| In Vitro | In vitro activity:
PF-04929113 is a small-molecule Hsp90 inhibitor based on the
6,7-dihydro-indazol-4-one scaffold. PF-04929113 developed by Serenex,
converts to SNX-2122, which is the active Hsp90 inhibitor form.
PF-04929113 exhibits potent effects on Her-2 stability and causes
expected up-regulation of Hsp70. PF-04929113 shows potent
antiproliferative activity against a broad range of cancer cell types,
e.g. MCF-7 (IC50=16 nM), SW620 (IC50=19 nM), K562 (IC50=23 nM), SK-MEL-5
(IC50=25 nM), and A375 (IC50=51 nM).
Kinase Assay:
Hsp90 from porcine spleen extract is isolated by affinity capture on a
purine-affinity media. The Hsp90 loaded media is then challenged with
PF-04929113 at a given concentration, ranging from 0.8 to 500 μM, and
the amount of Hsp90 liberated at each concentration is determined by
Bradford protein assay. The resulting IC50 values are corrected for the
ATP ligand concentration and presented as apparent Kd values.
Cell Assay:
All assays are done in 96-well plates. All cell lines are purchased
from ATCC. Proliferation rates are measured by seeding cells (MCF-7,
SW620, K562, SK-MEL-5 and A375 cancer cell lines) into 96-well plates,
followed by compound addition 24 h later. After addition of PF-04929113,
cells are allowed to grow for either an additional 72 or 144 h
depending on rate of growth. At harvest, media is removed and DNA
content for individual wells is determined using CyQuant DNA dye. Levels
of Hsp90 client proteins and phosphor-regulated proteins in A375 are
measured by high content analysis (HCA) using an ArrayScan 4.5
instrument after 24 hours of treatment with PF-04929113, followed by
methanol fixation. After fixation in 4% PBS-buffered formalin and
permeablization with 0.1% TX-100, cells are probed with anti-Her2,
antiphospho-S6 (pS6), antipERK, and anti-Hsp70 primary antibodies,
followed by TRITC or FITC conjugated secondary antibodies. Nuclei are
also stained with Hoechst DNA binding dye. For each well, 250-500
individual nuclei are identified along with the average staining
intensity for the client and phospho-proteins for each cell. Average
client staining intensities are then calculated for each well. |
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| In Vivo | PF-04929113 inhibits human MM cell growth in vivo, and
immuno-histochemical analysis shows PF-04929113 significantly inhibits
p-ERK and p-Akt in treated mice. Meanwhile, PF-04929113 treatment
significantly decreases the percentage of CD31+ cells and MVD,
consistent with an inhibitory effect on angiogenesis in vivo. A 50 mg/kg
administration of PF-04929113 delivered 3 times per week significantly
delays castrate-resistant LNCaP tumor growth and prolongs cancer
specific survival. Immuno-histochemical analysis indicates increased
SP70 expression, and decreases Ki67, Akt, and AR expression, after
treatment with PF-04929113. Inhibition of tumor progression by
PF-04929113 may result from a combination of decreased proliferative
(reduced Ki67 and Akt expression) or increased apoptosis (increased
ApopTag staining) rates. |
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