Abstract
K-Ras is a well-validated cancer target but is considered to be “undruggable” due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets.
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Acknowledgments
This work was supported by US National Institutes of Health (NIH) Grants DP1OD006933/DP1CA174419 (NIH Director’s Pioneer Award; S.W.F.), P50A095103-12 (NCI SPORE in GI Cancer; R.J. Coffey), and RC2CA148375 to L.J. Marnett as well as The Lustgarten Foundation Award (S.W.F.). The use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. The use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). The use of mass spectrometry core was supported by scholarship S000003067 from the Vanderbilt Ingram Cancer Center. The use of Vanderbilt NMR facility was supported in part by grants for NMR instrumentation from the US National Science Foundation (NSF) (0922862), NIH (S10 RR025677) and Vanderbilt University matching funds. A portion of the experiments described here used the Vanderbilt robotic crystallization facility, which was supported by NIH Grant S10 RR026915. The authors would like to thank Dr. Wade Calcutt in the Vanderbilt Mass Spectrometry Core Laboratory for his help in characterizing the covalently modified intact proteins by LC–MS.
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Sun, Q., Phan, J., Friberg, A.R. et al. A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand. J Biomol NMR 60, 11–14 (2014). https://doi.org/10.1007/s10858-014-9849-8
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DOI: https://doi.org/10.1007/s10858-014-9849-8