The research, led by E. Rosette Celsiya Mary and C. Sudandiradoss from Vellore Institute of Technology, systematically compared the binding resilience of first-generation IMiDs against next-generation CELMoDs—including iberdomide, mezigdomide, CC-885, CC-122, and CC-3060—across a panel of structurally prioritized deleterious CRBN variants. The team began by retrieving 409 missense single nucleotide polymorphisms (SNPs) of CRBN from the NCBI dbSNP database and then deployed a battery of functional prediction tools, including PolyPhen-2, PANTHER, MutationTaster, and SNPs & GO, alongside structural stability predictors DynaMut2 and DUET. From this comprehensive screen, six variants located within the CRBN Ubiquitin Ligase Targeting (CULT) domain—residues 318 to 426—emerged as particularly damaging: G344R, N351T, H353R, H357Q, S379I, W380R, and P382R. These residues sit squarely within the thalidomide-binding pocket, the very region that IMiDs and CELMoDs must engage to recruit CRBN into a functional ternary complex with the target protein.

What makes these findings clinically resonant is that several of the identified variants overlap with mutations experimentally documented in IMiD-treated patients. The P382R variant, for instance, has been reported in an IMiD-refractory or relapsed patient, while W380R sits within the most significant thalidomide-binding pocket. Other experimentally characterized variants such as C326Y, associated with pomalidomide refraction, and A347V, linked to lenalidomide resistance, further underscore the vulnerability of this domain. The researchers also noted that G344R was the only high-scoring variant found in the ClinVar database, flagged as a variant of uncertain significance—a detail that reinforces its potential clinical relevance even as most other high-impact variants remain absent from clinical genomic repositories.

To interrogate how these mutations affect ligand binding, the team constructed a homology model of CRBN using MODELLER with the UCSF Chimera 1.19 interface, anchored on a high-resolution crystal structure (PDB ID: 7BQU, resolved at 1.9 Å) and refined against an AlphaFold 2-predicted template. The resulting model achieved 100% sequence identity with the hybrid template, and Ramachandran plot analysis confirmed that approximately 89 to 90 percent of residues occupied favorable regions, with only 5 percent in disallowed zones. With this validated structural foundation in place, the researchers performed molecular docking using GNINA 1.0 integrated with AutoDock Vina, leveraging a convolutional neural network-based scoring function to predict binding affinities across all eight ligands against both wild-type and mutant CRBN proteins.

The docking results were striking. Among all tested compounds, CC-885—a next-generation CELMoD—demonstrated the most potent and consistent binding, with affinity scores ranging from −9.34 to −10.40 kcal/mol across all CRBN variants. Its best predicted affinity of −10.40 kcal/mol against wild-type CRBN substantially outperformed thalidomide (−7.80 kcal/mol), lenalidomide (−7.36 kcal/mol), and pomalidomide (−7.15 kcal/mol). Although CC-885’s ligand efficiency (−0.34) was lower than that of the smaller IMiDs—a consequence of its larger molecular size distributing binding energy across more heavy atoms—its absolute binding strength was unmatched. Mezigdomide and iberdomide occupied an intermediate tier, showing improved performance over traditional IMiDs but falling short of CC-885’s dominance. Notably, mezigdomide exhibited enhanced binding affinity in the S379I (−8.24 kcal/mol) and P382R (−8.15 kcal/mol) mutants compared to wild-type (−7.89 kcal/mol), hinting at some mutation-resilient properties among certain CELMoDs.

Interaction profiling using the Protein Ligand Interaction Profiler (PLIP) revealed that CC-885 engaged in a richer network of non-covalent contacts than its competitors, combining hydrogen bonds, hydrophobic interactions, and π-cation interactions across wild-type and mutant complexes. Residues such as ASP50, SER272, SER339, LEU261, TRP264, ASP265, LEU268, LEU273, and LEU342 featured prominently in these interactions, with LYS269 contributing a π-cation contact. By contrast, pomalidomide relied primarily on hydrogen bonds with ASP50, THR51, ASP265, and LYS269, while mezigdomide showed more variable and sometimes absent interaction patterns across mutants. This richer interaction landscape suggested that CC-885’s larger scaffold might provide a structural buffer against mutation-induced perturbations in the binding pocket.

