The Glue that Heals: St. Jude’s Researchers Unveil Molecular ‘Super-Glue’ Propelling Innovative Cancer Drug Development

Scientists at St. Jude Children’s Research Hospital have published their findings on SJ3149, a chemical with broad efficacy against various cancer types, particularly acute myeloid leukemia (AML). Scientists worked together to discover and improve a chemical molecule that functions as a molecular “super-glue” for a variety of tumors. SJ3149 binds to cancer-related protein casein kinase 1 alpha (CK1α), causing its elimination. The findings were published in the journal Nature Communications.

We have made a molecular super-glue. SJ3149 is the first-in-class potent and selective CK1α degrader, showing efficacy in both in vitro and in vivo cancer models.

— Senior co-corresponding author Zoran Rankovic, PhD, St. Jude Department of Chemical Biology and Therapeutics

Introduction

Have you ever wished you could just put a “Do Not Enter” sign on a faulty protein in a cancer cell? Thanks to molecular glue degraders, scientists are coming closer to doing just that. These ingenious tiny molecules function as matchmakers, bringing together a protein designated for destruction (the target) and the cellular executioner (an E3 ligase) in a lethal embrace. As a result, the target protein is tagged and disposed of, providing a fresh approach to cancer treatment.

Molecular glues work by hijacking the cell’s natural protein recycling system. The molecular glue binds the targeted protein to an enzyme, marking it for destruction via proteasomal degradation. For many cancer-related proteins that conventional small molecule inhibitors cannot effectively target, molecular glues may be a feasible therapeutic option. This prompted scientists at St. Jude to create a vast proprietary library of molecular glues and screen it against a variety of cancer cell lines, resulting in an early hit.

St. Jude’s researchers discovered a promising new glue named SJ3149. Screening a library of compounds against cancer cells revealed SJ3149’s ability to bind CK1α, a protein linked to cancer cell proliferation, to its executioner, CRBN. Rapid degradation of CK1α was observed in both lab and animal models. However, not all glues are created equal. Researchers improved SJ3149’s chemical structure to be more selective, binding to CK1α and not other proteins. This focused strategy reduces potential adverse effects, making SJ3149 a safer and more effective cancer treatment. After the researchers improved the found hit, the resulting SJ3149 molecule had greater efficacy and less off-target effects than comparable molecules. SJ3149 has shown impressive broad anti-cancer effectiveness, even for a molecular glue, earning it the nickname “super-glue.” The molecule also appears to share a profile with a class of licensed cancer medications known as murine double minute 2 (MDM2) inhibitors, indicating that it may have clinical usefulness.

Phenotypic Screening and Hit Identification:

• A library of 3630 molecular glues was tested against nine pediatric cancer cell lines, including the MOLM-13 cells.
• SJ7095 showed remarkable selectivity for MOLM-13 cells, with an IC50 of less than 1 µM.

Target identification and mode of action:

• SJ7095 was shown to work through CRBN-dependent protein degradation.
• TMT proteomics and immunoblotting identified CK1α, IKZF1, and IKZF3 as major degrades.
• CRISPR/shRNA experiments and DepMap data support MOLM-13’s dependence on CK1α and its participation in SJ7095’s impact.
• SJ7095 significantly increased CK1α degradation compared to lenalidomide.

Structure-guided optimization produces selective CK1α degraders:

• SJ7095’s C5 alterations produced powerful and selective CK1α degraders (SJ0040 and SJ3149).
• Both compounds outperformed lenalidomide for CK1α degradation rate and Dmax.
• SJ3149 emerged as the most potent, with a DC50 of 3.7 nM and a Dmax of 95%.
• Lead compounds exhibited decreased CRBN binding but increased ternary complex formation with CK1α, indicating their importance for degradation efficiency.

