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Nicholas J Pekas,1 Jason L Petersen,2,3 Monica Sathyanesan,1,2 Samuel S Newton1,2 1Department of Basic Biomedical Sciences, University of South Dakota Sanford School of Medicine, Vermilion, SD, USA; 2Sioux Falls VA Health Care System, Sioux Falls, SD, USA; 3Department of Internal Medicine, University of South Dakota Sanford School of Medicine, Vermillion, SD, USACorrespondence: Samuel S Newton Attn: Lee Medical Building, 414 E. Clark Street, Vermillion, SD 57069, USATel +1-605-658-6313Email Samuel.Sathyanesan@usd.eduIntroduction: Carbamoylated erythropoietin (CEPO) is a chemically engineered, nonhematopoietic derivative of erythropoietin (EPO) that retains its antidepressant and pro-cognitive effects, which are attributed to the increased expression of neurotrophic factors like brain derived neurotrophic factor (BDNF), in the central nervous system. However, the chemical modification process which produces CEPO from erythropoietin (EPO) requires pure EPO as raw material, is challenging to scale-up and can also cause batch-to-batch variability. To address these key limitations while retaining its behavioral effects, we designed, expressed and analyzed a triple, glutamine, substitution recombinant mimetic of CEPO, named QPO.Methods and Materials: We employ a combination of computational structural biology, molecular, cellular and behavioral assays to design, produce, purify and test QPO.Results: QPO was shown to be a nonhematopoietic polypeptide with significant antidepressant-like and pro-cognitive behavioral effects in rodent assays while significantly upregulating BDNF expression in-vitro and in-vivo. The in-silico binding affinity analysis of QPO bound to the EPOR/EPOR homodimer receptor shows significantly decreased binding to Active Site 2, but not Active Site 1, of EPOR.Discussion: The results of the behavioral and gene expression analysis imply that QPO is a successful CEPO mimetic protein and potentially acts via a similar neurotrophic mechanism, making it a drug development target for psychiatric disorders. The decreased binding to Active Site 2 could imply that this active site is not involved in neuroactive signaling and could allow the development of a functional innate repair receptor (IRR) model. Substituting the three glutamine substitution residues with arginine (RPO) resulted in the loss of behavioral activity, indicating the importance of glutamine residues at those positions.Keywords: carbamoylated erythropoietin, mimetic, antidepressant, innate repair receptor, erythropoietin receptor, nonhematopoietic

Nicholas J Pekas, Samuel S Newton Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA Background: Erythropoietin (EPO), a pleiotropic cytokine, binds to its receptor (EPOR) in bone marrow, activating a signaling cascade that results in red blood cell proliferation. A recently discovered naturally occurring EPO mutation (R150Q) at active site 1 (AS1) of the protein was shown to attenuate its canonical downstream signaling, eliminating its hematopoietic effects and causing a fatal anemia. The purpose of this work was to analyze the EPO–EPOR complex computationally to provide a structural explanation for this signaling change. Materials and methods: Computational structural biology analyses and molecular dynamics simulations were used to determine key interaction differences between the R150Q mutant and the wild-type form of EPO. Both were compared to another variant mutated at the same position, R150E, which also lacks hematopoietic activity. Results: The ligand–receptor interactions of the R150Q and R150E mutants showed significant variations in how they interacted with EPOR at AS1 of the EPO–EPOR complex. Both lost specific reported salt bridges previously associated with full complex activation. Conclusion: This work describes how the ligand–receptor interactions at AS1 of the EPO–EPOR complex respond to mutations at the 150th position. The interactions at AS1 were used to propose a potential mechanism by which the binding of EPO to the extracellular domain of EPOR influences its cytosolic domain and the resulting signaling cascade. Keywords: molecular dynamics, signaling cascade, binding affinity, hematopoiesis