KRAS Inhibitors & Degraders: Development & Validation of Biochemical & Cell-based Assays for Inhibitor Discovery & Development
Introduction
KRAS is an essential small GTPase that regulates biochemical signaling for cellular growth, proliferation, differentiation, and migration. Its function involves switching between a guanosine triphosphate (GTP)-bound “on” state and a guanosine diphosphate (GDP)-bound “off” state. Mutations in KRAS are among the most common oncogenic drivers in human cancers, particularly in lung, colorectal, and pancreatic cancers. These mutations, frequently occurring at codons G12, G13, and Q61, bias the protein toward the hyperactive “on” confirmation, driving aberrant cellular signaling and tumor formation.
For decades, KRAS was considered “undruggable”. However, recent clinical triumphs with the development of KRAS(G12C) small molecule inhibitors, such as MRTX849 (adagrasib) and AMG510 (sotorasib), have sparked further research into developing KRAS inhibitors. In addition, the manipulation of the ubiquitin-proteasome system has led to the development of targeted protein degraders (PROTACs), such as LC2 (a mutant-selective KRAS degrader), for cancer therapeutics.
Given the advances in targeting KRAS mutants, there is a need for new, robust, and high-throughput assay platforms to characterize next-generation inhibitors and degraders, particularly those that improve on potency, selectivity, and the ability to evade resistance mechanisms.
Scientists from Eurofins DiscoverX and Eurofins Discovery recently published an ACS publication describing the creation of complementary biochemical and cell-based assay screening platforms and characterized KRAS-targeting compounds across various mutants, including Wild-Type (WT), G12C, G12D, and G12V. This article reviews the publication focusing on the development and results of the assays.
Biochemical and Cell-based Assays Development & Results
Biochemical Competition Binding Assay
A novel high-throughput assay was developed to quantitatively measure the thermodynamic dissociation constant (KD) of small molecule inhibitors. This assay format features a qPCR-amplicon tagging technology, allowing for the sensitive and accurate quantification of KRAS binding. This format additionally offers several advantages over traditional methods like surface plasmon resonance (SPR) that can be limited by the tight-binding limit for high-affinity ligands. Table 1 shows dissociation constants measured for several KRAS inhibitors via the competitive binding assay.
| Protein | Compound Name | Average KD (nM) | Selectivity Profile |
|---|---|---|---|
| KRAS(G12D) | MRTX1133 | 0.40 ± 0.11 | High selectivity for G12D. This measurement (400 pM) resolves previous challenges with determining this ultra-high affinity by standard methods |
| KRAS(G12C) | MRTX849 | 9.59 ± 2.09 | Highly selective for G12C, with no binding detected to WT, G12D, or G12V up to 20μM |
| KRAS(G12C) | AMG510 | 220 ± 47 | Highly selective for G12C, with no binding detected to WT, G12D, or G12V up to 20μM |
Table 1. Thermodynamic dissociation constants measured for KRAS inhibitors targeting the switch II binding pocket.
The high selectivity for KRAS mutants G12D and G12C were observed, particularly for the G12C covalent switch II pocket-binding inhibitors MRTX849 and AMG510. The switch II pocket is a dynamic groove formed between the switch II region and the alpha 3 helix of the KRAS protein. This pocket is crucial for KRAS signaling and is the site for inhibitors to bind and prevent KRAS from interacting with downstream effectors.
Biochemical Activity Assay (Nucleotide Exchange Assay – NEA)
An established functional assay was used as a reference to determine the effect of compounds on KRAS’s nucleotide exchange activity. The assay confirmed the affinity rank order profile for inhibitor compounds, mainly the MRTX849 and AMG510 from biochemical studies.
These assays helped provide quantitative KD values for high-affinity ligands and the characterization of three key clinical-stage compounds: the G12D-selective inhibitor MRTX1133, and the G12C-selective inhibitors MRTX849 and AMG510.
