Generation of covalent adducts between nucleotides and proteins: applications as tools and modulators of activity
Final Report Abstract
Ras proteins play a pivotal role in the regulation of important cellular processes such as cell growth and survival. This intricate regulation is achieved by precisely tuned switching between inactive GDP-bound and active GTP-bound states. The importance of this regulation of Ras in cellular signalling becomes evident when considering that oncogenic mutations in these proteins, which shift the equilibrium between these states towards the active form, are found in 25% of human cancers. These mutations in Ras occur particularly often at positions G12, G13, and Q61. Directly targeting Ras has been challenging and after discovery of Ras in the early 1980s, no direct treatment options were available until recently, and first molecules targeting a particular mutant (KRasG12C) have entered the clinic only in the last few years. In this project, we developed modified nucleotide analogues that form a covalent bond with KRasG13C, another oncogenic variant causing cancer. We show that once modified, the protein is permanently locked in the respective state (e.g. inactive GDP- state) and cannot be activated, even in the presence of the nucleotide exchange factor SOS. In depth biophysical and kinetic studies show that the introduced modifications of the nucleotides do not affect the general binding affinity towards Ras, thus enabling the nucleotides to generally compete with high cellular concentrations of the natural nucleotides. Additionally, structural analysis via X-Ray protein crystallography allowed us to solve the first structure of this particular Ras mutant and showed, that the nucleotides adopt the anticipated binding pose in the active site of Ras. Finally, we could also show that oncogenic signalling is inhibited in cells upon covalent modification of Ras. In conclusion, we have developed nucleotide based small molecules that are capable of modifying a KRas mutant that has not directly been addressed successfully in the past. These nucleotides are currently being developed further to increase cell permeability and reactivity towards RasG13C and possibly other relevant oncogenic mutants.
Publications
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Mutant-Specific Targeting of Ras G12C Activity by Covalently Reacting Small Molecules. Cell Chemical Biology, 26(10), 1338-1348.
Goody, Roger S.; Müller, Matthias P. & Rauh, Daniel
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KRasG12C inhibitors in clinical trials: a short historical perspective. RSC Medicinal Chemistry, 11(7), 760-770.
Goebel, Lisa; Müller, Matthias P.; Goody, Roger S. & Rauh, Daniel
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Crystal Structure of KRasG13C in Complex with Nucleotide-based covalent Inhibitor bdaGDP. Worldwide Protein Data Bank. Worldwide Protein Data Bank.
Goebel, L.; Mueller, M.P. & Rauh, D.
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Crystal Structure of KRasG13C in Complex with Nucleotide-based Covalent Inhibitor edaGDP. Worldwide Protein Data Bank. Worldwide Protein Data Bank.
Goebel, L.; Mueller, M.P. & Rauh, D.
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Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors. eLife, 12.
Goebel, Lisa; Kirschner, Tonia; Koska, Sandra; Rai, Amrita; Janning, Petra; Maffini, Stefano; Vatheuer, Helge; Czodrowski, Paul; Goody, Roger S.; Müller, Matthias P. & Rauh, Daniel
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Targeting KRAS Diversity: Covalent Modulation of G12X and Beyond in Cancer Therapy. Journal of Medicinal Chemistry, 67(8), 6044-6051.
Kirschner, Tonia; Müller, Matthias P. & Rauh, Daniel
