Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor

Abstract

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues, including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury, while poor tissue availability of the protein limits its efficacy. Here, we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy) ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine), which selectively activates the tissue-protective EPO receptor, comprising an EPO receptor subunit (EPOR) and the common b-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR, CD131, and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations, STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer, lack of activity in off-target selectivity screening, and lack of functional effects in erythroleukemia cell line TF-1 and CD341 progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge, STS-E412 is the first nonpeptidyl, selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted.

Alternative Erythropoietin Receptors in the Nervous System

“…As one example, the human splice variant EV-3 which lacks the entire third exon of the full-length Epo transcript does not activate homodimeric EpoR, and hence does not stimulate erythropoiesis, but mediates neuroprotection of rat hippocampal and insect neurons [ 19 , 20 ]. In addition to natural splice variants, various Epo-derivatives (e.g., carbamylated Epo, Epobis, helix B surface peptide) and molecules completely unrelated to the Epo peptide sequence (e.g., STS-E412) have been identified as non-erythropoietic but neuroprotective specific Epo mimetics [ 154 , 155 , 156 , 157 , 158 ]. Recent studies identified three candidate receptors that are expressed in the nervous system and mediate cell protective and regenerative functions upon binding of Epo and/or one of its non-hematopoietic agonists.…”

“…In the nervous system, cell types that can co-express EpoR and βcR include neurons, astrocytes, microglia and vascular cells. Epo-mediated neuroprotection through EpoR/βcR has been demonstrated in various studies [ 158 , 162 , 167 , 168 ] including studies with βcR-deficient knock-out mice that lacked Epo-mediated neuroprotection [ 162 ] and relief of neuropathic pain [ 169 ]. In addition, Epo mediates cardio- and reno-protective effects through EpoR/βcR-signaling in vitro and in vivo [ 162 , 170 ].…”

Erythropoietin Promotes the Differentiation of Fetal Neural Stem Cells into Glial Cells Partly via EPOR-βCR/Syne-1/H3K9me3 Pathway

“…Previous studies have demonstrated that EPOR/βCR exist on primary human renal epithelial cells, endothelial progenitor cells and macrophages [15][16][17]. Within the brain at least three versions of the EPO receptor are now known to exist, including the βCR [18].…”

“…While the heteromic EPO receptor involving the βCR was initially thought to confer neuroprotection, evidence now supports a role for this heteromic EPO receptor and the homodimeric EPO receptor in the protection of neurons and glia [18]. It was suggested that nonpeptidyl compound STS-E412, a kind of EPO deprivate which selectively bind to EPOR/βCR heterodimer, could act as neuroprotective agents in the CNS, suggesting the neuroprotective role of EPOR/βCR heterodimer [15]. Our results demonstrated for the rst time that EPOR/βCR heterodimer also exist on primary NSCs/NPs.…”

Intranasal injection of recombinant human erythropoietin improves cognitive and visual impairments in chronic cerebral ischemia rats

“…Erythropoietin (EPO) is a single chain glycoprotein that promotes hematopoietic cell growth through binding to the EPO receptor; recombinant human EPO (rhEPO) has previously been successfully used in the treatment of severe anemia (1)(2)(3). EPO is primarily produced in the kidneys but is also detectable in other organs, such as the liver and the brain (4).…”