In a study published in the journal Molecular Diversity, Kader Sahin of Bahçeşehir University and Bandırma Onyedi Eylül University, Ajda Coker of Marmara University, and Serdar Durdağı of Bahçeşehir University describe a structure-based virtual screening campaign that combed through FDA-approved drugs in search of molecules capable of disrupting the interaction between β-catenin and T-cell factor 4, or Tcf4. This protein–protein interaction is the final, decisive step in Wnt signaling: when β-catenin accumulates in the cell nucleus and binds Tcf4, the complex switches on a battery of genes that drive tumor growth, survival, metastasis, and drug resistance. Block that binding, the logic goes, and the entire oncogenic program collapses.

The catch has always been the shape of the target. The β-catenin/Tcf4 interface sprawls across roughly 4,800 square angstroms, a vast and shallow surface with few of the deep, well-defined pockets that medicinal chemists traditionally exploit. For years, interfaces like this one were written off as undruggable. Yet structural biology has gradually revealed that even sprawling protein–protein interfaces concentrate their binding energy in a handful of so-called hot spots, short stretches of residues that contribute disproportionately to the affinity between the two partners. If a small molecule can be steered into those hot spots, the reasoning goes, it might achieve with a compact drug what would otherwise require a much larger molecule.

That is precisely the strategy the researchers pursued. Rather than screening synthetic chemical libraries filled with untested compounds, they turned to drug repurposing, focusing on medicines that have already passed safety scrutiny and are in clinical use. Repurposing offers a practical shortcut: known pharmacology, established safety profiles, and in many cases existing manufacturing and distribution infrastructure. The team first mapped the critical hot spot residues of β-catenin that engage Tcf4, then used molecular docking to position each candidate drug within the interface, asking which approved molecules could physically occupy and destabilize the contact points that hold the complex together.

The computational pipeline did not stop at docking. To separate plausible binders from artifacts, the researchers subjected the top candidates to physics-based molecular dynamics simulations, watching in atomic detail how each ligand behaved over time when bound to β-catenin. They then calculated binding free energies using the MM/GBSA method, a hybrid approach that combines molecular mechanics energies with implicit solvent models to estimate how strongly a ligand clings to its target. This layered filtering, docking followed by simulation followed by energetic scoring, is designed to weed out compounds that look good in a single static snapshot but fall apart under the thermal motion of a real cellular environment.

Three candidates emerged from the screen with stable binding to the critical hot spot residues of β-catenin, specifically asparagine 430, lysine 435, histidine 470, arginine 474, and lysine 508. They were Leucovorin, also known as folinic acid, a vitamin derivative routinely used to enhance chemotherapy; Carbenicillin, a penicillin-class antibiotic; and Ceforanide, a second-generation cephalosporin. All three are approved, well-characterized medicines, which means that if any of them proves effective against Wnt-driven cancers, the path from bench to bedside could be considerably shorter than for a brand-new chemical entity.

Computational predictions, however convincing, mean little until they are tested in living cells. The team moved their three hits into the laboratory, using HT-29 colorectal cancer cells, a widely studied cell line in which Wnt signaling is aberrantly active. The cells were treated with each compound and assessed for viability and for their ability to form colonies, a measure of long-term proliferative capacity that often correlates with tumorigenic potential. The results were striking: Carbenicillin and Ceforanide reduced cell viability and impaired colony formation, exhibiting superior anti-proliferative activity compared with the other candidates, including Leucovorin.

The convergence between the computational and experimental arms of the study is what gives the findings their weight. The two antibiotics that performed best in cell assays were the same molecules whose simulated binding poses anchored them most securely to the β-catenin hot spots. This consistency suggests that the anti-proliferative effects observed in HT-29 cells may indeed arise from disruption of the β-catenin/Tcf4 interaction, although the researchers are careful to frame the work as a proof of concept rather than a finished therapy. Further studies will be needed to confirm the mechanism directly, to characterize selectivity for cancer cells over healthy tissue, and to establish whether the compounds can achieve therapeutic concentrations in tumors.

Even so, the implications are considerable. Wnt/β-catenin signaling is implicated not only in colorectal cancer but in a broad family of malignancies, including hepatocellular carcinoma and other Wnt-driven tumors, and it also maintains cancer stem cell populations that fuel relapse and metastasis. A validated small-molecule inhibitor of the β-catenin/Tcf4 interaction would be a genuinely new class of anticancer agent, one that attacks the transcriptional engine of the tumor rather than a single downstream symptom. Prior efforts, including peptide mimetics and dedicated small molecules developed in academic labs, have struggled to reach the clinic, which makes the repurposing angle particularly attractive: these are drugs whose toxicity, metabolism, and drug interactions are already documented.