But static docking alone cannot capture the dynamic reality of protein-ligand recognition. To address this, the team turned to 200-nanosecond molecular dynamics simulations using GROMACS 2025.4 with the CHARMM36 force field, solvating each complex in a dodecahedral box with TIP3P water and 0.15 M NaCl to maintain physiological ionic strength. They focused on the wild-type, S379I, and P382R variants bound to pomalidomide, mezigdomide, and CC-885. The results were unambiguous: CC-885-bound complexes maintained superior conformational stability across triplicate independent simulations, with protein RMSD values ranging from approximately 0.39 to 0.56 nm, radius of gyration values consistently within 2.38 to 2.42 nm, and solvent-accessible surface area values remaining in a narrow band of roughly 230 to 270 nm². In contrast, pomalidomide complexes exhibited higher fluctuations and greater solvent exposure, particularly in the P382R mutant, while mezigdomide complexes showed intermediate behavior with notable ligand mobility in the S379I variant.

Ligand RMSD analysis further cemented CC-885’s advantage. The mean ligand RMSD values for CC-885 complexes ranged from approximately 0.26 to 0.57 nm across replicates, with the P382R mutant displaying particularly consistent ligand positioning. Pomalidomide and mezigdomide, by comparison, exhibited greater structural deviations and increased ligand mobility, with the P382R mutant showing the greatest fluctuation in the pomalidomide-bound complex. Hydrogen bond analysis revealed that CC-885 maintained persistent, continuous interactions throughout all simulation replicates, whereas pomalidomide and mezigdomide showed more intermittent bonding patterns. Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) calculations confirmed these observations, with CC-885 complexes exhibiting the most favorable binding free energies—reaching approximately −35 to −40 kcal/mol in certain replicates—compared to the weaker affinities of pomalidomide and mezigdomide complexes.

Perhaps most intriguingly, the S379I variant, which the variant impact analysis had classified as stabilizing rather than destabilizing, was confirmed through simulation to enhance protein compactness, as evidenced by consistently lower SASA values and favorable radius of gyration measurements across all ligand-bound complexes. Yet even this stabilizing mutation did not compromise CC-885’s binding performance. The P382R variant, clinically associated with IMiD resistance, showed clear signs of destabilization in the pomalidomide-bound complex—elevated RMSD and SASA values—but remained well-accommodated within the CC-885 binding environment. This differential response underscores a central thesis of the study: the choice of E3 ligase-recruiting ligand can determine whether resistance-causing mutations translate into therapeutic failure or remain structurally tolerated.

The implications of this work extend beyond the immediate finding that CC-885 outperforms first-generation IMiDs in mutation-resilient binding. By systematically mapping the structural consequences of CRBN variants within the CULT domain and demonstrating that next-generation CELMoDs can maintain stable ternary complex architecture despite these perturbations, the study provides a rational framework for future PROTAC design. Rather than treating resistance as an inevitable clinical endpoint, researchers can now consider ligand selection as a proactive strategy to preempt mutation-induced failure. The authors acknowledge that their findings remain at the computational stage and that experimental validation and clinical investigation will be essential to translate these insights into therapeutic practice. Nevertheless, the convergence of variant impact prediction, homology modelling, deep-learning-enhanced docking, and extended molecular dynamics simulation represents a powerful methodological template for identifying mutation-resilient degraders. As PROTAC therapy continues to gain momentum in cancer treatment since its clinical resurgence in 2015, studies like this one illuminate a path toward designing next-generation molecular glues and degraders that can withstand the evolutionary pressure of tumor mutation—turning the very mutations that once conferred resistance into targets for strategic ligand engineering.

