RAS mutations drive many tumors, including pancreatic cancer, colorectal cancer, and lung cancer. However, RAS was long considered difficult to drug because of its shallow structural pockets and tight GTP binding. The emergence of RAS(ON) inhibitors, molecular glues, and PROTACs has shifted RAS intervention from signal blockade toward clearance of oncogenic proteins.

Keywords: RAS; KRAS; NRAS; HRAS; RAS(ON); small GTPase; E3 ligase; molecular glue; PROTAC; protein degradation

1 Why RAS Is an Important Tumor Target

1.1 RAS Is Not a Pathway Name but a Protein Target

RAS targets usually refer to Ras proteins encoded by KRAS, NRAS, and HRAS. Ras proteins belong to the small GTPase family and control signal output through switching between GDP-bound and GTP-bound states. In normal cells, RAS activation is stimulus-dependent and transient. In tumor cells, oncogenic mutations can keep RAS in a long-lasting GTP-bound active state, continuously driving cell proliferation, survival, metabolic adaptation, migration, and invasion. Therefore, RAS is both a molecular switch and a protein target that can be recognized and intervened by inhibitors, molecular glues, or PROTACs.

1.2 Differences Among KRAS, NRAS, and HRAS

KRAS, NRAS, and HRAS all belong to the RAS proto-oncogene family, but they should not be simply substituted for one another in experimental design or drug screening. KRAS encodes K-Ras protein and is currently the most central member in RAS-targeted drug development. NRAS encodes N-Ras protein and is often associated with melanoma and hematologic malignancies. HRAS, Harvey Rat Sarcoma Viral Oncogene Homolog, encodes H-Ras protein and is one of the classical RAS family members. It is more commonly used in research on lipidation, membrane localization, and HRAS-selective intervention.

1.3 RAS Structure Determines Its Targeting Difficulty

Ras proteins contain a GTP-binding domain, Switch I region, Switch II region, and C-terminal membrane localization-related region. The GTP-binding domain determines its small GTPase property, but RAS binds GTP/GDP tightly, making direct competition at the nucleotide-binding site highly difficult. Switch I and Switch II undergo conformational changes during GDP/GTP state transitions, determining the ability of RAS to bind effector proteins such as RAF, PI3K, and RalGDS. They are also important regions for RAS(ON) drug recognition. The C-terminal hypervariable region and CaaX motif participate in lipid modification and membrane localization, making RAS signaling dependent on specific membrane compartments and spatial conformations.

2 How RAS Mutations Sustain Oncogenic Signaling

2.1 GDP/GTP Molecular Switch

The signaling state of RAS is determined by the GDP/GTP cycle. The GDP-bound state is usually a low-activity state, while the GTP-bound state, namely the RAS(ON) state, can bind downstream effector proteins and initiate signal transduction. GEFs such as SOS1 promote GDP release and GTP binding, driving RAS into the ON state. GAPs such as NF1 promote GTP hydrolysis, returning RAS to the OFF state. After oncogenic mutations weaken GTP hydrolysis or GAP-mediated inactivation, RAS remains in the active conformation for a prolonged period.

2.2 G12, G13, and Q61 Mutations

Oncogenic RAS mutations are mainly concentrated at G12, G13, and Q61. G12 and G13 are located near the GTP-binding pocket and can affect GAP-mediated GTP hydrolysis. Q61 directly participates in the catalytic process of GTP hydrolysis. KRAS G12C, G12D, G12V, G13D, NRAS Q61, and other mutations differ in structural conformation, downstream dependency, drug-binding pockets, and resistance routes. They should not be generally described only as “RAS mutations.”

2.3 Downstream Signal Output

Active RAS mainly regulates tumor cell behavior through the RAF-MEK-ERK, PI3K-AKT, and RalGDS-Ral branches. The RAF-MEK-ERK pathway is closely related to proliferation and transcriptional output and is commonly evaluated using p-MEK, p-ERK, DUSP6, and other indicators. The PI3K-AKT pathway participates in survival, metabolism, and anti-apoptosis and is often assessed using p-AKT, p-mTOR, and cell viability indicators. Ral and Rho/Rac-related branches participate in vesicle trafficking, cytoskeleton regulation, migration, and invasion. A single p-ERK readout cannot cover all biological effects of RAS and cannot directly prove whether RAS has been degraded.

