R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA
Introduction
The primary regulator of the growth and survival of erythroid progenitors, which mature into red blood cells, is the glycoprotein hormone erythropoietin (Epo) [1]. Epo exerts this effect by specifically interacting with a receptor present on the surface of progenitor cells which leads to receptor activation and initiation of an intracellular signal cascade. Over the last 10 years, the availability of recombinant Epo has led to its widespread use in stimulating red cell synthesis for the treatment of severe anaemia associated with acute and chronic disease [2]. In both chronic renal failure and cancer settings, severe anaemia and associated fatigue have a significant impact on the patient's quality of life. There have been a number of controlled trials in which patients receiving Epo demonstrated a significant improvement in quality of life scores and functional capacity.
While highly safe and efficacious, a desirable improvement sought for Epo therapy is in the mode of drug delivery [3]. The cost and inconvenience associated with chronic parenteral administration of Epo and other protein therapeutics have led a number of investigators to seek ways to deliver proteins orally, transdermally and by inhalation. However, the size and intrinsic lability of proteins has hindered progress along these lines. A second and perhaps even more difficult strategy to obtain orally administered agents has been to discover small molecule drugs that retain the full agonist activity of the larger protein molecules [4]. The motivation for attempting such a high-risk research strategy for Epo has been fueled by several potential benefits. In addition to the contemporary uses of Epo, an oral agent would potentially extend to therapeutic applications in less severe anaemia conditions associated with rheumatoid arthritis and other chronic inflammatory disorders. To this end, we and others have been pursuing small molecule mimetics of Epo and other protein hormones and cytokines. In the 10 years since Epo was first approved for use in man, we have not yet been successful in delivering an orally active Epo mimetic. However, a number of key steps have been reached in the search for small molecule agonists of the Epo receptor and a great deal of knowledge concerning Epo receptor structural biology has emerged from these efforts [5].
Peptide mimics of Epo
The Epo receptor (EPOR) is a member of a superfamily of cytokine receptors which includes receptors for growth hormone (GH), granulocyte-colony stimulating factor (G-CSF), thrombopoietin (TPO) and others [6]. The molecular arrangement of a prototypic member of this group would possess an extracellular ligand-binding domain, a single-pass transmembrane region and an intracellular signalling domain that interacts with members of a signalling cascade [7]. The receptors themselves have no intrinsic kinase activity.
In the case of EPOR, the mechanism for activation involves the binding and homodimerization of two receptor monomers by a single Epo molecule resulting in a 2 : 1 receptorhormone assembly. The dimerization of the receptor leads to cell signalling via the activation of Janus Kinase 2 molecules associated with the cytoplasmic domain [8].
The initial bias against finding small molecule mimetics of Epo was founded in the prevailing thought that the large surface area contacted by a glycoprotein the size of Epo (165 amino acids) to the dimeric receptor complex would be impossible to replicate with a much smaller molecular entity. However, both structural and mutagenesis studies with a related family member, GH receptor, showed that only a small subset of the ligandreceptor contacts accounted for the bulk of the binding energy [9,10]. The residues most influential in binding have been termed a functional epitope region, or a minimal area most important for ligand binding to the receptor.
Through combinatorial peptide screening techniques using phage display technology, we were able to isolate a novel 20 amino acid peptide mimetic (Epo Mimetic Peptide, EMP1Table 1) of EPO and subsequently to determine the structure as an EMP1EPOR complex [11,12]. The peptidereceptor assembly consisted of two peptides bound to two receptor monomers in a 2 : 2 ratio and in an almost perfectly symmetrical arrangement. The peptide has no sequence homology to that of Epo and was found to possess Epo mimetic action both in vitro and in animals albeit with less specific activity than that of Epo itself. Interestingly, we also found that the minimal active peptide sequence consists of only a 13 amino acid complement of the original 20 amino acid EMP1 sequence indicating that molecules even smaller than EMP1 can serve as receptor agonists [13].
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To further probe this concept, we have shown that preparation of peptide dimers using either defined chemical linkers [14] or larger polymeric PEG linkers [15] resulted in Epo mimetic molecules with greater specific activity. Significantly, it is also possible to convert inactive peptides that retain binding ability into weak agonists via ligand dimerization [15]. This suggests that binding alone can be exploited to provide the basis for receptor activation. However, not all inactive peptide sequences with retained binding ability could be converted into agonists, suggesting a conformational requirement that must also be satisfied before receptor activation can occur [15].
The discovery of a small peptidic molecule with the full agonist activity of Epo provided the impetus for others to reconsider the potential for the discovery of peptides and nonpeptidic mimetics of Epo and other cytokines. A number of other reports of peptide agonists for both the Epo [16,17] and TPO [18] receptors have subsequently been published.
The EPO mimetic peptide family reported in [16] is represented by a cyclic 18 amino acid peptide, termed ERB17, which was discovered in a fashion similar to EMP1 (Table 1). Although no structural characterization of this peptide bound to EBP has been reported it appears to be a complete agonist on Epo responsive cells. No report has emerged describing the activity of this peptide in animals. This discovery confirms the use of phage display technology in the discovery of novel ligands for cytokine receptors as does the report of TPO receptor agonists [18].