The study also offers a template for tackling other supposedly undruggable targets. By combining hot spot analysis, docking against a library of approved drugs, rigorous molecular dynamics validation, and energetic scoring, and only then moving to cell-based experiments, the researchers demonstrated a workflow that is both computationally disciplined and experimentally grounded. As the authors conclude, their findings provide compelling evidence that targeting the β-catenin/Tcf4 interaction with small molecules is feasible, and that Carbenicillin and Ceforanide may serve as promising repurposed candidates for therapeutic intervention in Wnt-driven malignancies. For patients with colorectal cancer, whose treatment options too often narrow as the disease progresses, the idea that answers might be hiding in plain sight among existing medicines is a hopeful one, and this study shows exactly how such hidden answers can be systematically found.

Subject of Research: Virtual drug repurposing to discover β-catenin/Tcf4 interaction inhibitors for colorectal cancer treatment

Article Title: Hot spot-driven discovery of β-catenin/Tcf4 interaction inhibitors via virtual drug repurposing study and experimental validation in colorectal cancer cells

Article References: Sahin, K., Coker, A., & Durdağı, S. (2026). Hot spot-driven discovery of β-catenin/Tcf4 interaction inhibitors via virtual drug repurposing study and experimental validation in colorectal cancer cells. Molecular Diversity. https://doi.org/10.1007/s11030-026-11720-1

Image Credits: AI Generated

DOI: 10.1007/s11030-026-11720-1

Keywords: β-catenin, Tcf4, Wnt signaling, colorectal cancer, drug repurposing, virtual screening, molecular docking, molecular dynamics, MM/GBSA, Carbenicillin, Ceforanide, protein-protein interaction inhibitors

At the forefront, the review article titled “From Large to Small Cytokine Receptor Antagonists” offers an insightful exploration into the transition from biological drugs to small molecule modulators targeting cytokine receptors. By examining the lessons learned from pivotal cytokines such as TNFα and IL-17, the article unpacks the challenges and opportunities in designing antagonists that can modulate immune signaling pathways effectively. It also discusses the future potential of novel therapeutic modalities, including extracellular degraders and oral peptides, which could revolutionize treatments for previously elusive cytokine targets like TSLP and TL1A.

In the realm of screening technologies, one of the original research pieces introduces a novel application of the enzyme-linked immunosorbent assay (ELISA) adapted for discovering protein-protein interaction inhibitors. Utilizing the spirochete flagellar hook as a test case, the research demonstrates how this assay effectively identifies compounds that disrupt lysinoalanine crosslinking between protein subunits. Screening a substantial chemical library of approximately 700 molecules, the study not only reaffirms the inhibitory effects of known compounds but also uncovers promising new candidates, including honokiol and zafirlukast. This innovative assay platform promises to accelerate the identification of small molecules capable of targeting intricate protein interfaces, which have historically been challenging to drug.

Another pioneering work addresses throughput bottlenecks in cellular thermal shift assays (CETSA), a technique used to confirm drug-target engagement in biological contexts. Two remarkable technological advancements are presented: an isothermal 1536-well ultra-high throughput screening (uHTS) platform featuring controlled thermal ramp-up combined with luminescence detection, and a Gradient Peltier Device that facilitates comprehensive melt curve analysis in a single assay plate. These technological strides overcome longstanding limitations of scale and resolution in thermal shift assays. Validation against fluorescence polarization data using the androgen receptor as a benchmark affirms the robustness and sensitivity of these improvements, underscoring their suitability for large-scale screening campaigns.

Further advancing drug-target engagement quantification, the MICRO-TAG method emerges as a novel fluorescence-based split-RNase S complementation assay. Unlike traditional thermal shift methods that rely on a single melting temperature, MICRO-TAG quantifies binding across programmable temperature series within living cells. Tested on key oncogenic and cellular proteins including MAPK1, KRAS, and UBE2N, this approach offers a sensitive, scalable, and physiologically relevant alternative to in vitro biophysical assays. Such advances could substantially improve early-stage drug discovery by providing accurate engagement data directly in cell-based contexts.