Figure 1. RTK-mediated RAS GDP/GTP cycle and major downstream signaling pathways

3 Why RAS Has Shifted from “Undruggable” to Targetable

3.1 Traditional Druggability Barriers

RAS was long considered difficult to drug mainly because its nucleotide-binding site is hard to compete with, the protein surface lacks deep pockets, the Switch regions are highly conformationally dynamic, and RAS function depends on membrane localization and effector protein interactions. Candidate molecules that can bind RAS in vitro may not necessarily access the correct conformation or effectively block signaling inside cells. Therefore, the key to RAS drug development has gradually shifted from “competing with GTP binding” to “recognizing mutant pockets, active conformations, and protein interaction interfaces.”

3.2 Mutation-Specific Inhibitors

Mutation-specific inhibitors use structural differences created by specific mutations to achieve selective binding. KRAS G12C inhibitors are representative of this direction, forming a covalent interaction with the mutant cysteine to restrict KRAS signal output. This strategy has strong selectivity and a clear mechanism, but its coverage is limited and it cannot be directly extended to all RAS mutation backgrounds such as G12D, G12V, and Q61. When evaluating this type of drug, mutant/wild-type selectivity, decreased RAS-effector protein interaction, and MAPK pathway inhibition should be confirmed, rather than only observing whether cell proliferation decreases.

3.3 RAS(ON) Active-Conformation Inhibitors

RAS(ON) inhibitors expand the recognition object from a single mutant pocket to the GTP-bound active conformation. The core of multi-selective RAS(ON) strategies such as daraxonrasib is to recognize active RAS and block the signaling connection between RAS and downstream effector proteins. The significance of this strategy is that it demonstrates the active RAS conformation itself can serve as an entry point for drug development. If a candidate only shows p-ERK reduction but does not prove GTP-state RAS selectivity or decreased RAS-effector protein interaction, it does not sufficiently support a RAS(ON)-targeting mechanism.

4 How E3 Ligases Advance RAS Intervention Toward Protein Degradation

4.1 Role of E3 Ligases in RAS Degradation

E3 ligases are not RAS family members and are not downstream pathways of RAS. They are substrate-selecting nodes in the ubiquitin-proteasome system. In RAS degradation research, RAS is the target protein to be cleared, while E3 ligases are responsible for introducing the target protein into the ubiquitination process. Small molecules binding RAS do not automatically cause degradation. Protein degradation may occur only when RAS and an E3 ligase form a spatial relationship favorable for ubiquitin transfer.

4.2 PROTAC and Molecular Glue Degradation Mechanisms

PROTACs use bifunctional molecules to connect a RAS-binding end with an E3 ligase ligand, enabling RAS to form a ternary complex with CRBN, VHL, or other E3 systems. Molecular glue degraders induce or stabilize protein interactions, allowing E3 ligases to recognize new substrates. Both depend on E3 ligases, but their modes of action are different. More importantly, molecular glue-like inhibitors and molecular glue degraders should not be confused. The former can block signaling but may not necessarily induce protein degradation.

(1) RAS-binding end

This determines whether the candidate recognizes KRAS, NRAS, HRAS, a specific mutant, or the RAS(ON) conformation.

(2) Linker or induced interface

This determines the spatial distance, orientation, and flexibility between RAS and the E3 ligase, directly affecting whether ubiquitin transfer is favorable.

(3) E3 ligase system

This determines whether the candidate recruits CRBN, VHL, or another E3 system, and affects degradation efficiency, cell-context dependence, and off-target risk.

