PROJECT SUMMARY/ABSTRACT Our recent discovery of orally brain permeable small molecules that mimic the bioactivity of Humanin (HN) peptide to enhance and normalize neuronal phospho-Akt (p-Akt) levels provides a unique opportunity for evaluating this new approach in Alzheimer's disease (AD). In this proposal, we will direct our efforts to optimize this new class of agents, focusing on screening additional hits, enhancing their potency, drug-like properties, solubility, oral brain permeability and efficacy in an AD model towards development of this novel therapeutic approach for AD. We will also use modeling to identify HN based peptidomimetics for testing. Our data show that these small molecule HN mimetics, like HN, can suppress neuronal death through its activation of the gp130 receptor and signaling via the PI3/Akt pathway and provide neuroprotection for primary hippocampal neurons against N-methyl D-aspartate (NMDA) and Aβ-induced neurotoxicity. HN is a naturally occurring mitochondrial-derived brain peptide that decreases with age and may act as a neuroprotective factor against AD-relevant neurotoxicity. Treatment of hippocampal neurons with our HN mimetic compound 2 resulted in an increase in p-Akt, and this correlated to its observed neuroprotective effects. In AD patients, a significant decrease in p-Akt has been reported. Similarly, in aged apolipoprotein E4 (ApoE4) mice, there is a significant decrease in p-Akt in the brain relative to age-matched ApoE3 mice suggesting that PI3/Akt signaling is affected by ApoE4, a risk factor in AD. Activation of PI3/Akt signaling can transcriptionally modulate genes related to memory such as choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and may also regulate postsynaptic proteins involved in neuroplasticity. AD is the most prevalent age-related dementia, currently afflicting more than 5.4 million people in the US. Given the urgent need for new therapeutic approaches for AD, these HN mimetics could provide promising lead candidates for therapeutic development. In Aim 1, we plan to evaluate small molecule HN mimetics and peptidomimetics for activation of gp130 and normalization of p-Akt along with their neuroprotection against Aβ and NMDA induced neurotoxicity. In Aim 2, we would conduct a design and synthesis campaign using current SAR and new docking/modeling data to identify small mimetics, peptides and peptidomimetics. We will optimize potency, drug-like properties, solubility and oral brain bioavailability for efficacy testing. The best analogs/peptidomimetics from Aims 1 and 2 will undergo in vitro ADMET profiling and pharmacokinetic (PK) studies, along with phosphoproteomics analyses, in Aim 3 to prioritize the optimal compounds for in vivo efficacy testing in the ApoE4(TR):5XFAD murine model of AD as part of Aim 4. The goal is to identify orally available HN-mimetics that enhance/normalize brain p-Akt levels and improve cognition. Like HN itself, they could also...