6392
Z. Qiao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6389–6392
5. Bawa-Khalfe, T.; Cheng, J.; Lin, S.; Ittmann, M.; Yeh, E. T. H. J. Biol. Chem. 2010,
285, 25859.
Thr499
6. Cheng, J.; Kang, X.; Zhang, S.; Yeh, E. T. H. Cell 2007, 131, 584.
7. Yamaguchi, T.; Sharma, P.; Athanasiou, M.; Kumar, A.; Yamada, S.; Kuehn, M. R.
Mol. Cell Biol. 2005, 25, 5171.
Val532
Gly531
His529
8. Hoeller, D.; Dikic, I. Nature 2009, 458, 438.
9. Goldenberg, S. J.; McDermott, J. L.; Butt, T. R.; Mattern, M. R.; Nicholson, B.
Biochem. Soc. Trans. 2008, 36, 828.
Phe498
10. Colland, F.; Formstecher, E.; Jacq, X.; Reverdy, C.; Planquette, C.; Conrath, S.;
Trouplin, V.; Bianchi, J.; Aushev, V. N.; Camonis, J.; Calabrese, A.; Borg-Capra, C.;
Sippl, W.; Collura, V.; Boissy, G.; Rain, J.; Guedat, P.; Delansorne, R.; Daviet, L.
Mol. Cancer Ther. 2009, 8, 2286.
11. Schneekloth, A. R.; Pucheault, M.; Tae, H. S.; Crews, C. M. Bioorg. Med. Chem.
Lett. 2008, 18, 5904.
Trp465
Ile528
Phe496
Thr495
Pro527
12. Shen, L.; Tatham, M. H.; Dong, C.; Zagorska, A.; Naismith, J. H.; Hay, R. T. Nat.
Struct. Mol. Biol. 2006, 13, 1069.
Cys603
Trp534
13. Micale, N.; Kozikowski, A. P.; Ettari, R.; Grasso, S.; Zappala, M.; Jeong, J.; Kumar,
A.; Hanspal, M.; Chishti, A. H. J. Med. Chem. 2006, 49, 3064.
14. Dourlat, J.; Liu, W.; Gresh, N.; Garbay, C. Bioorg. Med. Chem. Lett. 2007, 17, 2527.
15. Feytens, D.; Cescato, R.; Reubi, J. C.; Tourwe, D. J. Med. Chem. 2007, 50, 3397.
16. Micale, N.; Vairagoundar, R.; Yakovlev, A. G.; Kozikowski, A. P. J. Med. Chem.
2004, 47, 6455.
17. Sherrill, R. G.; Sugg, E. E. J. Org. Chem. 1995, 60, 730.
18. Spectroscopic data for 8: 1H NMR (400 MHz, CDCl3) d 9.67 (s, 1H), 7.65–7.34 (m,
8H), 7.17 (d, J = 8.0 Hz, 1H), 4.78–4.60 (m, 3H); 13C NMR (100 MHz, CDCl3): d
195.78, 170.42, 166.52, 141.70, 138.18, 131.95, 130.61, 130.37, 129.73, 129.63,
128.31, 124.92, 121.43, 70.10, 57.71; HRMS (ESI) [M+H]+ C17H16N3O2 calcd
294.1243, found 294.1220.
Figure 2. Binding model of 38 in SENP1 catalytic site. Cyan sticks-38, yellow
ribbons-SENP1 protein, yellow lines-SENP1 residues, magenta dashed line-hydrogen
bond.
19. The DNA of the catalytic domain of SENP1 (SENP1c, aa419–aa643) was
amplified from PC3 cell cDNA library, cloned into pET28a(+) vector, and
hydrogen bond between C(3)–NH and Gly531;
Trp465 with C(5)- and diazepine-fused phenyls;
p
–
p
interaction of
p–p interaction
expressed in Escherichia coli BL21 by induction with 0.2 mM isopropyl b-D-
of C(3)NH-phenyl and His529; nonpolar interaction between
C(5)-phenyl and Val532. The phenyl group of the Cbz moiety in-
serts into a hydrophobic pocket consisting of residues Phe496,
Thr495, Thr499, Phe498, Ile528 and Pro527. In general, these
observations were corroborative with the structure–activity
relationship discussed above.
The compounds with the best SENP1 inhibitory activity were
tested against prostate cancer cells (PC3) to evaluate their ability
to inhibit cancer cell growth in vitro. The lead SENP1 inhibitors
thiogalactoside at 25 °C for 12 h. Cell pellets were resuspended in buffer A
(300 mM NaCl, 50 mM pH 8.0 PBS, 10 mM imidazole, 0.2 mM PMSF and 2 mM
b-mercaptoacetic ethanol) and lysed by sonication on ice. The his-tagged
protein was purified by affinity chromatography using Ni-NTA-agarose
(QIAGEN) and eluted with 250 mM imidazole in buffer A. The SUMO–
D
RanGAP plasmids were kindly provided by Dr. Jinke Cheng (SJTU School of
Medicine). Expression and purification of SUMO-
methods.