Subject of Research: Computational evaluation of next-generation CRBN E3 ligase modulators as alternatives to immunomodulatory drugs for overcoming CRBN mutation-driven resistance in PROTAC therapy

Article Title: CRBN E3 ligase modulators (CELMoDs) as potential alternative for immunomodulatory drug (IMiD) resistance in CRBN-based targeted therapy

Article References: Mary, E. R. C., & Sudandiradoss, C. (2026). CRBN E3 ligase modulators (CELMoDs) as potential alternative for immunomodulatory drug (IMiD) resistance in CRBN-based targeted therapy. Results in Chemistry, 31, Article 103972. https://doi.org/10.1016/j.rechem.2026.103972

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103972

Keywords: PROTACs, CRBN, CELMoDs, IMiDs, drug resistance, molecular docking, molecular dynamics, CC-885, mezigdomide, CULT domain, multiple myeloma, targeted protein degradation

The core problem the researchers set out to solve is a bottleneck familiar to anyone working in targeted protein degradation. Proteomics-based approaches can, in principle, reveal every protein whose abundance changes when a compound is applied, but they are expensive and slow, making them impractical as a first-pass screen for large chemical libraries. Target-based biochemical assays such as AlphaLISA or TR-FRET require purified proteins and, crucially, prior knowledge of which target-E3 pair to test—an assumption that defeats the purpose of hunting for glues against unknown or classically undruggable substrates like transcription factors and scaffold proteins. Phenotypic screens, meanwhile, are biologically rich but cannot easily distinguish a true degrader from an ordinary inhibitor, forcing laborious downstream mechanistic deconvolution. What the field needed was a cheap, fast, and mechanistically informative primary readout.

The team’s answer was to make the transcriptome itself the universal readout. Gene expression signatures are high-dimensional proxies for cellular state, a principle demonstrated at scale by the L1000 platform and made affordable by DRUG-seq, which skips RNA purification and uses direct cell lysis with multiplexed library preparation. The researchers employed an optimized version, DRUG-seq2, which they report offers superior sensitivity for low-abundance regulatory transcripts—a critical feature when the signals of interest come from immature, weak molecular glues. In their workflow, Hep3B liver cancer cells were seeded in 96-well plates, treated with compounds at a uniform concentration of 1 micromolar for 24 hours, and processed through the miniaturized sequencing protocol. The library comprised 117 compounds: 11 commercial molecular glues serving as validated benchmarks and 106 in-house candidates synthesized by two collaborative research groups, all rationally designed to modulate the cereblon (CRBN) E3 ligase.

A deceptively simple but essential innovation lies in how the baseline is defined. Because DRUG-seq2 relies on direct lysis, technical noise can swamp the subtle transcriptional ripples produced by nascent glues. Before any analysis, the team performed systematic pairwise Pearson correlation analysis across all DMSO vehicle wells on each plate, excluding any replicate with a correlation coefficient below 0.95. By constructing the reference transcriptome solely from high-consensus control wells, they established a robust, self-consistent zero-point that maximizes statistical power to resolve low-magnitude perturbations. Downstream, differential expression analysis with DESeq2—flagging genes with absolute log2 fold change greater than 1 and adjusted p-value below 0.05—was paired with gene set enrichment analysis (GSEA) built from six curated collections, including Hallmark, Reactome, KEGG, Gene Ontology Biological Process, C6, and the C3 transcription factor target set. This two-layer design means even compounds with few or zero individual differentially expressed genes can still betray coordinated pathway-level activity.

The mechanistic heart of the platform is a genetic benchmark: an isogenic pair of Hep3B cell lines, one wild-type and one in which CRBN has been knocked out via CRISPR-Cas9. The researchers established a stable Cas9-expressing parent line, introduced single-guide RNAs targeting CRBN, and isolated single-cell clones. Clone 3 emerged as the definitive knockout after rigorous validation. Treatment with CC-90009, a clinical molecular glue that degrades the translation termination factor GSPT1, provided the proof: in wild-type cells the drug rapidly and almost completely eliminated GSPT1 protein, while in the knockout the effect vanished entirely, along with the drug’s cytotoxic potency, which showed a pronounced right-shift in the IC50 curve. Any compound whose transcriptional signature collapses in the knockout is therefore flagged as CRBN-dependent—a hallmark of a genuine glue—whereas signals that persist likely reflect off-target toxicity, conventional inhibition, or hijacking of a different E3 ligase such as VHL or DCAF16.

