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 microM) 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 mm3 and mean of treated group = 232 mm3; difference = 1466 mm3, 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.
Gyurkocza
Boglarka
B
Department of Cancer Biology and Cancer Center, University of Massachusetts Medical School, LRB428, 364 Plantation Street, Worcester, MA 01605, USA.
Plescia
Janet
J
Raskett
Christopher M
CM
Garlick
David S
DS
Lowry
Philip A
PA
Carter
Bing Z
BZ
Andreeff
Michael
M
Meli
Massimiliano
M
Colombo
Giorgio
G
Altieri
Dario C
DC
D000208:Acute Disease; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005998:Glycine; D018922:HL-60 Cells; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D020014:K562 Cells; D007951:Leukemia, Myeloid / Q000188:drug therapy*; D008239:Lysine; D008297:Male; D051379:Mice; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000494:pharmacology*; D012107:Research Design; D000077022:Survivin; D014183:Transplantation, Heterologous; D020298:U937 Cells
D000970:Antineoplastic Agents; C107489:BIRC5 protein, human; D018841:HSP90 Heat-Shock Proteins; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D000077022:Survivin; C503112:shepherdin; D008239:Lysine; D005998:Glycine