Volume 38 also prominently features a Special Issue editorial and collection focusing on “Protocols in 3D Biology: Technologies and Methodologies Reshaping 3D Cell Culture.” This thematic cluster presents an integrated vision where bioprinting, automated bioreactors, and artificial intelligence-driven analytical tools coalesce to push 3D cell culture beyond experimental setups into practical, high-throughput New Approach Methodologies (NAMs). These cutting-edge platforms have demonstrated efficacy in modeling complex human tissues and disease states — ranging from patient-derived cancer organoids to engineered skeletal muscle spheroids and organotypic barrier models. The synergistic deployment of these approaches heralds a new era of predictive, human-relevant platforms for pharmaceutical development, promising to reduce reliance on traditional animal models and improve translational outcomes.

The integration of enzyme complementation techniques, sophisticated thermal profiling, and scalable screening platforms within living cellular environments represents a notable advance in drug discovery sciences. These innovations collectively enhance the fidelity, throughput, and applicability of target engagement assessments, crucial parameters for optimizing lead compounds during the drug development pipeline. By embracing these technologies, scientists can better characterize molecular interactions and pharmacodynamics within physiologically relevant systems, accelerating the path from hit identification to clinical candidates.

Through its rigorous editorial standards and focus on translational impact, SLAS Discovery continues to cement its role as a pivotal journal at the intersection of advanced technology and biology in drug discovery. Volume 38 represents a vibrant testament to the journal’s commitment to publishing high-impact research that bridges the gap between fundamental biological insights and applied therapeutic innovation. Researchers, academicians, and industry professionals alike will find the curated content invaluable for navigating the evolving landscape of drug discovery tools and methodologies.

This volume not only equips researchers with new experimental tools but also illustrates the importance of adopting automated, integrated workflows in modern biological research. The application of large-scale screening approaches combined with precise quantification methods underscores a broader trend towards data-rich, high-throughput experimentation. These capabilities are particularly transformative in the context of 3D cell cultures, where replicating physiological conditions poses unique challenges that conventional two-dimensional cultures cannot address.

SLAS Discovery’s focus on enveloping emerging technologies such as AI-driven analysis within biological protocols reflects an acute understanding of the future trajectory of drug discovery. Automation, high-content imaging, and machine learning algorithms are poised to revolutionize data analytics, enabling unprecedented resolution and predictive modeling in complex biological systems. The special issue protocols illuminate these trends, demonstrating practical platforms that incorporate real-time data acquisition, temperature regulation, and biophysical assessment for a mechanistic understanding of drug action.

As 3D cell culture platforms become increasingly integrated with scalable automation and analytical tools, their utility in personalized medicine and disease modeling expands. The research featured in this volume showcases patient-derived organoid systems and tissue models that recapitulate disease heterogeneity and microenvironmental factors, fostering improved drug screening and therapeutic development workflows. By embracing these sophisticated technologies, the field moves closer to realizing more human-relevant testing platforms that can replace or complement traditional animal models, thereby enhancing ethical standards and translational fidelity.

In summary, SLAS Discovery Volume 38 provides a vibrant snapshot of state-of-the-art innovations pushing the frontiers in drug discovery and 3D biology. From novel assay platforms capable of tackling protein-protein interactions, to advanced thermal profiling techniques and the systematic deployment of automation in 3D cell culture, this issue exemplifies the dynamic, interdisciplinary nature of contemporary biomedical research. It sets a benchmark for future studies striving to unravel molecular complexity within biologically relevant contexts and underscores the critical role of technology in shaping next-generation therapeutic discovery.

Subject of Research: Advances in drug discovery technologies, 3D cell culture methodologies, and cellular assays for protein-target engagement.

Article Title: SLAS Discovery Volume 38: Protocols and Innovations Transforming 3D Biology and Drug Discovery.

News Publication Date: 2024

Web References:

• SLAS Discovery Journal
• Review – From Large to Small Cytokine Receptor Antagonists
• ELISA for Protein-Protein Interaction Inhibitors
• Streamlining Cellular Thermal Shift Assay
• MICRO-TAG Enzyme Complementation

Image Credits: SLAS Publishing

Keywords: Drug discovery, High-throughput screening, Protein-protein interaction inhibitors, Cellular thermal shift assay, 3D cell culture, Bioprinting, Automation, Artificial intelligence, Organotypic models, Small molecule therapeutics.