Screening the full library revealed a strikingly polarized activity spectrum. Roughly 20 percent of the compounds were transcriptionally inert, producing zero differentially expressed genes under the test conditions. At the other extreme, the high-activity cluster was dominated by known glues: SJ3149, which targets CK1α, elicited the most profound response, followed by lenalidomide, BMS-986397, and a MYC degrader. For most of these, transcriptional activity was almost completely abolished in the CRBN knockout, exactly as expected for classic glues. Encouragingly, one in-house compound, LC-02-047-P1, landed among the potent performers, marking it as a promising novel candidate. Conversely, compounds such as LC-02-105 and LC-02-033 displayed a paradoxical knockout-amplified phenotype, indicating CRBN-independent mechanisms and allowing the team to filter out molecules whose intrinsic scaffold toxicity overwhelmed any degradation function.

The pathway-level analysis added mechanistic texture that raw gene counts alone could not provide. Potent GSPT1 degraders such as CC-90009 upregulated regulators of the integrated stress response—DDIT3 (CHOP), GDF15, TRIB3, DDIT4, and PPP1R15A—consistent with the biology of translation termination failure: ribosomes stall at stop codons, collide, and trigger the ZAKα-mediated ribotoxic stress response, which in turn activates ATF4 and suppresses the translational apparatus to mitigate proteotoxic stress. GSEA of CC-90009 confirmed enrichment of integrated stress response, proteasome, and apoptosis pathways alongside downregulation of translation machinery. LC-02-047-P1, by contrast, showed metabolic rather than stress-related regulatory characteristics, hinting that its degraded target might be an upstream switch governing multiple metabolic pathways. The enrichment layer also rescued latent hits: LC-02-051 performed modestly in the differential expression analysis but ranked near the top in pathway enrichment, and its signal was completely blocked by CRBN knockout—suggesting a genuine but weak glue worth optimizing. Even CC-92480 yielded an instructive anomaly, showing slightly expanded enrichment in knockout cells, which the authors attribute to the absence of its high-affinity target IKZF1 in Hep3B, pushing the compound toward non-canonical substrates or generic xenobiotic stress.

To translate these multidimensional data into decisions, the researchers built a scatterplot integrating transcriptional potency (differential gene counts) with functional breadth (enriched pathway counts) across both wild-type and knockout conditions, then stratified the library into four tiers. Tier one comprises potent glues with high wild-type activity that nearly vanishes upon CRBN loss—the highest priorities for optimization. Tier two captures intermediate and latent leads whose coordinated functional signals suggest seed scaffolds needing structural refinement. Tier three contains active but E3-independent perturbators, likely conventional inhibitors, and tier four collects inert compounds. The spatial logic is intuitive: strong glues such as SJ3149, lenalidomide, CC-90009, and LC-02-047-P1 occupy the upper-right quadrant and collapse toward the origin in the knockout, while pharmacologically inert molecules cluster near the origin in both conditions and target-independent agents stubbornly refuse to move. The thresholds, the authors note, can be customized for different libraries.

Proteomic cross-validation demonstrated that the transcriptomic tiers predict real degradation events. Tandem mass tag quantitative proteomics of LC-02-047-P1-treated Hep3B cells revealed marked downregulation of CSNK1A1 (CK1α), and western blotting across Hep3B, Huh7, and HepG2 hepatocellular carcinoma lines confirmed near-complete, selective CK1α degradation with no effect on GSPT1. A decisive control sealed the case: cells engineered to express a non-degradable GSPT1 mutant abolished CC-90009’s activity but left LC-02-047-P1’s CK1α degradation untouched, proving the candidate acts entirely independently of GSPT1. The weaker tier-two compound LC-02-051 produced only minor proteomic perturbation, with TPM3 changes failing statistical validation—consistent with its latent classification. On scalability, the economics are compelling: the platform costs less than 100 RMB (roughly 14 US dollars) per sample, about 2 percent of standard TMT proteomics, with an end-to-end turnaround of three to four weeks even beyond 1,000 compounds.

