L. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3039–3043
3043
Figure 5. Docking of (SRS)-5 to the Nrf2 binding site of Keap1 Kelch domain. (A) Top view with (SRS)-5 (shown in stick model) bound to Keap1 Kelch domain shown in ribbon
graphics and interacting Keap1 residues in VDW surface. (B) Overlay of the (SRS)-5 colored by element type with Nrf2 peptide in green as docked onto the Nrf2 binding site of
Keap1 Kelch domain (rotated ꢀ90° around the X-axis in A/B for a better view of the interactions with Keap1 residues shown at the bottom.
4. Baird, L.; Dinkova-Kostova, A. T. Arch. Toxicol. 2011, 85, 241.
5. Calkins, M. J.; Johnson, D. A.; Townsend, J. A.; Vargas, M. R.; Dowell, J. A.;
previously reported indirect inhibitors of Keap1–Nrf2 interaction,
we exposed (SRS)-5 to a high concentration (50 lM) of glutathione
Williamson, T. P.; Kraft, A. D.; Lee, J. M.; Li, J.; Johnson, J. A. Antioxid. Redox
Signal. 2009, 11, 497.
6. Jeong, W. S.; Jun, M.; Kong, A.-N. T. Antioxid. Redox Signal. 2006, 8, 99.
7. Lau, A.; Villeneuve, N. F.; Sun, Z.; Wong, P. K.; Zhang, D. D. Pharmacol. Res. 2008,
58, 262.
8. Yates, M. S.; Kensler, T. W. Drug News Perspect. 2007, 20, 109.
9. Li, J.; Ichikawa, T.; Janicki, J. S.; Cui, T. Expert Opin. Ther. Targets 2009, 13, 785.
10. Yu, X.; Kensler, T. Mutat. Res. 2005, 591, 93.
11. Zhao, J.; Redell, J. B.; Moore, A. N.; Dash, P. K. Biochem. Biophys. Res. Commun.
2011, 407, 501.
12. Steel, R.; Cowan, J.; Payerne, E.; O’Connell, M. A.; Searcey, M. ACS Med. Chem.
Lett. 2012, 3, 407.
13. Williamson, T. P.; Amirahmadi, S.; Joshi, G.; Kaludov, N. K.; Martinov, M. N.;
Johnson, D. A.; Johnson, J. A. Chem. Biol. Drug Des. 2012, 80, 810.
14. Pergola, P. E.; Krauth, M.; Huff, J. W.; Ferguson, D. A.; Ruiz, S.; Meyer, C. J.;
Warnock, D. G. Am. J. Nephrol. 2011, 33, 469.
15. Pergola, P. E.; Raskin, P.; Toto, R. D.; Meyer, C. J.; Huff, J. W.; Grossman, E. B.;
Krauth, M.; Ruiz, S.; Audhya, P.; Christ-Schmidt, H.; Wittes, J.; Warnock, D. G. N.
N. Engl. J. Med. 2011, 365, 327.
16. Nagaraj, S.; Youn, J. I.; Weber, H.; Iclozan, C.; Lu, L.; Cotter, M. J.; Meyer, C.;
Becerra, C. R.; Fishman, M.; Antonia, S.; Sporn, M. B.; Liby, K. T.; Rawal, B.; Lee, J.
H.; Gabrilovich, D. I. Clin. Cancer Res. 1812, 2010, 16.
17. Egner, P. A.; Chen, J. G.; Wang, J. B.; Wu, Y.; Sun, Y.; Lu, J. H.; Zhu, J.; Zhang, Y.
H.; Chen, Y. S.; Friesen, M. D.; Jacobson, L. P.; Munoz, A.; Ng, D.; Qian, G. S.; Zhu,
Y. R.; Chen, T. Y.; Botting, N. P.; Zhang, Q.; Fahey, J. W.; Talalay, P.; Groopman, J.
D.; Kensler, T. W. Cancer Prev. Res. 2011, 4, 384.
18. Shapiro, T. A.; Fahey, J. W.; Dinkova-Kostova, A. T.; Holtzclaw, W. D.;
Stephenson, K. K.; Wade, K. L.; Ye, L.; Talalay, P. Nutr. Cancer 2006, 55, 53.
19. Prestera, T.; Holtzclaw, W. D.; Zhang, Y.; Talalay, P. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 2965.
20. Inoyama, D.; Chen, Y.; Huang, X.; Beamer, L. J.; Kong, A.-N. T.; Hu, L. J. Biomol.
Screen. 2011, 17, 435.
21. Chen, Y.; Inoyama, D.; Kong, A.-N. T.; Beamer, L. J.; Hu, L. Chem. Biol. Drug Des.
2011, 1014, 78.
that we used to mimic cysteine residues. No thiol addition or
decomposition could be detected over 48 h, indicating that (SRS)-
5 inhibits the Keap1–Nrf2 protein–protein interaction by non-
covalent binding and thus is a first-in-class direct inhibitor of
Keap1–Nrf2 interaction. (SRS)-5 was designated as a probe with
the NIH Molecular Libraries Program (ML334) due to these proper-
ties and its potency as an inhibitor of Keap1–Nrf2 interaction.
In summary, we have discovered, through high-throughput
screening of the NIH MLPCN library of small molecules, a first-
in-class direct inhibitor of the Keap1–Nrf2 protein–protein interac-
tion. It was found that only one stereoisomer among the possible
eight isomers is active. The different binding activity between
the stereoisomers is expected of structurally specific ligand–target
interactions. The strong binding affinity of (SRS)-5 (ML334,
LH601A) over its stereoisomers and the preliminary, yet tractable,
structure–activity relationships provide further evidence of its
direct binding to Keap1 and support its use as a lead for structure
optimization.
Acknowledgments
We thank Dr. Suzanne Forry-Schaudies from the National Can-
cer Institute for her advice and continuing efforts in overseeing the
success of this project. We gratefully acknowledge the financial
support of grants CA133791 and MH093197 (to L.H.) from the Na-
tional Institutes of Health. This work was funded in part by the
NIH-MLPCN program (1 U54 HG005032-1 awarded to S.L.S.).
22. Bolm, C.; Schiffers, I.; Atodiresei, I.; Hackenberger, C. P. R. Tetrahedron:
Asymmetry 2003, 14, 3455.
23. Christov, C.; González-Bulnes, P.; Malhaire, F.; Karabencheva, T.; Goudet, C.;
Pin, J.-P.; Llebaria, A.; Giraldo, J. ChemMedChem 2011, 6, 131.
24. Invitrogen. Cell Sensor™ ARE-bla Hep G2 Cell-based Assay Protocol, 2006; p 8.
25. DiscoveRx. PathHunterÒ Keap1–Nrf2 Functional Assay for chemiluminescent
detection of activated Nrf2. Product Booklet: 93-0821C3, 2011; p 12.
References and notes
1. Magesh, S.; Chen, Y.; Hu, L. Med. Res. Rev. 2012, 32, 687.
2. Nguyen, T.; Yang, C. S.; Pickett, C. B. Free Radical Biol. Med. 2004, 37, 433.
3. Stewart, J. D.; Hengstler, J. G.; Bolt, H. M. Arch. Toxicol. 2011, 85, 239.