Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues.
Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.
Sharp
Swee Y
SY
Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, United Kingdom.
Prodromou
Chrisostomos
C
Boxall
Kathy
K
Powers
Marissa V
MV
Holmes
Joanna L
JL
Box
Gary
G
Matthews
Thomas P
TP
Cheung
Kwai-Ming J
KM
Kalusa
Andrew
A
James
Karen
K
Hayes
Angela
A
Hardcastle
Anthea
A
Dymock
Brian
B
Brough
Paul A
PA
Barril
Xavier
X
Cansfield
Julie E
JE
Wright
Lisa
L
Surgenor
Allan
A
Foloppe
Nicolas
N
Hubbard
Roderick E
RE
Aherne
Wynne
W
Pearl
Laurence
L
Jones
Keith
K
McDonald
Edward
E
Raynaud
Florence
F
Eccles
Sue
S
Drysdale
Martin
M
Workman
Paul
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D018360:Crystallography, X-Ray; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D005260:Female; D045325:HCT116 Cells; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D019073:HT29 Cells; D006801:Humans; D007555:Isoxazoles / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011720:Pyrazoles / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D016896:Treatment Outcome; D023041:Xenograft Model Antitumor Assays
D015415:Biomarkers; D018841:HSP90 Heat-Shock Proteins; D007555:Isoxazoles; D011720:Pyrazoles; C520150:VER-49009; C520149:VER-50589; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D000251:Adenosine Triphosphatases