The authors are candid about limitations. The single 24-hour window captures an integrated cellular state—primary degradation consequences blended with secondary cascades and stress responses—rather than the immediate transcriptional footprints of neosubstrate depletion, and future iterations will incorporate time-resolved profiling. Transcriptomic signatures also remain an indirect proxy for protein-level events, so candidate leads must ultimately be confirmed by quantitative proteomics and structural biology demonstrating direct ternary complex formation. The choice of screening cell line matters too, since E3 and substrate abundance dictate sensitivity; the authors suggest abundance-matched models, such as VHL-high or DCAF15-high backgrounds, for targeted campaigns. Even so, the GPS platform fills a genuine gap, bridging high-volume chemical screening and low-throughput mechanistic validation, and offering the field a practical roadmap for converting molecular glue discovery from a lucky accident into an engineering discipline.

Subject of Research: High-throughput transcriptomic screening for the discovery of CRBN-dependent molecular glue degraders

Article Title: A scalable, high-throughput glue perturbation screening platform for molecular glue discovery

Article References: Huang, D., Shuang, Z., Chen, L., Ouyang, H., Yang, P., Huang, L., Lu, W., Shi, M., Ding, X., Jiang, B., & Wu, W. (2026). A scalable, high-throughput glue perturbation screening platform for molecular glue discovery. iScience, 29(10), Article 117600. https://doi.org/10.1016/j.isci.2026.117600

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117600

Keywords: molecular glue degraders, targeted protein degradation, CRBN, DRUG-seq2, transcriptomics, gene set enrichment analysis, CRISPR knockout, E3 ubiquitin ligase, proteomics, drug discovery, GSPT1, CK1α

The study, led by Maja Louise Arendt of the University of Copenhagen and Jennifer R. S. Meadows of Uppsala University and SciLifeLab, focused on cereblon, the protein that thalidomide binds. Cereblon was only identified as the drug’s primary target in 2010, when researchers showed that thalidomide attaches to this substrate receptor of the cullin-4 E3 ubiquitin ligase complex, known as CUL4-RBX1-DDB1. When thalidomide occupies cereblon, the degradation machinery’s specificity changes: it begins recruiting, ubiquitinating and destroying an unnatural set of proteins called neo-substrates, including transcription factors such as SALL4, p63, Ikaros and Aiolos. These proteins govern critical developmental and immunological processes, which helps explain both the drug’s teratogenic tragedy and its therapeutic benefits. At the same time, native substrates such as MEIS2 are no longer degraded, adding further biological consequences. To date, cereblon remains the only described mammalian target of thalidomide.

The crux of the new findings lies in a single amino acid position within the cereblon protein. The C-terminal region of human cereblon, spanning roughly amino acids 318 to 426, forms the thalidomide-binding CULT domain, and variation within this 109-amino-acid stretch is known to determine how different species respond to the drug. In humans, the amino acid at position 388 is a valine, abbreviated V388. In mice, the equivalent position holds an isoleucine instead. This seemingly minor substitution does not prevent thalidomide from binding to cereblon, but it creates steric hindrance that blocks neo-substrates from entering the binding pocket, preventing their ubiquitination and degradation. The result is that thalidomide and its derivatives are pharmacologically ineffective in mice, a fact that contributed to the drug’s original licensing as a supposedly safe sedative, since rodent tests failed to predict its devastating effects in humans.

When the researchers mined the Dog10K consortium datasets, which include single nucleotide and structural variant calls from 1,929 and 1,879 canids respectively, they found that every single dog examined encodes an isoleucine at the position equivalent to human V388. Across the roughly 30-kilobase CRBN gene region, 384 variable sites were detected, but only three had any potential to alter the protein’s coding sequence, and each of these was extraordinarily rare, with allele frequencies below 0.2 percent. Two structural deletions overlapping the gene were also identified, both at frequencies below 0.5 percent. In other words, the canine cereblon protein is essentially uniform across the species, and it uniformly carries the mouse-like isoleucine that has been shown to abolish thalidomide’s neo-substrate degradation activity.

To place this finding in evolutionary context, the team turned to two massive comparative genomics resources: the Zoonomia alignment of 240 mammalian species and the TOGA alignment covering 344 eutherian mammals. At the nucleotide level, the codon encoding position 388 showed little evolutionary constraint, with phyloP conservation scores of 0.40, 4.66 and -1.74 across its three bases; only the second position showed purifying selection. This wobbling tolerance is mirrored by amino acid variability across the mammalian tree. Among 344 species, 72 percent encode an isoleucine at the equivalent position, including all 55 available species of the order Carnivora, to which dogs belong. Only bats, rodents and rabbits, and the order Primates, which includes humans, were found to encode both valine and isoleucine at this site. Notably, rabbits and guinea pigs carry a valine, consistent with decades of toxicology data showing that rabbits, unlike mice, do develop thalidomide-induced birth defects similar to those seen in humans.