4.3 Selection of CRBN and VHL

CRBN and VHL are commonly used E3 ligase systems in targeted protein degradation research. They differ in ligand structure, complex conformation, substrate spectrum, and cellular expression background. Therefore, after changing the E3 system, changes in degradation efficiency may not necessarily come from the RAS-binding end itself, but may instead result from ternary complex orientation, accessibility of ubiquitination sites, or differences in E3 expression. RAS degrader development cannot only optimize target affinity; E3 dependence and proteasome dependence must also be verified.

5 How to Distinguish RAS Inhibition from RAS Degradation

5.1 Functional Inhibition Does Not Equal Protein Degradation

The core of RAS functional inhibition is to reduce RAS signal output, while total RAS protein can remain unchanged. Mutation-specific inhibitors and RAS(ON) inhibitors can reduce RAS-RAF interaction, p-MEK, p-ERK, and cell proliferation, but this does not mean that RAS has been cleared. RAS-targeted degradation must show reduced total KRAS, NRAS, or HRAS levels and further demonstrate increased ubiquitination, E3 dependence, and proteasome dependence.

5.2 Key Experimental Evaluation Points

RAS inhibitor evaluation should cover target binding, GTP/GDP conformational selectivity, RAS-effector protein interaction, and downstream pathway output. RAS degrader evaluation must additionally include E3 recruitment, RAS ubiquitination, total RAS reduction, proteasome dependence, and E3 dependence validation. Observing only reduced cell proliferation or p-ERK reduction cannot prove that targeted degradation has occurred.

6 RAS-Targeting Resistance and Combined Intervention

6.1 Inhibitor Resistance

Resistance to RAS inhibitors often arises from target alteration and pathway compensation. At the target level, mechanisms include secondary mutations, altered drug-binding pockets, and changes in RAS conformation. At the pathway level, mechanisms include RTK feedback activation, increased SHP2/SOS1 dependence, MAPK pathway restoration, and PI3K-AKT compensation. If the original target is inhibited but p-ERK or p-AKT recovers, bypass RTKs, SHP2/SOS1, and downstream pathway mutations should be examined first.

6.2 Degrader Resistance

RAS degraders may also be limited by the proteostasis system. Reduced CRBN or VHL expression, abnormal E3 complex function, altered proteasome activity, changes in the RAS-binding epitope, or altered intracellular localization may all weaken degradation efficiency. This type of resistance does not necessarily present as a failure of the candidate to bind RAS. It may also present as failed E3 recruitment, insufficient ubiquitination, or reduced proteasome processing capacity.

6.3 Directions for Combined Intervention

Combined intervention should select nodes according to the source of resistance. When upstream feedback is obvious, intervention can focus on RTK, SHP2, or SOS1. When downstream MAPK or PI3K-AKT signaling recovers, MEK, ERK, PI3K, or AKT-related nodes should be evaluated. When degradation efficiency is insufficient, E3 ligase expression, linker structure, ternary complex stability, and proteasome dependence need to be reassessed. Combination strategies should not rely only on reduced cell viability, but should determine whether the resistance-driving node is truly suppressed.

7 Products Related to RAS-Targeted Research

The key focus of RAS-targeted research has expanded from “whether downstream signaling can be inhibited” to “whether active conformations can be recognized and oncogenic proteins can be cleared.” RAS GTPase determines target status and conformational recognition, while E3 ligases determine whether ubiquitination and proteasomal degradation occur. Establishing experimental systems around these two core tool categories is fundamental for understanding the mechanisms of RAS(ON) inhibitors, molecular glues, and PROTACs.

For more related articles, please see below:

[1] Three Key Components of PROTAC Design: E3 Ligase Ligands, Linkers, and Target Protein Ligands (with an Aladdin Reagent Selection Guide)

[2] Polyethylene Glycol (PEG) and Its Derivatives: End-Group Design and Applications in PROTACs, ADCs, and Medical Device

[3] Targeted protein degradation technology: proprietary drugs are not difficult, drug resistance is no longer

[4] KRAS Inhibitors — The "Undruggable Target"? A Thing of the Past