1 for the treatment of AD and MCI.