The researchers also considered whether cancer itself might change the equation. Tumors accumulate mutations that can alter drug sensitivity, so the team searched for non-silent, protein-altering mutations in the CRBN gene within two published canine tumor-normal sequencing datasets, comprising 55 mammary tumors and 43 diffuse large B-cell lymphoma cases, as well as 723 samples from the cBioportal Canine Cancer Genome Atlas. The search yielded almost nothing: a single non-synonymous mutation, p.CRBN A10T, was found in one lymphoma sample, affecting the N-terminal region far from the thalidomide-binding CULT domain. No mutations were identified that would plausibly make canine cancer cells more susceptible to thalidomide than normal cells. In human multiple myeloma, somatic CRBN mutations are likewise reported to be neutral or to confer resistance, never enhanced sensitivity.

These genetic findings cast a revealing light on the veterinary literature. Thalidomide has been tested in dogs with splenic hemangiosarcoma, mammary carcinoma, lung carcinoma, lymphoma and multiple myeloma, usually in combination with other therapies, making efficacy difficult to assess. One recent multicentre retrospective study reported that 71 percent of seven dogs with relapsed multiple myeloma maintained or achieved complete remission on thalidomide as a single-agent rescue therapy, with moderate lethargy in two dogs as the only recorded side effect. Strikingly, a 53-week toxicology study in beagles using doses up to 1,000 milligrams per kilogram daily, roughly fifty times the human clinical dose for a ten-kilogram dog, found no clinically significant adverse effects, no hematological or biochemical changes and no evidence of the peripheral neuropathy that plagues human patients. The authors argue that this remarkable tolerance is itself a warning sign: a drug with no side effects at massive doses may simply have no pharmacological activity.

There are caveats. The sedative effect of thalidomide appears to be independent of cereblon, and one canine study reported reduced vascular endothelial growth factor staining in metastatic tissue from treated dogs, hinting that anti-angiogenic mechanisms independent of the CRBN-CUL4-RBX1-DDB1 complex might operate in dogs. A pharmacokinetic study in tumor-bearing dogs also reported a range of clinical signs, though the authors of the new analysis note these could reflect advanced cancer rather than drug toxicity. The researchers are careful not to claim that thalidomide is definitively useless in dogs; rather, they conclude that the molecular function of the drug in this species must be directly investigated before its therapeutic role in canine cancer can be understood or justified.

Beyond its immediate veterinary implications, the study is a compelling demonstration of how publicly available genomic data can inform clinical pharmacology. Because DNA-damaging chemotherapy agents tend to work comparably across humans and dogs, veterinary oncologists have reasonably borrowed human anticancer drugs. But for targeted agents such as thalidomide, whose action depends on the precise structure of a single protein, species differences in that target can silently nullify the drug’s effect. Given thalidomide’s tragic history and its strict regulation in human medicine, the authors advocate that molecular evidence of function should be established before the drug is used therapeutically in dogs, a lesson that applies equally to the growing arsenal of cereblon-targeting protein degraders now entering human clinical development.

Subject of Research: Genetic variation in the canine cereblon protein and its implications for thalidomide therapy in dogs with cancer

Article Title: Genetic evidence supports that thalidomide should not be used therapeutically in dogs

Article References: Arendt, M. L., & Meadows, J. R. S. (2026). Genetic evidence supports that thalidomide should not be used therapeutically in dogs. Veterinary Oncology, 3(1), Article 14. https://doi.org/10.1186/s44356-026-00067-2

Image Credits: AI Generated

DOI: 10.1186/s44356-026-00067-2

Keywords: thalidomide, cereblon, CRBN, dogs, canine cancer, veterinary oncology, comparative genomics, neo-substrates, ubiquitin ligase, drug repurposing, teratogenicity, Dog10K