[supplementary informantion]
New non-quinone geldanamycin analogues from genetically engineered
Streptomyces hygroscopicus
Cheng-Zhu Wu, An Na Moon, Jae hyuik Jang, Dongho Lee, Sun-Young Kang, Joon-Tae
Park, Jong Seog Ahn, Bang Yeon Hwang, Young Ho Kim, Hong-Sub Lee and Young-Soo
Hong*
EXPERIMENTAL SECTION
Colorimetric determination of ATPase activity
Histidine-tagged yeast Hsp90 protein was purified by metal affinity chromatography 1, 2. The
protein was used after dialysis with assay buffer. ATPase activity was monitored by
phosphate hydrolysis using malachite green, as previously reported 3. Briefly, 1 μM Hsp90
was incubated for 2.5 hours at 37oC with 0.2 mM ATP and with either of the novel
compounds or a solvent control (DMSO). After incubation, a malachite green-molybdate
solution was added, followed by quenching with 34% sodium citrate. The absorbance was
measured at 620 nm in a spectraphotometer (Molecular Devices, Sunnyvale, CA). The IC50
values for these compounds under optimized conditions were determined from a range of
inhibitor concentrations (0.1–100 μM).
ATP-Sepharose binding assay with human Hsp90α
The assay was performed as previously described
4
and according to the manufacturer’s
instructions (Jena Bioscience GmbH, Jena, Germany) with minor modifications. A total of 1
ug of human Hsp90α protein (Stressgen, Victoria, BC, Canada) with DMSO, novel
compounds, or geldanamycin was incubated on ice in 100 μl ATP-containing binding buffer.
After 30 minutes, 50 μl of pre-equilibrated C10-linked aminophenly-ATP-Sepharose was
added to the tubes, which were then incubated at 4oC for another 3 hours with frequent
mixing to resuspend the resin. Following incubation, sepharose was washed, pelleted and
analyzed by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The bound proteins were analyzed by western blotting with Hsp90 antibody (Stressgen).
Cell viability assay
Cells of human ovarian cancer cell line A2780 and breast cancer cell lines SK-Br3 and
BTB474 were obtained from the Korean Cell Line Bank. Cells were maintained in DMEM
(Hyclone, Logan, UT) containing 10% fetal bovine serum (Hyclone) and 1%
penicillin/streptomycin (Gibco, Franklin Lakes, NJ) in a humidified 5% CO2 atmosphere at
37oC. Upon reaching confluence, the cells were detached using a trypsin-EDTA solution. The
cells were seeded in wells of 96-well plates at a concentration of 1 × 104 cells/well 1 day
before the start of treatment. Cells were treated with various concentrations of the two
compounds for 72 hours. Cell viability was detected by water-soluble tetrazolium WST-8
assay (Dojindo Molecular Technologies; Gaithersburg, MD). The amount of formazan
formed was measured from the absorbance at 450 nm with a reference wavelength of 620 nm
by using a microplate reader (Biotek, Winooski, VT).
Western blot analysis
After incubation with drugs, the cells were harvested, washed twice in phosphate buffered
saline, and lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 1% triton X-100, 150 mM NaCl,
and a cocktail of protease inhibitors (Sigma-Aldrich)]. Proteins in the lysate were resolved by
8% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking, the
blot was incubated overnight with antibody to c-Raf, Her2, or β-actin (Cell Signaling
Technology, Cambridge, MA) followed by secondary antibody coupled to horseradish
peroxidase. The bound antibody was visualized by enhanced chemiluminescence (INTRON
Biotechnology, Seoul, Korea).
S. Figure 1 Key 1H-1H COSY (bold) and HMBC correlations of DHQ7 (4) and DHQ8
(5)
S. Figure 2 Representative competitive binding assay between compound and ATP for
human Hsp90α
Human Hsp90 protein was mixed with vehicle (DMSO as control), ATP, and compounds
at indicated concentrations and incubated with C 10-linked-aminophenyl-ATP sepharose.
Material bound to the beads was analyzed by western blotting with antibodies against
Hsp90.
References
1. Kim W, et al. Rational biosynthetic engineering for optimization of geldanamycin
analogues. ChemBiochem. 10(7):1243-1251 (2009)
2. Panaretou B, et al. ATP binding and hydrolysis are essential to the function of the Hsp90
molecular chaperone in vivo. EMBO J. 17(16):4829-4836 (1998)
3. Rowlands MG, Newbatt YM, Prodromou C, Pearl LH, Workman P, Aherne W. Highthroughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Anal.
Biochem. 327(2):176-183 (2004)
4. Wu CZ, et al. 6-Alkylsalicylic Acid Analogues Inhibit In Vitro ATPase Activity of Heat
Shock Protein 90. Archives of Pharmacal Research. 33(12):1997-2001 (2010)