A breakthrough in targeting RNF43 offers new hope for inhibiting Wnt signaling in cancer treatment
Within every cell in our body, a delicate molecular dance determines whether cells grow, divide, or remain quiet. Among the most crucial orchestrators of this dance is the Wnt signaling pathway—an ancient communication system that guides embryonic development and maintains tissue health throughout our lives. When this pathway functions properly, it keeps tissues like our intestinal lining constantly renewing. But when it goes awry, it can become a powerful driver of cancer progression.
For years, scientists have recognized that a protein called RNF43 acts as a critical brake on the Wnt signaling pathway. This transmembrane E3 ubiquitin ligase keeps Wnt signaling in check by marking its receptors for destruction.
Unfortunately, cancer often disables this brake system—inactivating mutations of RNF43 frequently appear in colon, pancreatic, stomach, and endometrial cancers, removing this crucial constraint on cell growth and opening the door to tumor development.
Despite RNF43's importance in cancer biology, researchers have struggled to study it directly due to a fundamental limitation: the absence of selective molecular tools capable of detecting or manipulating endogenous RNF43 in its natural cellular environment. Without precise instruments to target RNF43 specifically, our understanding of its functions has remained limited. Now, a breakthrough study published in ACS Central Science has changed this landscape dramatically, introducing a powerful new peptide tool that promises to unlock RNF43's secrets and potentially pave the way for innovative cancer therapies 1 5 9 .
To appreciate this scientific advance, we first need to understand how Wnt signaling works and where RNF43 fits into this complex system.
The Wnt/β-catenin pathway operates like a sophisticated thermostat for cell growth. When Wnt proteins (the "on" signal) bind to Frizzled receptors and LRP-5/6 co-receptors on the cell surface, they trigger a cascade that ultimately allows β-catenin to accumulate and travel to the cell nucleus, where it activates genes responsible for cell proliferation and survival 2 .
In the absence of Wnt signals (the "off" state), a destruction complex that includes Axin, APC, GSK-3β, and CK1α captures β-catenin and marks it for proteasomal degradation. This ensures that cell division occurs only when appropriate signals are present 2 .
RNF43 and its relative ZNRF3 serve as critical negative regulators in this system. These transmembrane E3 ubiquitin ligases act like molecular brakes by ubiquitinating Frizzled receptors, sending them for degradation and thus reducing the cell's ability to respond to Wnt signals 1 6 . This prevents excessive Wnt pathway activation.
Complicating this picture are R-spondins—proteins that powerfully enhance Wnt signaling. They work by binding to LGR4/5/6 receptors and RNF43/ZNRF3, effectively clearing these E3 ligases from the cell membrane and preventing them from degrading Frizzled receptors. With the brakes disengaged, Wnt signaling can proceed unimpeded 2 9 .
Interactive visualization of Wnt signaling pathway and RNF43's regulatory role
In healthy tissues, this intricate balance between activation and inhibition maintains proper tissue homeostasis. But in cancer, this system is often hijacked.
The significance of RNF43 in cancer became evident when genomic studies revealed frequent inactivating mutations across multiple cancer types. Interestingly, different mutations have different functional consequences:
Truncating mutations in the RING domain (e.g., R132X, R145X) completely eliminate the enzyme's activity 3 .
Phosphorylation-site mutations prevent RNF43 activation by kinases like CK1, abolishing its ability to regulate Frizzled receptors 6 .
The most common mutation G659Vfs*41 does not impair RNF43's function but appears frequently in tumors with defective DNA mismatch repair systems 3 .
This complex mutational landscape highlighted the need for precise tools to study different RNF43 variants and their roles in specific cancer contexts. The development of selective RNF43 binders represents a crucial step toward achieving this precision.
The research team at Genentech employed an innovative approach to target RNF43: disulfide-constrained peptides (DCPs).
Unlike small molecules that may lack specificity or antibodies that are too large for certain applications, peptides offer an ideal middle ground. They can be engineered for high affinity and specificity while remaining small enough to access challenging cellular targets. The disulfide bonds create a rigid, stable structure that can withstand the harsh environment inside cells and tissues, making them perfect candidates for therapeutic development 9 .
The researchers used a powerful technique called phage display, which allows them to screen vast libraries of potential peptide binders. They created highly diverse DCP libraries based on 13 different natural peptide scaffolds, each with variations in the loop regions that determine target recognition 9 .
By "panning" these libraries against the extracellular domain of RNF43, they could fish out peptides that showed any binding ability. The initial screening identified a promising candidate called GUR-1, derived from the gurmarin scaffold—a natural peptide originally isolated from the Indian plant Gymnema sylvestre 9 .
The use of disulfide-constrained peptides modeled after natural cystine-knot peptides provided exceptional stability and diverse functionality, overcoming limitations of previous approaches.
