Kinga Nyíri1,2, Anett Stéger1,2, Gergely Koppány1,2, Judit Matejka1,2, Beáta G. Vértessy1,2
1 Dept. Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest
2 Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest
Introduction: KRAS protein belongs to the small G-proteins family and acts as a molecular switch regulating crucial signaling pathways in cell growth and differentiation. The GTP bound form of KRAS can activate numerous functionally different downstream effector proteins, while the GDP-KRAS complex is inactive. Alternation between these two states requires co-factors, since GDP/GTP exchange and GTP hydrolysis would be very slow in the intrinsic way. Thus GTPase activating proteins (GAPs) accelerate the cleavage of the nucleotide to switch off signaling. Considering its essential role in signaling mutations influencing KRAS function have long been associated with cancer. One of the most frequent oncogenic mutations is G12C, which induce drastic decrease of the hydrolysis rate due to clash of cysteine-12 with GAP. The resulting lower turn-off rate of the active GTP-KRAS-G12C leads to uncontrolled cell growth and differentiation. According to recent results, specific treatment of such tumors would be possible with a covalent inhibitor that can interact selectively with the GDP bound form of KRAS-G12C and retard its activation. Nevertheless, the cellular efficiency of the reported inhibitors must be improved to enable clinical application.
Aims: Thus we, together with our collaborators in Rasopathy consortium, set out to develop a more potent inhibitor based on this scaffold. Our plan is to modify the reactive, so-called “warhead” group of the most potent inhibitor (ARS-853) available currently to enhance effectivity.
Method: Towards this end we established the production and purification of the G-domain (1-169) of the G12C mutant KRAS protein in our laboratory. Then we have compared the effectivity and selectivity of two warheads to that of ARS-853 by differential scanning calorimetry (thermofluor), fluorescent nucleotide exchange kinetics measurements, native and trypsinolysis mass spectrometry (MS). We also quantitated the free thiols after treatment of the mutant protein with the warheads and the reference inhibitor.
Results: Our first results showed that all compounds reacted with only one out of the four cysteines of the protein, although warhead fragments were less effective than the reference compound. In case of ARS-853 we were able to verify that only Cys-12 was reacting with the inhibitor. The thermofluor, nucleotide exchange and thiol quantitation assays were conclusive for the reference compound, however for the analysis of warheads the sensitivity of the assays should be improved.
Conclusion: We successfully established in vitro inhibitor testing methods for oncogenic mutant KRAS protein target.
This project has been supported by the NVKP_16-1-2016-0020 grant.
Doctoral School: George Oláh Doctoral School
Program: Chemistry and Chemical Technology
Supervisor: Beáta G. Vértessy
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