TMT proteomics confirms selective CK1α degradation:

• SJ0040 and SJ3149 selectively destroyed CK1α in MOLM-13 cells without significantly affecting other proteins.
• Kinetic studies suggest that CK1α degradation has downstream effects.
• Lead compounds that degraded CK1α faster also stabilized β-catenin and p21 sooner.
• Rescue trials confirm CK1α as the cause of cytotoxicity.
• CK1α G40N mutant expression in MOLM-13 cells inhibited SJ3149’s cytotoxic impact. 

Methods

Library screening and hit identification:

• A 3630-compound molecular glue library was screened against nine pediatric cancer cell lines (including MOLM-13) in 384-well plates.
• Cell viability was assessed after 72 hours using the CellTiter-Glo luminescent assay.
• Dose-response curves were generated to determine IC50 values for hits.
• Clustering analysis was used to identify compounds with distinct activity profiles. 

Target identification and mode of action:

• Ligand competition experiments with lenalidomide and CRBN mutant cells validated SJ7095’s CRBN-dependent activity.
• TMT proteomics in MOLM-13 cells revealed damaged proteins following SJ7095 treatment.
• Immunoblotting confirmed SJ7095’s ability to degrade CK1α and increase p21 levels.
• DepMap data and CRISPR/shRNA studies evaluated CK1α reliance in MOLM-13 cells.
• The immunoblotting analysis evaluated the degradation of CK1α by SJ7095 and lenalidomide.

Structure-guided optimization of CK1α degraders:

• Molecular dynamics simulations guided C5 substitutions on SJ7095.
• A series of C5-substituted amid analogs were developed and evaluated in MOLM-13 cells.
• SJ0040 and SJ3149 were found to be effective and selective CK1α degraders.

Biochemical and biophysical characterization of lead compounds:

• The CRBN fluorescence polarization (FP) displacement assay assessed CRBN binding affinity.
• CK1α and IKZF1 HiBiT degradation tests in HEK293 and Jurkat cells assessed degradation kinetics and Dmax.
• CellTiter Glo 2.0 measured cell viability in conjunction with degradation assays.
• The NanoBRET test in CK1α HiBiT cells with HaloTag-CRBN fusion detected ternary complex formation.

Validation of CK1α selectivity and downstream effects:

• TMT proteomics in MOLM-13 cells examined the wider degradation patterns of SJ0040 and SJ3149.
•  Kinetic monitoring of HiBiT-β-catenin in live cells comparing β-catenin stability and CK1α degradation.
• SJ3149-induced p21 induction and IKZF2 degradation were examined by immunoblotting in MOLM-13 cells.
• Overexpression of the CK1α G40N mutant in rescue studies demonstrated that SJ3149’s cytotoxicity is dependent on this protein.

Key Takeaways

• Molecular glue degraders such as SJ3149 target specific proteins for degradation, providing a fresh strategy for cancer treatment.
• SJ3149 significantly destroys CK1α in cancer cells, reducing tumor development.
• The medication exhibits wide anti-cancer efficacy and possible synergy with existing treatments.
• This study sets the door for the future development of tailored medicines for a range of malignancies.

Conclusion

SJ3149 was developed by extensive collaboration. The work included developing, manufacturing, and screening the molecular glue library, as well as structure-guided medicinal chemistry optimization and testing in patient-derived cancer cells. It was only possible because of the joint experience of the Rankovic, Fischer, and Klco labs, as well as internal and external collaborators. The method can now be utilized as the foundation for further investigation.

The search for CK1α has led to a promising future in cancer therapy, where precision “protein glue” like SJ3149 may reduce cancer’s roar. This molecule’s potency, specificity, and combination with TP53 provide us with possibilities for treating hitherto untreatable malignancies. Challenges remain, but each one overcome takes us closer to a world in which cancer is a silent melody arranged by the balance of protein breakdown. Molecular glues have ushered in a new era in chemical biology. With this discovery, researchers have created a pipeline for identifying potentially promising molecular glues for cancer treatment.

This isn’t just a triumph; it’s a symphony of hope, rewriting the cellular code one protein, one “glue,” along with one hope at a time.

Article Source: Reference Paper | Reference Article

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