Hsp90 Inhibitors Identified from a Library of Novobiocin
Analogues.
Yu XM, Shen
G, Neckers
L, Blake H, Holzbeierlein
J, Cronk
B, Blagg
BS.
Department of Medicinal Chemistry and The Center for Protein Structure and
Function, The University of Kansas, 1251 Wescoe Hall
Drive, Malott 4070, Lawrence, Kansas 66045-7582,
Urologic Oncology Branch, National Cancer Institute, NIH, Rockville, Maryland
20850, and Department of Urology, The University of Kansas Medical Center,
Kansas City, Kansas 66103.
Novobiocin is a C-terminal inhibitor of the Hsp90
protein folding machinery, which is responsible for the conformational
maturation of numerous proteins involved in cancer growth and survival. Due to novobiocin's poor inhibitory activity (
approximately 700 muM), very little attention
has been paid toward the development of novobiocin
analogues for Hsp90 inhibition. In this study, a parallel library of 20 novobiocin derivatives was prepared and the biological
activity of each evaluated by Western blot analysis of Hsp90 client proteins.
A4 was found to be a potent inhibitor of Hsp90 as determined by its ability to
cause the degradation of several Hsp90 client proteins in both breast and
prostate cancer cell lines. In the presence of 1 muM A4, several Hsp90 client proteins were degraded,
including AKT, Her2, Hif-1alpha, and the androgen receptor.
PMID: 16159253 [PubMed - as supplied by publisher]
Bradykinin mimics ischemic
preconditioning by generating reactive oxygen species (ROS).
To identify intermediate steps leading to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red that becomes fluorescent after exposure to
ROS. Fluorescence intensity in treated cells was expressed as % of that in
paired untreated cells. Bradykinin (500nM) caused
51+/-16% increase in ROS generation (p<0.001). Co-incubation with either bradykinin B2 receptor blocker HOE140 (5 micro M) or free
radical scavenger N-(2-mercaptopropionyl) glycine
(1mM) prevented this increase confirming the response was receptor-mediated and
ROS were actually being measured. Closing mitochondrial KATP (mKATP)channels with
5-hydroxydecanoate (5HD, 1mM) prevented increased ROS generation. Bradykinin-induced ROS generation was blocked by L-NAME
(200 micro M) implicating nitric oxide as an intermediate. Blockade of guanylyl cyclase with ODQ (10
micro M) aborted bradykinin-induced ROS generation,
but not that from diazoxide, a direct opener of mKATP channels. The PKG blocker 8-Br-cGMPS (25 micro M) eliminated bradykinin's
effect. Conversely, direct activation of PKG with 8-pCPT-cGMP (100 micro M)
increased ROS generation (39+/-15%, p<0.004) similar to bradykinin.
This increase was blocked by 5HD. Finally, the nitric oxide donor SNAP (1 micro
M)increased ROS by 34+/-6%. This increase was also
blocked by 5HD. In intact rabbit hearts bradykinin
(400nM) decreased infarction from 30.5+/-3.0% of the risk zone in control
hearts to 11.9+/-1.4% (p<0.01). This protection was aborted by either L-NAME
(200 micro M) or ODQ (2 micro M)(35.4+/-5.7% and 30.4+/-3.0%
infarction, resp., pNS vs control). Hence, bradykinin
preconditions through receptor-mediated production of nitric oxide which
activates guanylyl cyclase.
The resulting cGMP activates PKG that opens mKATP. Subsequent release of ROS triggers cardioprotection.