Increased potency of an erythropoietin peptide mimetic through covalent dimerization

Wrighton, Nicholas C.; Palaniappan, Balasubramanian; Barbone, Francis P.; Kashyap, Arun K.; Farrell, Francis X.; Jolliffe, Linda K.; Barrett, Ronald W.; Dower, William J.

doi:10.1038/nbt1197-1261

Cited by 116 publications

(64 citation statements)

References 18 publications

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“…It would also be interesting to test combinations of peptides binding to A-and B-class Eph receptors involved in a particular pathological process, such as EphB4 and EphA2 in cancer (5,8,49,50). Finally, the affinity of the EphB receptorbinding peptides may be greatly increased by dimerization through a flexible linker (36,42,43). However, it is possible that the dimerized peptides may become agonists.…”

Section: Fig 5 Peptides As Ephb Receptor-targeting Agentsmentioning

confidence: 99%

“…Phage display is a powerful approach to identify competitive antagonists and ligand mimics, but the peptides isolated are typically of low affinity and often need to be optimized by further "affinity maturation" (29,30,39,40), introduction of conformational constraints (30,41), or cross-linking to increase valency (36,42,43). To optimize the TNYL peptide, we have used a different approach.…”

Section: Fig 5 Peptides As Ephb Receptor-targeting Agentsmentioning

confidence: 99%

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EphB Receptor-binding Peptides Identified by Phage Display Enable Design of an Antagonist with Ephrin-like Affinity

Koolpe

Burgess

Dail

et al. 2005

Journal of Biological Chemistry

131

157

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The Eph receptor tyrosine kinases are overexpressed in many pathologic tissues and have therefore emerged as promising drug target candidates. However, there are few molecules available that can selectively bind to a single Eph receptor and not other members of this large receptor family. Here we report the identification by phage display of peptides that bind selectively to different receptors of the EphB class, including EphB1, EphB2, and EphB4. Peptides with the same EphB receptor specificity compete with each other for binding, suggesting that they have partially overlapping binding sites. In addition, several of the peptides contain amino acid motifs found in the G-H loop of the ephrin-B ligands, which is the region that mediates high-affinity interaction with the EphB receptors. Consistent with targeting the ephrin-binding site, the higher affinity peptides antagonize ephrin binding to the EphB receptors. We also designed an optimized EphB4-binding peptide with affinity comparable with that of the natural ligand, ephrin-B2. These peptides should be useful as selective inhibitors of the pathological activities of EphB receptors and as targeting agents for imaging probes and therapeutic drugs.

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Section: Fig 5 Peptides As Ephb Receptor-targeting Agentsmentioning

confidence: 99%

Section: Fig 5 Peptides As Ephb Receptor-targeting Agentsmentioning

confidence: 99%

EphB Receptor-binding Peptides Identified by Phage Display Enable Design of an Antagonist with Ephrin-like Affinity

Koolpe

Burgess

Dail

et al. 2005

Journal of Biological Chemistry

131

157

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“…The establishment of this proof of concept for NGF loop 1 along with the emerging role of peptidomimetics as a key intermediate-stage strategy in small molecule drug design (8, 26 -28) will contribute to rational screening and design programs for producing potent NGF small molecule mimetics with agonist activity. Our NGF dimeric peptidomimetic strategy has been further encouraged by the synthesis of small peptides mimicking the 34-kDa dimeric protein erythropoietin and the finding that erythropoietin receptor activation by peptidomi-metics also requires the dimeric form (30). These erythropoietin peptidomimetics have served as starting points for the design of erythropoietin small molecule compounds (31).…”

