Erythropoietin (EPO) Mimicry

1 July/20 May, 1999

Erythropoietin (EPO) Mimicry

Activation of transmembrane receptors for cytokines occurs when oligomerization of receptor chains is induced by binding of a protein ligand (cytokine) to a specific ligand-binding domain on the receptor. The resulting clustering of tyrosine kinase domains on the cytoplasmic side of the receptor initiates signal transduction events. Receptors may also be activated by bivalent receptor antibodies and by dimeric peptides that interact with the ligand binding domain. Thus activation of receptors by small, peptidyl molecules might make it possible to develop cytokine mimetics that may replace natural counterparts that are now on the market for therapy. This is illustrated here for erythropoietin (EPO). EPO, used for stimulating production of red blood cells, is a major product in biotechnology with world-wide sales of $2.6 billion already in 1995. Thus there has been an increasing interest in molecular mimicry with EPO potency for obviously huge commercial implications. In general, the steps in this direction are to fish out active peptide domains from EPO, synthesize their derivatives, and to discover non-peptide small mimetics resistant to proteolytic digestion with good permeability and ideally for oral administration. The research for such mimetic agonists with equivalent potency to EPO is exploratory at this moment but a significant advance has been made lately for this target.
A three-way collaboration between researchers at Affymax, RW Johnson Pharmaceutical and the Scripps Research Institute has led to the discovery of a 20-amino acid peptide (GGTYSCHFGPLTWVCKPQGG) with an affinity (Kd) of 200 nM, compared to 200 pM for EPO (Wrighton NC et al. 1996: uid=8662529). It is remarkable that this peptide activates the EPO receptor in a variety of cell-based and animal assays. The EPO receptor requires dimerization for activation, much like the growth hormone receptor. Indeed, the crystal structure at 2.8 A resolution of a complex of this mimetic agonist peptide with the extracellular domain of EPO receptor (EPO binding protein or EPB) reveals that a peptide dimer induces an almost perfect twofold dimerization ot the receptor (Livnah O et al. 1996: uid=8662530). This 20-amino acid peptide has a b-sheet structure and is stabilized by the C-C disulfide bond. The ingenious screening system appears to be the key for this success.

The researchers here employed phage display where random peptides were exposed on coat proteins of filamentous phage. A library of random peptide-phage was allowed to bind to and subsequently eluted from EPB. They used weak-binding system to first fish out EPB-weak-binding (Kd 10 mM) CRIGPITWVC as the consensus sequence. With further clever manipulation for selecting the higher affinity peptides, the group isolated the aforementioned 20-amino acid agonist peptide, the sequence of which does not actually exist in the native EPO. For details of the methods, refer to the paper quoted above.

Evidently there are similar research activities elsewhere but the results refereed to here are most notable and helped dispel the skepticism by showing that small peptides can be found that minic large polypeptide hormones such as EPO.

Several hurdles should be cleared before EPO-mimetic agonists be developed as therapeutics. These include:

Molecular weight of peptide agonists should be even smaller than the one (MW 2000) isolated above

Binding to EPO receptor should be equivalent in affinity to EPO. The peptide isolated above is still weak by a factor of 1000 than that of native EPO in vitro and even less potent in animal studies.

Agonists should be non-peptide EPO-mimetics to avoid possible proteolytic digestion.

EPO-mimetics should be administered orally.

More cenently a chemically defined, dimeric form of an EPO mimetic peptide (EMP) was reported that displayed 100-fold increased affinity for the EPOR and correspondingly elevated potency in cell-based assays and in mice (Wrighton, NC et al, 1997: uid=9359108). In fact the half-maximal response in a cellular proliferation assay is evoked at an native EPO concentration of 10 pM, 10(-2) of its Kd value for EPO-EPOR binding site 1 (Kd approximately equal to nM), and 10(-5) of the Kd for EPO-EPOR binding site 2 (Kd approximately equal to 1 uM). Since overall half-maximal binding (IC50) of cell-surface receptors is produced with ca 0.18 nM EPO, only approximately 6% of the receptors would be bound in the presence of 10 pM EPO. Actually the crystal structure of EPO-EPOR extracellular ligand-binding domains at 1.9 A shows that EPO imposes a unique 120 degrees angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways (Syed, RS et al, 1998: uid=9774108). The efficiency of signalling through EPOR seems to depend critically on receptor orientation.

The researchers at RW Johnson Pharmaceutical further extended their work to discover the minimal structural elements required for EPO mimetic action, namely a 13 amino acid peptide (EPO-mimetic peptide EMP20). The Tyr4 and Trp13 appeared to be essential for mimetic action (Johnson DL et al, 1998: uid=9521688). It was shown previously that Phe93 in the extracellular domain of EPOR is crucial for binding EPO. With 20-mer EMP1 containing EMP20 sequence, the analysis of EPO- and EMP1-EPOR crystal structures and amino acid substitutions indicate that for both EPO and EMP, Phe93 and Phe205 are important binding determinants, even though these ligands share no sequence or structural homology (Middleton SA et al, 1999: uid=10318834). The potency of EMP1 is less than native EPO, and the potency could be improved through the covalent dimerization of two peptide molecules to a considerable extent. In another line of research, a peptide (192-QRVEILEGRTECVLSNLRGRTRY-216) in the dimerization site of EPOR was found to activate the receptor in the absence of EPO (Naranda T et al, 1999: uid=10377456). The peptide does not bind to the EPO binding site, and neither the peptide nor EPO altered the affinity of the other for the receptor. In addition, the peptide and EPO were strongly synergistic (>10 times) in the activation of the signal transduction.

An interesting alternative approach is being taken towards enhancing the potency of EPO-mimetics. In the EPO signal transduction, intracellular signalling molecules such as hematopoietic cell phosphatase (HCP or also known as SHP1) come in the cascade. Inhibiting HCP causes responsive cells to be hypersensitive to EPO (Barbone FP et al, 1999: uid=10334672). The study of EPO mimetics is advancing at a remarlable speed. It is quite likely that the combination of EPO mimetics with the dimerization peptide and/or small-molecular HCP inhibitors achieve the EPO potency similar to that of native EPO.

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