An Epo mimetic peptide with an alternative activation site has also been reported [17] (see Table 1). This linear peptide sequence was derived from a homology search method based on earlier work with the insulin receptor with a foundation in the concept of dimerization domains. The resultant peptide, termed ERP, is a short peptide identical to a stretch of the EPOR sequence and, based on the design paradigm, would be expected to activate the receptor via an interaction distal to the Epo and EMP1 binding site. This peptide appears to activate the signalling pathway utilized by Epo itself and was found to be active in animals. While no crystal structure has been reported for this molecule, such a report would provide very interesting information on a potentially unique EPOR activation mechanism.
Taken together, these peptide EPOR agonist ligands have proven to be extremely valuable tools for probing the role of subunit assembly in receptor activation. However, as therapeutics they are still lacking in the ability to be delivered orally to treat anaemia. To accomplish this feat, a non-peptidic small molecule will most probably be required. Such an orally available molecule would likely have to a molecular weight in the range of 150600 to have suitable properties for intestinal absorption [19]. The Epo mimetic peptides reported to date all have molecular weights in excess of ca. 1500, which when coupled with inherent instability of peptides make them poor candidates for an oral therapeutic [19].
Non-peptide mimetics
The discovery of peptide mimics of cytokines led to an accelerated interest in screening for non-peptide or small molecules that could activate receptor and function as a cytokine mimic. There have been two basic strategies used to discover active molecules, (i) converting antagonists to agonists and (ii) isolating molecules that bind outside the ligand binding site as shown above for peptides ligands.
The first case involves screening for competitive binders (antagonists) to the ligand binding site of the receptor and then chemically linking the two small molecule binders together into a dimer or higher multimer. These minimally bivalent dimers should then be able to bring together two monomers of receptor, hopefully into an active dimer configuration. The earliest demonstration of the soundness of the method was demonstrated for Epo receptor binding peptides [15]. As described above, two inactive peptides were linked with a bivalent polyethylene glycol linker molecules and converted a peptide sequence with binding ability into a weak agonist. This same principle has been expanded to obtain a non-peptidic EPOR agonist [20]. The process here was one in which chemical libraries were screened to isolate a molecule that acts as a competitive inhibitor of EpoEBP binding. The identified compound (compound 1) was not capable of supporting the proliferation of Epo responsive cells but when it was used to decorate a Starburst dendritic linker polymer (containing eight small molecule attachment sites) an agonist compound was obtained (compound 5). Compound 1 demonstrated an IC50 of the Epo-EBP association at around 60 µM. Upon conversion, the multivalent compound 5 has a molecular weight approximately 6400 daltons, binds receptor with an IC50 of 4 µM and activates EPOR with an EC50 of 5 µM in a cell proliferation assay. The maximum activity for compound 5 is 30% of the maximum activity observed for Epo and so, in turn, full agonist behaviour was not obtained. Concentrations of compound 5 above this level appeared to be toxic to the Epo responsive cells used in the study. While there were no in vivo experiments reported, however, it is doubtful, considering the size of compound 5, that it is orally available. While compound 5 has low potency and most likely low oral availability, it is an exciting milestone towards proof of the concept that non-peptidic molecules can mimic the activity of Epo.
While not directly related to the EPOR, there have been at least two additional discoveries with significance towards small molecule hormone and cytokine mimicry worth mention here. These include small molecule agonists of both the insulin receptor [21] and the murine G-CSF receptor [22]. The second is especially notable in that the agonist molecule targets an activation site other than the hormone binding domain and that the activation ability appears species restricted to the murine receptor and therefore would have no utility in man. These findings are especially notable as evidence for continuing our search for small molecule agonists of the EPOR since they illustrate that molecules with such potential exist.
Conclusions
The goal remains to identify a small molecule that will act as an orally available agonist of the Epo receptor. A number of key milestones have been achieved including the discovery of both peptide and non-peptide mimics of Epo. This has even been extended to create Epo mimetic fusion proteins which contain the EMP1 sequence [23]. However, it remains to be seen if small defined linkers can be designed to create molecules which will not only bind the EPOR but which will also be capable of promoting an active receptor assembly. This remains a significant technical hurdle and is complicated by recent findings that suggest that overall geometric constraints can influence the magnitude of EPOR activation [5,24]. As we have shown, it is also possible to isolate receptor antagonist molecules which have the ability to dimerize the EPOR in a way that does not lead to receptor activation [25]. The structural difference between the agonist and antagonist structures is modest and is an additional complication that must be faced as we strive to discover small molecule agonists of the EPOR with clinical potential [5]. Further, evidence has emerged that some fraction of the EPOR extant on the cell surface exists as a dimer of two EPOR molecules in the resting state and introduces an additional hurdle to be crossed by the theoretical small molecule agonist [26,27]. A small molecule mimetic would then also be required to promote rearrangement of the inactive dimer to the active state. Even in light of these complications, the growing understanding of the structural and cellular biology of the EPOR will likely provide the basis for additional advances. While no molecules with the desired Epo mimetic potential and oral bioavailability have arrived in the clinic, many encouraging discoveries have already occurred to suggest that someday the goal will be obtained.
Notes
Correspondence and offprint requests to: Dr Linda K. Jolliffe, R. W. Johnson Pharmaceutical Research Institute, 1000 Route 202, Box 300, Raritan, NJ 08869, USA.
References