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confidence: 99%

Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects

Xie

Tisi

Yeo

et al. 2000

Journal of Biological Chemistry

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Previous work indicating that nerve growth factor (NGF) protein loops 2 and 4 interact with TrkA receptors raise the possibility that small molecule mimetics corresponding to TrkA-interacting domains that have NGF agonist activity can be developed. We applied our previously developed strategy of dimeric peptidomimetics to address the hypothesis that loop 4 small molecule dimeric mimetics would activate TrkA-related signal transduction and mimic NGF neurotrophic effects in a structure-specific manner. A loop 4 cyclized peptide dimer demonstrated NGF-like neurotrophic activity, whereas peptides with scrambled sequence, added or substituted residues, or cyclized in monomeric form were inactive. Activity was blocked by the TrkA inhibitors K252a and AG879 but not by NGF p75 receptor blocking antibody. Dimeric, but not monomeric, peptides partially blocked NGF activity. This profile was consistent with that of a NGF partial agonist. ERK and AKT phosphorylation was stimulated only by biologically active peptides and was blocked by K252a. The ERK inhibitor U0126 blocked the neurite-but not the survival-promoting activity of both NGF and active peptide. These studies support the proof of concept that small molecule NGF loop 4 mimetics can activate NGF signaling pathways and can mimic death-preventing and neurite-promoting effects of NGF. This finding will guide the rational design of NGF single-domain mimetics and contribute to elucidating NGF signal transduction mechanisms.Nerve growth factor (NGF) 1 acts via TrkA and p75 receptors to regulate neuronal survival, promote neurite outgrowth, and up-regulate certain neuronal functions such as mediation of pain and inflammation (1-5). These actions suggest that NGF agonists or antagonists might be useful in regulating these processes (6 -8). Factors limiting therapeutic applications of the NGF protein include restricted penetration of the central nervous system and the poor medicinal properties characteristic of most proteins (9, 10). The development of small molecule mimetics with favorable chemical properties that function as agonists or antagonists that mimic or inhibit NGF functions in the appropriate biological context will be critical in advancing potential in vivo applications of NGF. Moreover, in settings in which NGF might contribute to neuronal death, pain, or inflammatory mechanisms, NGF antagonists may be particularly relevant. Creation of single domain NGF mimetics will also constitute a powerful approach for linking specific NGF domains with specific patterns of intracellular signal transduction.A NGF mimetic (agonist or antagonist) would be expected to contain structural determinants of one or more NGF active sites that interact with NGF receptors. Multiple techniques have been used to deduce which domains of the NGF protein interact with NGF receptors (11). A peptide mapping approach in which synthetic peptides with sequences corresponding to specific NGF regions were tested for their ability to inhibit NGF activity pointed to residues 29 -35 as a key active ...

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“…19 It was found that the dimerization of 2 Epo mimetic peptides strongly increased their activity, though it remained inferior to that of native Epo. 20,21 The association of 2 Epo molecules obtained by either chemical cross-linking or recombinant DNA-mediated fusion of coding regions resulted in a more stable protein than the native monomer with an increased in vivo life span. 22,23 Based on evidence that the bridging of 2 adjacent Epo receptors triggers a conformational change that initiates signal transduction 24,25 and that high-and low-affinity sites for the Epo receptor are present on Epo, [26][27][28] we hypothesized that the fusing of 2 Epo molecules might confer an increase in intrinsic activity by providing 2 closely associated high-affinity domains.…”

Section: Introductionmentioning

confidence: 99%

Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo

Dalle

Henri

Rouyer-Fessard

et al. 2001

Blood

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High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34 ؉ human hematopoietic cells, the human dimer induced a 3-to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in ␤-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO element and of a plasmid encoding the tetracycline controlled transactivator tTA. Administration of tetracycline completely inhibited the expression of the mEpo dimer. On tetracycline withdrawal, expression of the Epo-Epo dimer resumed, thereby resulting in a large and sustained hematocrit increase in ␤-thalassemic mice. No immunologic response against the dimer was apparent in mice because the duration of the hematocrit increase was similar to that observed with the monomeric form of mouse erythropoietin. ( IntroductionErythropoietin (Epo) is a 34-kd glycoprotein produced mainly by kidney paratubular cells in response to reduced oxygen delivery. 1,2 Epo stimulates erythroid progenitor cell proliferation, differentiation, and maturation, and it inhibits cell apoptosis, which results in increased erythrocyte production. [3][4][5] The recombinant human erythropoietin (rhEpo) hormone is widely used to compensate for the reduced production of endogenous Epo in renal failure and to correct the associated anemia. Administration of rhEpo alleviates the necessity for blood transfusion and greatly improves the quality of life for patients. [6][7][8] Clinical studies have shown that rhEpo can also be effective in the treatment of other chronic anemias, 9 especially when endogenous Epo levels are inappropriately low. When injected into ␤-thalassemic mice, rhEpo has been shown to restore balanced globin chain synthesis and to improve the erythrocyte phenotype. 10 In patients with ␤-thalassemia, serum Epo levels are often lower than expected, 11 and injections of high doses of rhEpo have been shown to increase blood hemoglobin levels and occasionally to alleviate the need for transfusion. [12][13][14][15] Although high doses of Epo may be beneficial for patients with ␤-thalassemia, the cost of such a treatment prevents its use in long-term controlled therapeutic trials and in routine clinical practice. Consequently, various strategies have been pursued to increase the yield of Epo produced by gene transfer and to enhance its intrinsic activity. For i...

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Increased potency of an erythropoietin peptide mimetic through covalent dimerization

Cited by 116 publications

References 18 publications

EphB Receptor-binding Peptides Identified by Phage Display Enable Design of an Antagonist with Ephrin-like Affinity

EphB Receptor-binding Peptides Identified by Phage Display Enable Design of an Antagonist with Ephrin-like Affinity

Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects

Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo

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