Design and Development of a Behaviorally Active Recombinant Neurotrophic Factor

Abstract

Introduction: 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: antidepressant; carbamoylated erythropoietin; erythropoietin receptor; innate repair receptor; mimetic; nonhematopoietic.

Figure 1

Behavioral Assays and Hematocrit. (A) The treatment and testing schedule for the FST and OFT is shown. (B) The cumulative immobile duration over the scored portion of the FST is shown for both QPO-treated and vehicle-treated mice. QPO treatment resulted in a significantly decreased immobile duration compared to vehicle-treated controls (p < 0.05, N = 6). (C) The cumulative distance moved in the OFT is shown for QPO-treated and vehicle-treated mice. QPO showed no significant difference in total distance moved during the OFT compared to vehicle-treated mice (p > 0.05, N = 6). (D) The treatment and testing schedule for the ORMT is shown. (E) The preference of QPO-treated vs vehicle-treated mice for a novel object instead of a trained object is shown here as a discrimination index. QPO showed a significantly increased preference for the novel object compared to vehicle-treated controls (p < 0.05, N = 6 for treated, 8 for vehicle control). (F) The comparison of total hematocrit (as a percentage) between QPO-treated mice (10 doses, 40µg/kg i.p., over 14 days) and vehicle-treated mice is shown. QPO treatment caused neither a significant increase nor decrease in hematocrit. (G) Treatment and testing schedule for mice undergoing Novelty-Induced Hypophagia Testing. (H) NIHT results for QPO-treated mice are presented in comparison to vehicle-treated controls in terms of the duration of time between introduction of sweetened condensed milk and the first drink in seconds. Statistical significance is denoted by *p < 0.05.

Figure 2

Design of QPO from EPO-EPOR complex. (A) EPO (green ribbon) is shown bound to EPOR (molecular surface representation) – PDB ID-1EER. The high-affinity active site 1(AS1) is colored salmon and the low-affinity active site 2 (AS2) is colored blue. (B) The front view in A is rotated 90̊ toward the viewer. (C) Magnified view of boxed region in A is shown. The 3 amino acid residues that were chosen for substitution mutagenesis are indicated by red spheres and the corresponding residue number in the sequence is shown. The distance between the residue atoms and the nearest receptor atom in the active sites are indicated in red. (D) Magnified view of the boxed region in the top view is shown.

Figure 3

Purification Silver Stain and Western Blot Analyses. (A) A silver stain depicting each step of the purification procedure is shown. From left to right with the well # in parentheses: (1) broadband protein ladder (2) the insoluble fraction of the whole cell lysate (3) the soluble fraction of the whole cell lysate (4) the eluate of the amylose binding column, containing a mixture of MBP-QPO and MBP (5) cleavage of MBP-QPO using factor Xa, resulting in the disappearance of the MBP-QPO band, and appearance of the QPO band at approximately 22 kDa (6) eluate from immobilized metal affinity chromatography column, containing the histidine-tagged QPO polypeptide with imidazole (7) dialysis of QPO polypeptide into 1X phosphate-buffered saline (B) A Western blot in which QPO’s reactivity with an anti-EPO antibody was compared with ngEPO, dgCEPO, EPO, and CEPO is shown. (C) A Western blot in which QPO’s reactivity with an anti-6X histidine antibody was compared with ngEPO, dgCEPO, EPO, and CEPO is shown.

Figure 4

Behavioral analysis of arginine substitution recombinant. (A) Discrimination index of RPO and vehicle in the novel object recognition test. (B) Immobility time in the forced swim test. (C) Distance moved in the open field test in vehicle and RPO treated mice. RPO was dosed at 40µg/kg/day i.p for 4 days (N = 6).

Figure 5

Binding Affinity Assessment and Dissociation Constant Calculation. This figure presents the average calculated free energies of binding (ΔG_B) of ngEPO and QPO to (A) Active Site 1 of EPOR/EPOR and (B) Active Site 2 of EPOR/EPOR. Statistical significance is denoted by *p < 0.05 by Student’s T-test. (C) This table provides calculated dissociation constants (nM) for ngEPO and QPO. Experimentally determined EPO dissociation constants taken from Goldwasser/Wilson and Jolliffe. (D) The fold change in expression levels of BDNF mRNA in neuronally differentiated PC-12 cells are shown in comparison to vehicle-treated controls. BDNF shows a significant upregulation in expression after treatment with QPO (p < 0.01, N = 5 vehicle/6 treated). (E) The fold change in the expression of BDNF in the hippocampus of BALB/c mice. BDNF shows a statistically significant 50% increase in expression compared to vehicle-treated controls (p < 0.05, N = 5).