The researchers employed a stepwise affinity maturation process to transform their initial, modest-affinity hit into a powerful molecular tool 9 :
The team screened their DCP phage libraries against the extracellular domain of human RNF43, identifying GUR-1 as a starting point with approximately 1 μM affinity.
Using "soft randomization" of loop 4 (the same loop modified in GUR-1), they created a new library that yielded GUR-1.6, which showed a 10-fold improvement in binding affinity.
The researchers then constructed a larger library with randomized loops 1, 2, and 4 simultaneously. From this library, they identified GUR-1.6.12, which contained changes in loops 1 and 2.
By combining beneficial mutations from previous rounds, the team created GUR-1.6.12.2—their ultimate lead compound with single-digit nanomolar affinity for RNF43.
Throughout this process, the researchers used surface plasmon resonance (SPR) to precisely measure binding affinity and specificity, ensuring that each iteration represented a genuine improvement.
The team conducted extensive characterization of their lead peptide:
| Peptide Version | Key Structural Features | Binding Affinity (KD) | Key Improvements |
|---|---|---|---|
| GUR-1 (initial hit) | Extended loop 4 compared to wild-type gurmarin | ~1 μM | Initial binding activity |
| GUR-1.6 | Three residue changes in loop 4 | ~100 nM | 10-fold affinity improvement |
| GUR-1.6.12 | Additional changes in loops 1 and 2 | ~10-100 nM | Enhanced specificity |
| GUR-1.6.12.2 (final) | Combined beneficial mutations | Single-digit nM | Optimal affinity and specificity |
| Application | Method Used | Key Finding |
|---|---|---|
| Binding affinity measurement | Surface plasmon resonance (SPR) | Single-digit nanomolar affinity |
| Structural characterization | NMR spectroscopy and computational modeling | Defined structure and binding interface |
| Cellular localization | Immunofluorescence in mouse intestines | Detected RNF43 in intestinal crypts |
| Specificity testing | Binding assays against related proteins | No cross-reactivity with ZNRF3 |
The researchers confirmed that GUR-1.6.12.2 showed no cross-reactivity with ZNRF3, despite the high similarity between these two E3 ligases. This exceptional specificity enables researchers to study RNF43 functions without confounding effects from its relative 9 .
| Reagent/Tool | Function/Application | Significance in RNF43 Research |
|---|---|---|
| GUR-1.6.12.2 peptide | Selective RNF43 binding | Enables detection and manipulation of endogenous RNF43 |
| Biotinylated GUR-1.6.12.2 | Immunofluorescence and detection | Allows visualization of RNF43 localization in tissues |
| Hexavalent RNF43-DCP | Wnt signaling inhibition | Blocks R-spondin binding to suppress Wnt signaling |
| Phage display libraries | Peptide discovery platform | Facilitated identification of initial RNF43 binders |
| RNF43 extracellular domain | Binding assays | Served as target for peptide screening and characterization |
| Surface plasmon resonance | Affinity measurements | Quantified binding strength and specificity of peptides |
The most promising application of these RNF43-binding peptides lies in their potential to develop new cancer treatments. The researchers created a hexavalent version of GUR-1.6.12.2—essentially connecting six copies of the peptide together—which significantly enhanced its ability to remain bound to RNF43 1 9 .
This multivalent peptide acts as a functional inhibitor of Wnt signaling by competing with R-spondin for binding to RNF43. By blocking this interaction, the peptide prevents R-spondin from clearing RNF43 from the membrane, allowing RNF43 to continue degrading Frizzled receptors and thus suppressing Wnt signaling 1 5 9 .
This approach is particularly relevant for cancers that depend on sustained Wnt signaling for growth but have lost their natural braking mechanisms through RNF43 mutations. By restoring control over Wnt signaling, these peptide binders offer a potential pathway to suppress tumor growth in cancers characterized by dysregulated Wnt activity.
Six copies of GUR-1.6.12.2 connected together create a multivalent binder with enhanced avidity and functional potency.
The development of selective peptide binders for RNF43 represents more than just a technical achievement—it opens the door to fundamentally new understanding and potential interventions in cancer biology. These molecular tools allow scientists to visualize RNF43 in its native environment, manipulate its activity with unprecedented precision, and dissect its unique functions separate from its relative ZNRF3.
The specificity of GUR-1.6.12.2 for RNF43, without cross-reactivity to ZNRF3, suggests a path toward more selective interventions that could modulate Wnt signaling in diseased tissues while sparing healthy ones.
As research with these tools advances, we can anticipate deeper insights into how RNF43 functions in different cancer contexts and how we might design smarter therapeutics that restore the natural brakes on cancer growth.
The molecular arms race inside our cells continues, but now scientists have a powerful new weapon in their arsenal.
This article is based on research findings from "Selective and Potent Peptide Binders of RNF43 for Wnt Signaling Inhibition" published in ACS Central Science (2025).