RAS (rat sarcoma) family of oncogenes, KRAS, HRAS, NRAS, encode guanosine triphosphatase (GTPase) proteins play an important role in cell proliferation, differentiation, and regulation by acting as a regulatory switch that is turned on and off by the GTP and GDP molecules (See Figure 1). When mutated, oncogenes cause normal cells to become cancerous. In the rapidly evolving field of oncology, RAS mutations have emerged as pivotal biomarkers, driving advancements in targeted therapies and personalized treatment strategies.
Frequent RAS mutations associated with cancers:
- Pancreatic, lung, and colorectal cancers are highly driven by KRAS G12 mutations, the most prevalent of these being KRAS G12C, G12D, G12V mutations
- Melanoma is highly associated with NRAS Q61 mutations
- HRAS mutations represent around 7% of all RAS-driven cancers and are commonly found in thyroid and kidney cancers.
Eurofins Discovery offers robust RAS assays that include wild-type and clinically important drug target mutants of RAS for cancer therapeutic intervention. These assays are designed to:
- Identify therapeutics that affect RAS nucleotide exchange and assess selectivity of compound binding to SWITCH II pockets.
- Evaluate potency and decipher mechanism-of-action of compound hits from primary screens.
- Identify lead molecules through further investigations in cell-based, biophysical, translational, in vivo integrated solutions.
Figure 1. Overview of RAS pathways.
Assessing Nucleotide Exchange Activity of RAS Isoforms
Our RAS Human Variant LeadHunter® Panel covers the most common RAS variants (Table 1) performed via HTRF-based nucleotide exchange assay (NEA), which directly measures GDP to GTP exchange activity. This panel enables concurrent potency and selectivity assessment to rapidly identify direct and indirect RAS inhibitors. Each NEA-based RAS assay is robust and well-validated by clinical and tool references (Figure 2). The identified compounds can be orthogonally confirmed via KRAS-SOS1 protein-protein interaction (PPI) and biophysics platforms (e.g., SPR).
Figure 2. Demonstrative results by NEA-based RAS variant assays. A. KRAS G12D inhibitor MRTX-1133 showed > 100-fold stronger potency than BI-2865, a pan-KRAS inhibitor, in the KRAS G12D assay. B. Similar potencies were obtained between the above-mentioned two RAS inhibitors in the KRAS G12V assay. C. MRTX-1133 showed slightly left-shifted potency compared to BI-2865 in the KRAS G12R assay. AMG-510, a selective KRAS G12C inhibitor, exhibited no inhibition against these three KRAS G12 mutations. N.D.: Not detected.
Table 1. The selectivity and potency of reported KRAS and SOS1 inhibitors were successfully determined. AMG-510 and MRTX-1133 displayed selective and strong inhibition against KRAS G12C and KRAS G12D, respectively. BI-2865 displayed high potent activity across all KRAS variants compared to NRAS and HRAS. BI-3406, a SOS1 inhibitor, showed moderate inhibition against most RAS mutations. ND: Not detected at 100 μM.
Discovering RAS SWITCH-II Pocket Binders
KRAS drug discovery has been traditionally labelled a “challenging to drug” or “undruggable” target due to the belief that the target lacks pharmacologically targetable pockets. Recent advances in small molecule discovery targeting the switch II pocket have introduced new avenues to selectively target mutant variants of this enzyme.
Table 2. KRAS inhibitor binding selectivity against wild-type and mutant protein using Eurofins Discovery’s KRAS assays. MRTX-1133 is a selective inhibitor for KRAS mutants over wild-type.
Evaluating In vitro and In vivo efficacy of RAS Inhibitors
The drug sensitivity and efficacy can be seamlessly evaluated via translational 2D- and 3D-based cell proliferation assays against 60+ OncoPanel® RAS mutant cancer cell lines (Figure 3). Anticancer responses can be further examined via in vivo RAS mutant oncology models by our partner lab Pharmacology Discovery Services (PDS) with 10+ cell line-derived (CDX), patient-derived (PDX), and orthotopically implanted xenograft models harboring a variety of RAS variants (see Features).
Figure 3. OncoPanel RAS mutant cancer cell lines. A. Number of cancer cell lines bearing KRAS (40+), NRAS (18+), and HRAS (7+) mutations. B. Number of cancer cell lines bearing KRAS mutations including G12D (9+), G12V (10+), G12C (4+), and G13D (4+). The cell proliferation assays can be performed in 2D and 3D formats with flexible treatment designs (3-, 5-, 10-day) and multiple readouts (HCA and CTG).
Let Eurofins Discovery advance your RAS drug discovery program and help you discover the next druggable RAS inhibitors through our comprehensive RAS drug discovery solutions.