DRanGAP followed similar
20. Nicholson, B.; Leach, C. A.; Goldenberg, S. J.; Francis, D. M.; Kodrasov, M. P.;
Tian, X.; Shanks, J.; Sterner, D. E.; Bernal, A.; Mattern, M. R.; Wilkinson, K. D.;
Butt, T. R. Protein Sci. 2008, 17, 1035.
21. SUMO-CHOP-reporter fluorescence assay: SENP1 (20 nM) was preincubated with
compounds for 10 min at 37 °C before the SUMO–reporter–enzyme and the
reporter substrate were added and incubated for 30 min. The fluorescence
intensity was determined on the Synergy H4 Hybrid Microplate Reader plate
reader with excitation and emission at 475 and 555 nm, respectively, at room
temperature. The relative activity of SENP1 was determined by measuring the
RFU value. Net RFUs were determined by subtracting the blank RFU value (the
reporter substrate in assay buffer) from each data point. Percentage inhibition
was calculated using the following equation [1ꢀ(RFUcompd/RFUDMSO)] ꢁ 100.
IC50 values were determined using a sigmoidal dose–response (variable slope)
model.
36 and 38 showed IC50 values of 13.0 and 35.7 lM against prostate
cancer cells, respectively.
In summary, we designed and synthesized a series of SENP1
inhibitors based on benzodiazepine scaffold. They showed inhibi-
tory activity as good as IC50 = 9.2 lM. This is the first report on de-
sign and synthesis of SENP1 inhibitors. Further study is underway
to obtain inhibitors with improved potency and druggability and to
eventually utilize them to validate the novel therapeutic target.
22. SUMO-
for 10 min at 37 °C, SUMO-
reaction was stopped by boiling in sample buffer. The proteins were separated
D
RanGAP cleavage assay: After preincubation of SENP1 with compounds
D
RanGAP was added and incubated for 30 min. The
Acknowledgments
by SDS–PAGE and visualized by coommassie blue stain for SUMO-
cleavage.
DRanGAP
We thank National Science Foundation of China (20702031;
91013008), Ministry of Science and Technology of China
(2009CB918404; 2010CB912104), E-Institutes of Shanghai
Universities (EISU) Chemical Biology Division, and National
Comprehensive Technology Platforms for Innovative Drug R&D
(2009ZX09301-007) for financial support of this work.
23. Spectroscopic data for 36: 1H NMR (300 MHz, CDCl3) d 9.66 (s, 1H), 7.91–7.86
(m, 3H), 7.65-7.56 (m, 3H), 7.51–7.29 (m, 11H), 7.21 (d, J = 8.1 Hz, 1H), 6.87 (s,
1H), 5.86 (d, J = 8.1 Hz, 1H), 5.22 (s, 2H), 4.74 (s, 2H) ppm; 13C NMR (100 MHz,
CDCl3)
d 195.17, 168.08, 168.01, 166.39, 152.92, 141.29, 138.07, 135.78,
132.24, 130.89, 130.82, 129.96, 129.76, 128.65, 128.60, 128.45, 128.36, 125.47,
121.71, 117.88, 67.45, 67.25, 57.71 ppm; HRMS (ESI) [M+1]+ C32H27N4O5 calcd
547.1981, found 547.1982.
24. Spectroscopic data for 38: 1H NMR (400 MHz, CDCl3) d 9.66 (s, 1H), 7.93 (d,
J = 8.0 Hz, 1H), 7.80–7.79 (m, 2H), 7.65–7.57 (m, 4H), 7.50–7.28 (m, 10H), 7.22
(d, J = 8.4 Hz, 1H), 6.81 (s, 1H), 5.85 (d, J = 8.0 Hz, 1H), 5.22 (s, 2H), 4.74 (s, 2H)
ppm; 13C NMR (100 MHz, CDCl3) d 195.23, 168.09, 167.89, 166.69, 153.23,
141.27, 138.34, 138.03, 135.94, 134.67, 132.24, 130.87, 130.82, 129.91, 129.77,
129.43, 128.60, 128.31, 128.28, 125.44, 122.02, 121.72, 117.65, 67.55, 67.10,
57.70 ppm; HRMS (ESI) [M+1]+ C32H27N4O5 calcd 547.1981, found 547.1980.
References and notes
1. Hochstrasser, M. Nature 2009, 458, 422.
2. Heun, P. Curr. Opin. Cell Biol. 2007, 19, 350.
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