Biology of erythropoietin

Molineux, Graham; Sinclair, Angus M.

doi:10.1007/978-3-7643-8698-6_3

Cited by 3 publications

(9 citation statements)

References 89 publications

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“… Notes: ( A ) H3-thymidine incorporation assays in UT-7/EPO cells demonstrates the inverse correlation between molecules with increasing numbers of N linked carbohydrate (rHuEPO = 3; darbepoetin alfa = 5; AMG 114 and AMG 205 = 7) and decreasing in vitro proliferation activity. Proliferation is measured as counts CPM of H3-thymidine incorporation into newly synthesized DNA and EC 50 values of a representative experiment are shown.…”

Section: Figurementioning

confidence: 99%

“…Proliferation is measured as counts CPM of H3-thymidine incorporation into newly synthesized DNA and EC 50 values of a representative experiment are shown. ( B ) Competitive binding assays were performed in UT-7/EPO cells whereby I 125 -rHuEPO was bound to cells and competed with increasing concentration of ESA for 2–3 hours.…”

Section: Figurementioning

confidence: 99%

“…Along with the benefits of each of these treatment options, there are potential safety risks that are reviewed elsewhere 1,2. Red blood cell production, the process of erythropoiesis, occurs in the bone marrow and is controlled by the natural hormone erythropoietin (EPO), a 165 amino acid glycoprotein primarily produced by interstitial fibroblasts in the kidney 3. The EPO molecule has a compact globular structure that contains one O-linked and three N-linked carbohydrate side chains that constitute approximately 40% of its mass 3.…”

Section: Introductionmentioning

confidence: 99%

“…Red blood cell production, the process of erythropoiesis, occurs in the bone marrow and is controlled by the natural hormone erythropoietin (EPO), a 165 amino acid glycoprotein primarily produced by interstitial fibroblasts in the kidney 3. The EPO molecule has a compact globular structure that contains one O-linked and three N-linked carbohydrate side chains that constitute approximately 40% of its mass 3. EPO production is regulated at the transcriptional level by hypoxia inducible transcription factor (HIF) subunits HIF-1α and HIF-2α, part of the cellular oxygen sensory machinery 4.…”

Section: Introductionmentioning

confidence: 99%

“…3 Hematopoietic stem cells resident in the bone marrow differentiate into multiple myeloid and lymphoid lineages, including the erythroid lineage. The earliest committed erythroid progenitors are classified as burst forming units-erythroid.…”

Section: Introductionmentioning

confidence: 99%

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Erythropoiesis stimulating agents approaches to modulate activity

Sinclair¹

2013

BTT

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Recombinant human erythropoietin (rHuEPO), such as the approved agents epoetin alfa and epoetin beta, has been used successfully for over 20 years to treat anemia in millions of patients. However, due to the relatively short half-life of the molecule (approximately 8 hours), frequent dosing may be required to achieve required hemoglobin levels. Therefore, a need was identified in some anemic patient populations for erythropoiesis stimulating agents with longer half-lives that required less frequent dosing. This need led to the development of second generation molecules which are modified versions of rHuEPO with improved pharma-cokinetic and pharmacodynamic properties such as darbepoetin alfa, a hyperglycosylated analog of rHuEPO, and pegzyrepoetin, a pegylated rHuEPO. Third generation molecules, such as peginesatide, which are peptide mimetics that have no sequence homology to rHuEPO have also recently been developed. The various molecular, pharmacokinetic, and pharmacodynamic properties of these and other erythropoiesis stimulating agents will be discussed in this review.

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Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Erythropoiesis stimulating agents approaches to modulate activity

Sinclair¹

2013

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Development of an in vitro Bioassay for Recombinant Human Erythropoietin (rHuEPO) Based on Proliferative Stimulation of an Erythroid Cell Line and Analysis of Sialic Acid Dependent Microheterogeneity: UT-7 Cell Bioassay

et al. 2017

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Determination of biological activity and its comparison with clinical behavior is important in the quality assessment of therapeutic glycoproteins. In vivo studies are usually employed for evaluating bioactivity of these glycomolecules. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with in vivo studies. Negatively charged sialic acid residues are known to be critical for in vivo bioactivity of rHuEPO. To address this need, we employed the human acute myeloid leukemia cell line UT-7 for the determination of proliferative stimulation induced by rHuEPO. Relative potencies of various intact and sugar-trimmed rHuEPO preparations were estimated using the International Standard for Human r-DNA derived EPO (87/684) as a reference for bioactivity. The cellular response was measured with a multi-channel photometer using a colorimetric microassay, based on the metabolism of the Resazurin sodium by cell viability. For a resourceful probing of physiological features of rHuEPO with significance, we obtained partly or completely desialylated rHuEPO digested by the neuraminidase enzyme without degradation of carbohydrates. Two-fold higher specific activity was shown by asialoerythropoietin in in vitro analysis compared with the sialoerythropoietin. Further, computational studies were also carried out to construct the 3D model of the erythropoietin (EPO) protein structure using standard comparative modeling methods. The quality of the model was validated using Procheck and protein structure analysis (ProSA) server tools. N-glycan units were constructed; moreover, EPO protein was glycosylated at potential glycosylation amino acid residue sites. The method described should be suitable for potency assessments of pharmaceutical formulations of rHuEPO (European Pharmacopeia, 2016).

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A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

Štambuk

Manojlović

Turčić

et al. 2014

IJMS

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Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

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Biology of erythropoietin

Cited by 3 publications

References 89 publications

Erythropoiesis stimulating agents approaches to modulate activity

Erythropoiesis stimulating agents approaches to modulate activity

Development of an in vitro Bioassay for Recombinant Human Erythropoietin (rHuEPO) Based on Proliferative Stimulation of an Erythroid Cell Line and Analysis of Sialic Acid Dependent Microheterogeneity: UT-7 Cell Bioassay

A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

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