Biology Reference
In-Depth Information
molecules can be identified that can activate cytokine receptors and retain
properties suitable for oral delivery.
EPO mimetics without homodimerization
Difficulties with development of small molecules that activate EPOR by
dimerization can be bypassed by targeting a different mechanism. One report
describes a peptide that activates EPOR by binding to a domain on EPOR sim-
ilar to major histocompatibility complex (MHC) peptides (Fig. 2). This 23-
amino acid peptide is reported to have both
in vitro
and
in vivo
EPO activity
[62]. The molecule appears to activate by binding EPOR at a region distal to
its binding site (transmembrane domain), suggesting that it activates different-
ly than does rHuEPO. The mechanism may be similar to that of the virus enve-
lope protein, gp55, that also activates EPOR by an interaction with the EPOR
transmembrane region [67, 68]. The nature of how activation EPOR occurs by
gp55 is not understood.
Another approach to mimetic discovery is to modulate steps downstream
from receptor activation such as by inhibiting hematopoietic cell phosphatase
(HCP) [69, 70]. HCP is an enzyme that dephosphorylates JAK-2, a kinase that
is part of the EPO signal transduction cascade [5]. JAK-2 is normally activat-
ed (phosphorylated) as a consequence of EPOR activation. HCP binds to acti-
vated (phosphorylated) EPOR and then dephosphorylates JAK-2, terminating
signal transduction. Truncated EPOR lack the HCP binding site, and thus HCP
cannot dephosporylate JAK-2, resulting in hypersensitivity of the receptor to
EPO. EPOR truncations have been described in humans whereby the affected
individuals have increased hemoglobin concentrations but very low EPO con-
centrations due to a hypersensitive EPOR [71, 72]. These observations suggest
that small molecule antagonists of HCP may result in increased EPOR activi-
ty that increases erythropoiesis in the absence of added EPO.
One concern of HCP inhibitors relates to observations associated with HCP
mutations in mice. These mice (motheaten) have a defective
HCP
gene [73]
and have multiple hematopoietic abnormalities, including increases in
macrophages, lymphocytes, and erythrocytes. HCP is a negative regulator for
several different cytokine receptors besides EPOR [69, 70, 74, 75]. Although
HCP inhibitors may be effective at increasing erythroid cell number, increases
in cell number of other hematopoietic cells may limit the usefulness of these
compounds.
Gene therapy
Controlled delivery of
EPO
genes to humans is another promising approach
for EPO therapy. Early work in this field depended on direct injection of plas-
mid DNA containing constitutively active
EPO
genes into the muscles of mice
