Auburn Research Day 2022

B i omed i ca l Resea rch | Gr adua te/Undergr adua te St udent Design and Development of Novel Dual Ppar δ/α Agonist for Neurodegenerative Disease

50

(1) Fajar Setyo Wibowo; (1) Rania Mohamedelhassan; (1) Manoj Govindarajulu; (1) Priyanka Das Pinky; (1) Vishnu Suppiramaniam; (3) Sieun Yoo; (3) Juming Zong; (2) Darren Beck; (2) Danielle McCullough; (1) Forrest Smith; (1) Rajesh H. Amin (1) Department of Drug Discovery and Development, Harrison School of Pharmacy; (2) Edward Via College of Osteopathic Medicine; (3) Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors responsible for promoting the expression of genes associated with energy regulation. PPARs exist in three main isoforms ( γ, α , and δ ) with activity specific to their cellular compartmentalization. Insulin sensitizing properties of PPAR agonists improve glucose utilization and lower circulating lipid levels. Current PPAR agonists such as the thiazolidinedione ( γ agonists) and fibrates (α agonists) show promise in animal models for mitigating progression of Alzheimer’s disease (AD), including pathologies and neurodegeneration. However, many of these compounds are associated with harmful effects on human health such as myocardial infarctions. While animal models for AD therapies have been promising, all have failed in clinical trials, thus casting doubt for this class of nuclear receptors use as an intervention in AD. Although PPAR γ is extensively investigated as a therapeutic target for AD therapy, PPAR δ is the most abundant isoform expressed in the brain. Therefore, our goal was to improve upon the therapeutic effects of PPAR agonists and design compounds that can avoid side effects associated with PPAR γ , while maintaining full activation of PPAR δ and partial activation of PPAR α . Methods: Our lead compound, AU-403 was designed computationally with an emphasis on ADME using Schrodinger and GastroPlus modeling software.

Our in-silico design was formulated around reduced interactions with the activated function 2 (AF2) tyrosine-473 residue, a key ligand contact involved in full activation of PPAR γ . To validate PPAR activity we compared the impact of full PPAR agonists with AU-403 using reporter activity assays, lipid accumulation in 3T3-L1 adipocytes, and the ability to reduce inflammatory signaling in macrophages. Lastly, we tested AU403’s ability to improve memory deficits, and neuroinflammation in an advanced AD mouse model (3xTgAD) Results: AU-403 demonstrated the following results: a decreased ability to induce lipid accumulation in 3T3-L1 adipocytes relative to full γ agonist Rosiglitazone, similar activation of PPAR δ relative to full δ agonist GW-0742, partial activation of PPAR α relative to α agonist Fenofibrate, improvement in behavioral deficits and markers associated with neuroinflammation including Amyloid beta levels. and lastly an improvement in insulin signaling and mitochondrial markers after one month of daily AU-403 (5mg/kg) treatment. Our future aims are to validate our in-silico models of ligand binding to each PPAR isoform determined by x-ray crystallographic protein-ligand complexes, determine specificity for co-activator/co-repressor recruitment utilizing TR-FRET assays, and test the ability to mitigate deficits with insulin signaling and energy regulation in a model of AD.

Casey L. Sexton 1 , Joshua S. Godwin 1 , Bradley A. Ruple 1 , Mason C. McIntosh 1 , Shelby C. Osburn 1 , Blake R. Hollingsworth 1 , Philip J. Agostinelli 1 , Andreas N. Kavazis 1 , Tim N. Ziegenfuss 2 , Hector L. Lopez 2 , Ryan Smith 3 , Kaelin C. Young 1,4 , Varun B. Dwaraka 3 , C. Brooks Mobley 1 , Adam P. Sharples 5 , Michael D. Roberts 1,4 1 Auburn University; 2 The Center for Applied Health Sciences, Canfield, OH; 3 TruDiagnostic, Lexington, KY; 4 Edward Via College of Osteopathic Medicine-Auburn Campus; 5 Institute for Physical Performance, Norwegian School of Sport Sciences, Oslo, Norway B i omed i ca l Resea rch | Gr adua te/Undergr adua te St udent Global DNA Methylation Status in Relation to Resistance Training with High VS Low Loads to Failure 51

Objective: Our objective was to determine how global DNA methylation status of skeletal muscle differs with resistance training (RT) to failure in trained participants using either 80% of their estimated one-repetition maximum (Est. 1-RM) (80FAIL) or 30% of their Est. 1-RM (30FAIL). Hypotheses: We hypothesize there will be a significant decrease in global DNA methylation of DNA with both RT conditions, but that 30FAIL RT will elicit a greater magnitude of hypomethylation than 80FAIL RT. Methods: Previously trained college-aged males (n = 11, age 23 ± 4 years, percent fat 11.4 ± 6.4%, training experience 4 ± 3 years, squat strength relative to body weight 1.7 ± 0.3) voluntarily underwent two bouts of RT. Participants completed both the 80FAIL and 30FAIL conditions (separated by one week). For each bout of RT, participants completed 4 sets of back squats and 4 sets leg

extensions with either the 80FAIL or 30FAIL training conditions. Muscle biopsies were collected from the vastus lateralis before (PRE), 3 hours (3hPOST), and 6 hours (6hrPOST) after each RT bout. DNA was then batch-isolated from muscle tissue and submitted for analysis using the Illumina MethylationEPIC array. Results: Total number of repetitions performed were significantly higher for 30FAIL training vs 80FAIL training (p < .001), however total training volume (sets x reps x load) was not significantly different between conditions (p= 0.571). Global methylation changes between PRE, 3hPOST and 6hPOST are presented herein. In addition, significant differences between conditions are emphasized for each post-exercise time point. Conclusions: With this study we expand our understanding of how the manipulation of RT variables affect epigenetic modifications in skeletal muscle.

30