Antileukemic activity of shepherdin and molecular diversity of Hsp90 inhibitors

Background. Heat shock protein 90 (Hsp90) is a molecular chaperone that
is involved in signaling pathways for cell proliferation, survival, and
cellular adaptation. Inhibitors of Hsp90 are being examined as cancer
therapeutic agents, but the molecular mechanism of their anticancer
activity is still unclear. We investigated Hsp90 as a therapeutic target
for acute myeloid leukemia (AML) by use of the Hsp90 inhibitor
shepherdin (a novel peptidyl antagonist of the interaction between Hsp90
and survivin, which is a regulator of cell proliferation and cell
viability in cancer). Methods: We studied protein interactions by
molecular dynamics simulations and conducted competition experiments by
use of enzyme-linked immunosorbent assay (ELISA). Shepherdin[79-83], a
novel variant carrying the survivin sequence from Lys-79 through Gly-83,
or its scrambled peptide was made permeable to cells by adding the
antennapedia helix III carrier sequence. Apoptosis, Hsp90 client protein
expression, and mitochondrial dysfunction were evaluated in AML types
(myeloblastic, monocytic, and chronic myelogenous leukemia in blast
crisis), patient-derived blasts, and normal mononuclear cells. Effects
of shepherdin on tumor growth were evaluated in AML xenograft tumors in
mice (n = 6). Organ tissues were examined histologically. Results:
Shepherdin[79-83] bound to Hsp90, inhibited formation of the
survivin-Hsp90 complex, and competed with ATP binding to Hsp90.
Cell-permeable shepherdin[79-83] induced rapid (within 30 minutes) and
complete (with concentrations inducing 50\% cell death of 24-35 mu M)
killing of AML types and blasts, but it did not affect normal
mononuclear cells. Shepherdin[79-83] made contact with unique residues
in the ATP pocket of Hsp90 (Ile-96, Asp-102, and Phe-138), did not
increase Hsp70 levels in AML cells, disrupted mitochondrial function
within 2 minutes of treatment, and eliminated the expression of Hsp90
client proteins. Shepherdin[79-83] abolished growth of AML xenograft
tumors (mean of control group = 1698 mm(3) and mean of treated group =
232 mm(3); difference = 1466 mm(3), 95\% confidence interval = 505.8 to
2426; P = .008) without systemic or organ toxicity and inhibited Hsp90
function in vivo. Conclusions: Shepherdin is a novel Hsp90 inhibitor
with a unique mechanism of anticancer activity.