Cell-based Assays for Targeted Protein Degradation & Compound Target Engagement
Cell lines were developed using the Enzyme Fragment Complementation (EFC) technology primarily to ensure that the targets are expressed at physiologically relevant (endogenous) levels for accurate potency assessment for protein degraders. Using CRISPR technology, an enzyme donor (ED) fragment of β-galactosidase was knocked into the endogenous KRAS locus to create an ED-target fusion protein. The cell lines created were KRAS(WT), KRAS(G12C), and KRAS(G12D) and were used to perform targeted protein degradation and compound target engagement assays.
Figure 1. Schematic showing a CRISPR-Cas9-mediated knock-in and the protein degradation assay using EFC. A. The KRAS cell lines were generated by introducing a ribonucleoprotein complex containing gRNA and Cas9 protein along with a dsDNA donor comprised of the ED (an enzyme donor (ED) fragment of β-galactosidase) tag flanked by homologous recombination sequences into cells. Events leading to the in-frame knock-in needed to generate the ED-target fusion proteins expressed at endogenous levels in a disease-relevant cell model are not shown. B. ED-target fusion proteins expressed at high or medium levels (basal EFC signal) are brought into proximity with an E3 ligase via a bifunctional molecule (such as a PROTAC). Subsequently, the target protein is ubiquitinated and degraded by the proteasome. Upon addition of EA (the complementary enzyme acceptor (EA) fragment of β-galactosidase), chemiluminescent substrate and detection reagents, loss of EFC signal is recorded.
Targeted Protein Degradation Assay (SPRINTer)
To assess the effect of degraders on endogenous KRAS protein levels, the novel, homogeneous SPRINTer™ cell-based targeted protein degradation assay was performed using the EFC cell lines. The LC2 PROTAC, a KRAS(G12C)-specific degrader consisting of an MRTX849 warhead linked to an E3 ligase ligand was tested. Results demonstrated selectivity confirmation wherein the assay successfully demonstrated that LC2 selectively induced a loss of signal (protein degradation) in the KRAS(G12C) cells (DC50 of 1.9 μM and Dmax of 69%), while showing no significant degradation in KRAS(WT) and minimal degradation in KRAS(G12D) cells. These results align with the published selectivity data of LC2.
Compound Target Engagement Assay (InCELL Pulse)
The EFC cell lines were also adapted for the InCELL Pulse™ Compound Target Engagement assay to interrogate the binding of inhibitors to the KRAS mutants within the native cellular environments. Results demonstrated inhibitor selectivity to the KRAS(WT) and mutant KRAS proteins. MRTX849 and AMG510 showed high affinity to the KRAS(G12C) protein, while MRTX1133 appears to have high selectivity for the KRAS(G12D) protein and some interactions with the KRAS(G12C) and KRAS(WT) proteins.
Conclusion and Future Significance
The comprehensive assay suite described here, and in more detail in the ACS publication, includes the development of novel biochemical binding, endogenous expression cell-based degradation, and target engagement assays for the pharmacological characterization of KRAS inhibitors.
By allowing for the accurate and quantitative determination of binding affinity (KD) for high-affinity ligands, functional inhibition (IC50), and degradation potency (DC50) in a high-throughput format, this suite of assays is poised to support the discovery and optimization of next-generation KRAS inhibitors and degraders. These assays are crucial for developing more tailored and effective therapeutic strategies that can address resistance mechanisms and target KRAS mutants beyond G12C and G12D.
Related Resources
- Medhanie Kidane, Rene M. Hoffman, Jennifer K. Wolfe-Demarco, Ting-Yu Huang, Chi-Ling Teng, Saheli Samanta, Luis M. Gonzalez Lira, Jennifer Lin-Jones, Gabriel Pallares, Jane E. Lamerdin, Nicole B. Servant, Chun-Yao Lee, Chao-Tsung Yang, and Jean A. Bernatchez. Suite of Biochemical and Cell-Based Assays for the Characterization of Kirsten Rat Sarcoma (KRAS) Inhibitors and Degraders. ACS Pharmacology & Translational Science, 2024 7 (12), 3921-3934
- SPRINTer Cell-based Assays for Protein Degradation: discoverx.com/applications/targeted-protein-degradation
- InCELL Cell-based Compound Target Engagement Assays: discoverx.com/applications/target-engagement
- SPRINTer KRAS Protein Turnover Cell Line Assay Kits