A high-throughput assay to identify compounds that can induce dimerization of the erythropoietin receptor.

Erythropoietin induces dimerization of the erythropoietin receptor on the surface of erythroid progenitor cells, promoting the differentiation of these cells into mature red blood cells. To facilitate screening of large chemical collections for identification of compounds that can dimerize erythropoietin receptor, we have developed a novel, high-throughput in vitro assay to detect compounds that can cause dimerization of the erythropoietin receptor in solution. To develop this assay, amino acid sequences corresponding to the extracellular domain of erythropoietin receptor were expressed in Escherichia coli as erythropoietin-binding protein (rEBP). A modified version of this protein ((33)P-rEBP) containing a protein kinase A substrate site incorporated into the rEBP was also expressed in E. coli and labeled in vitro using protein kinase A and ¿gamma-(33)PATP. An erythropoietin mimetic peptide (EMP-1), that induces dimerization of rEBP in solution was used to demonstrate dimerization of (33)P-rEBP and rEBP in a 96-well microtiter plate format. EMP-1 induced dimerization of rEBP in this assay with an EC(50) of approximately 245 nM and had a maximal effect at 0.5-2 microM and required the presence of rEBP immobilized on the plate capable of binding EMP-1. EMP-1-induced dimerization of (33)P-rEBP and rEBP was reversed by excess unlabeled rEBP and was not masked by complex mixtures such as whole cell fungal extracts. These data demonstrate the ability of (33)P-rEBP to dimerize with rEBP in vitro in a format that is fully compatible with high-throughput screening.

Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.