64P - Isothermal chemical KRAS denaturation assay for monitoring stability and inhibitors interactions

Background

KRAS is a frequent oncogene in human cancer and is an interesting therapeutic target due to its critical function in many malignancies. Although RAS was previously considered undruggable, recent successful identification of covalent inhibitors has shed light on the potential for cancer treatment. In drug research, RAS enzymatic activity is often measured, but methods based on RAS thermal stability (TSA) are also found useful. In these assays, RAS binding ligands stabilizes the structure over the native conformation and shifts its denaturation temperature. Among TSAs, differential scanning fluorimetry (DSF) has gained the most attention due to method’s good throughput. However, DSF lacks the needed sensitivity and results are markedly TSA dye-dependent. In addition, ligand behavior might vary when measured over the physiological temperature. In some cases, binding affinities obtained using isothermal chemical denaturation (ICD) have been found more reliable. Commonly used chemical denaturants like urea and guanidium chloride can be used to provide reliable information on ligand binding in a concentration dependent manner. However, these denaturants often require long incubation time and may affect the affinity of ionic compounds at high concentrations.

Methods

To overcome the difficulties in TSA and ICD techniques, we have developed a modification of the Protein-Probe (PP) technique, to measure isothermal protein stability at mild ICD conditions over time. The principle of this method is based on designed dual-labeled peptide-probe, and time-resolved Förster resonance energy transfer (TR-FRET) monitoring.

Results

As a model KRAS mutation, we studied KRAS(G12C) (50 nM) interaction with the most promising covalent inhibitors. Inhibitor induced stabilization of KRAS(G12C) can be monitored directly from the signals in a dose dependent manner in less than 60 min. The same effect was not seen with G12V.

Conclusions

ICD combined with the novel FRET-probe is a prominent option for TSAs, as no special equipment are needed. Isothermal concept can also potentially provide more understanding of how these proteins behave in vivo.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Academy of Finland and Otto A. Malm Foundation.

Disclosure

H. Härmä, K. Kopra: Financial Interests, Personal, Ownership Interest: QRET Technologies Oy. All other authors have declared no conflicts of interest.