Molecular glues for an oncology target — discovered directly in living cells
Using DELs in Cells – Molecular Glue Direct, Vipergen screened an oncology target in triplex with two E3 ubiquitin ligases and identified 89 hits with 2 clusters being potential molecular glues in a single multiplexed screen. Four resynthesized compounds were carried into validation and were all confirmed to induce the target–E3 ligase interaction in an orthogonal co-immunoprecipitation assay.
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Directly screening for glue chemistry in a living cell
Targeted protein degradation harnesses the cell’s own ubiquitin–proteasome machinery to remove a protein of interest (POI) rather than merely inhibiting it, hereby expanding the druggable proteome. It depends on bringing the POI into productive contact with one of the 600+ E3 ubiquitin ligases encoded by the human genome. Molecular glues defined as small molecules that induce or strengthen a protein–protein interaction through cooperative binding are an elegant way to do this, yet they have proven notoriously difficult to discover by conventional methods.
Vipergen’s DELs in Cells – Molecular Glue Direct service screens for molecular glues directly inside living cells. Built on our cellular Binder Trap Enrichment (cBTE) technology in a multiplexed format, it co-expresses the POI and an E3 ligase in the same cell and reads out the compounds binding each protein and, crucially, the compounds shared between them. Those shared hits are the candidate glues. Because targets are expressed as an intrinsic part of the project, no purified protein is required; only the amino acid sequence of the POI and the E3 ligase. The service is applied to an oncology target in the case described here.
A degradation-ready oncology target in need of novel glue chemistry
For this program, targeted protein degradation was the preferred pharmaceutical mode of action against an oncology target. That made molecular glues an attractive modality: compact, drug-like molecules capable of recruiting the POI to an E3 ligase and triggering its ubiquitination and degradation.
The difficulty is discovery. Molecular glues act at a protein–protein interface that often does not exist until the compound is present, so there is no pre-formed pocket to screen against. Traditional biochemical and high-throughput approaches struggle to surface this chemistry, and they typically demand purified proteins and pre-validated interaction pairs. What the program needed was a way to interrogate the POI against a relevant panel of E3 ligases directly — and to separate true interface-binding glues from compounds that merely bind the target alone.
One multiplexed screen. One POI. Two E3 ligases.
Vipergen screened the oncology target using DELs in Cells – Molecular Glue Direct, co-expressing the POI together with two E3 ligases in a single triplex screen. Each protein was tagged with its own barcoding DNA, allowing compound binding to be mapped independently to the POI and to each E3 ligase within the same living cell. Molecular glues were read out as the compounds shared between the POI and an E3 ligase.
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Construct design & balanced expression
The POI and both E3 ligases were expressed in Xenopus oocytes, eliminating the need for purified protein. Because human E3 ligases are active in this system, upstream ubiquitination inhibitors were included to prevent target ubiquitination and degradation, and expression was balanced across the three proteins. -
Multiplexed cBTE screen
The DNA-encoded library and the protein Bait-DNAs were introduced into the cell. cBTE traps compound–target complexes so that binding events are captured for the POI and each E3 ligase simultaneously. -
Encoding & readout
Binding events are encoded by DNA ligation, then read out by DNA sequencing and decoding. Frequent hitters and nonspecific binders are removed, leaving only high-quality hits. -
Hit mapping & clustering
Decoded hits were mapped to the POI and to each E3 ligase, then grouped into chemical series by pairwise Tanimoto similarity. Compounds shared between the POI and an E3 ligase were flagged as candidate molecular glues.
Figure 1.
Pairwise Tanimoto similarities of the hits identified in the Molecular Glue Direct screen (left). Blue lines (right) denote mapping of the hits to the POI and the E3 ligases, respectively.
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89 hits, six series, and glues confirmed by Co-IP
The triplex screen returned 89 hits. Pairwise Tanimoto similarity analysis grouped them into six distinct chemical series. Of these, clusters 1 and 4 comprised hits shared between the POI and one of the two ligases, E3L-01 — the signature of a molecular glue that bridges the two proteins.
To confirm that these shared hits genuinely induced the target–ligase interaction, compounds were resynthesized off-DNA and tested in an orthogonal co-immunoprecipitation (Co-IP) assay measuring the interaction between the POI and E3L-01. All four resynthesized compounds [Compounds 1-4] induced the interaction. A fifth, chemically unrelated compound included as a control [compound 5]) produced no interaction, confirming the glue effect was specific.
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Figure 2.
Verification of compounds in a Co-IP assay. Compounds 1–4 induced the interaction between the POI and E3 ligase. Almost no interaction was seen when a chemically unrelated compound (5) was assayed.
Direct molecular glue discovery, validated in one workflow
This case shows DELs in Cells – Molecular Glue Direct doing what conventional glue discovery struggles to do: surface interface-binding chemistry against a therapeutically relevant target, map it cleanly to a specific E3 ligase, and only during confir
Key takeaways for your program
- No purified protein required. The POI and both E3 ligases were expressed as an intrinsic part of the project, from the target amino acid sequence alone.
- Multiplexing does the triage. Barcoding lets a single screen distinguish POI-only binders from genuine POI–E3 ligase glues, so the shortlist is enriched for real glues before any follow-up.
- Screen hits translate to glue activity. Four of four shared hits induced the POI–E3L-01 interaction in Co-IP.
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