Journal of Medicinal Chemistry
Article
column (5 μm, 2.1 × 12.5 mm) (Agilent Technologies, Santa Clara,
CA, USA). The following mobile phases were used for the separation:
solvent A, water/0.1% formic acid; and solvent B, methanol/0.1%
formic acid. The gradient used for separation was 95 to 5% solvent A
over 10 min at a flow rate of 250 μL/min. Using the mass of 496.2569
0.005, optimized positive mode ESI conditions were found as
follows: sheath and auxiliary gas, 60 and 4 arbitrary units, respectively;
spray voltage, 4 kV; capillary temperature and voltage, 325 °C and 49
V, respectively; tube lens voltage, 115 V. Full scan high-resolution
mass spectra were collect at 60000 Hz from m/z 150 to 2000. The full
scan data for standard curve samples and unknowns were processed
with Xcalibur software to determine concentrations (Thermo Fisher
Scientific).
C4-2 Conditioned Medium Assay. C4-2 and BT-549 cells were
cultured as described above. 11 was added to the medium with C4-2
cells and incubated for 6 h at 37 °C in a humidified atmosphere (5%
CO2) in serum-free medium. Medium was then collected and added to
BT-549 cells for 3 h. Inhibition of the PI3 kinase pathway was followed
by monitoring the p-Akt T308 levels by Western blotting.
Cleavage of Compound 11 with Purified PSA. Compound 11
was incubated in 50 mM Tris-HCl−0.1 M NaCl, pH 7.8, buffer for 6 h
at 37 °C with purified active human PSA (Merck, Darmstadt,
Germany) and then added to BT-549 cells for 30 min, cultured as
previously described. Inhibition of PI3 kinase was followed by
monitoring p-Akt T308 levels by Western blotting.
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Statistical Analysis. Probability (p) values were calculated using
the ANOVA one-way test; p values ≤0.05 were considered to be
statistically significant. The results are expressed as the mean SEM, n
= 3.
AUTHOR INFORMATION
■
Corresponding Author
(11) LeBeau, A. M.; Banerjee, S. R.; Pomper, M. G.; Mease, R. C.;
Denmeade, S. R. Optimization of peptide-based inhibitors of prostate-
specific antigen (PSA) as targeted imaging agents for prostate cancer.
Bioorg. Med. Chem. 2009, 17, 4888−4893.
*(M.E.W.) Phone: +1-(336)-758-3898. E-mail: welker@wfu.
edu (G.K.) Phone: +1-(336)-713-7650. E-mail: gkulik@
(12) Vlahos, C. J.; Matter, W. F.; Brown, R. F.; Traynor-Kaplan, A.
E.; Heyworth, P. G.; Prossnitz, E. R.; Ye, R. D.; Marder, P.; Schelm, J.
A.; Rothfuss, K. J.; Serlin, B. S.; Simpson, P. J. Investigation of
neutrophil signal transduction using a specific inhibitor of
phosphatidylinositol 3-kinase. J. Immunol. 1995, 154, 2413−2422.
(13) Holmes, D. PI3K pathway inhibitors approach junction. Nat.
Rev. Drug Discov. 2011, 10, 563−564.
(14) Garlich, J. R.; De, P.; Dey, N.; Su, J. D.; Peng, X.; Miller, A.;
Murali, R.; Lu, Y.; Mills, G. B.; Kundra, V.; Shu, H. K.; Peng, Q.;
Durden, D. L. A vascular targeted pan phosphoinositide 3-kinase
inhibitor prodrug, SF1126, with antitumor and antiangiogenic activity.
Cancer Res. 2008, 68, 206−215.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Marcus W. Wright (Wake Forest University,
Department of Chemistry) for LC-ESI-MS analysis, Tina
Snider (Department of Urology, Wake Forest Baptist Health)
for performing ECLIA tPSA assays, and Karen Kline for editing.
This work was supported by WFU Cross-Campus Collabo-
rative Research Program and R01CA118329 to G.K.
(15) Gharbi, S. I.; Zvelebil, M. J.; Shuttleworth, S. J.; Hancox, T.;
Saghir, N.; Timms, J. F.; Waterfield, M. D. Exploring the specificity of
the PI3K family inhibitor LY294002. Biochem. J. 2007, 404, 15−21.
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W. E.; Belew, R. K.; Olson, A. J. Automated docking using a
lamarckian genetic algorithm and and empirical binding free energy
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(17) Abbott, B. M.; Thompson, P. E. Synthetic studies of the
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ABBREVIATIONS USED
■
BAD, Bcl-2-associated death promoter; Bcl-2, B-cell lymphoma
2; Bcl-XL, B-cell lymphoma extra-large; DCC, N,N′-dicyclohex-
ylcarbodiimide; DCM, dichloromethane; DMAP, 4-dimethyla-
minopyridine; DMSO, dimethyl sulfoxide; DTT, dithiothritol;
PARP, poly(ADP-ribose) polymerase; PBS, phosphate buffer
saline; PI3K, phosphatidylinositol-3-kinase; PSA, prostate-
specific antigen; PTEN, phosphatase and tensin homologue
deleted on chromosome 10
(18) Zha, J.; Harada, H.; Yang, E.; Jockel, J.; Korsmeyer, S. J. Serine
phosphorylation of death agonist BAD in response to survival factor
results in binding to 14-3-3 not BCL-X(L). Cell 1996, 87, 619−628.
(19) Datta, S. R.; Dudek, H.; Tao, X.; Masters, S.; Fu, H.; Gotoh, Y.;
Greenberg, M. E. Akt phosphorylation of BAD couples survival signals
to the cell-intrinsic death machinery. Cell 1997, 91, 231−241.
(20) del Peso, L.; Gonzalez-Garcia, M.; Page, C.; Herrera, R.; Nunez,
G. Interleukin-3-induced phosphorylation of BAD through the protein
kinase Akt. Science 1997, 278, 687−689.
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