Molecular glues (MG) are small mono-valent ligands without a linker, that bind to both the target protein and the E3 ligase simultaneously, allowing the functional activity of the ligase to ubiquitinate the target protein.
The advantage of a molecular glue is that they are smaller than a PROTAC in molecular weight (thereby obeying Lipinski’s principles) and easier to develop in terms of medicinal chemistry (no linkerology required). It is therefore easier to screen large numbers of compounds for molecular glue activity.
Screening for molecular glues requires assay formats that detect the association of the ligase and target protein. These formats include HTRF and AlphaLISA for higher throughput, and immunoblotting for lower throughput screening.
Molecular glues may also be used to discover new ligase activity. There are thought to be, 500 to 600 ligases in the human genome. It is assumed that there is differential- tissue, time-dependent and disease-related expression of these ligases that can be used as selectivity and specificity tools to “aim” the depletion of target proteins in appropriate cell types at specific times in the cell life cycle.
Molecular Glues Assay Development
For molecular glues, there is a significant degree of serendipity in detecting a small molecule that binds to and brings together both the target and a ligase to drive degradation. Therefore, higher throughput assays may be required, in which case we would develop HTRF, AlphaLISA, or Lumit reagents. These are designed to either detect the interaction between target protein and ligase or to measure the quantity of target protein present in cell lysates.
In HTRF, AlphaLISA or Lumit, these technologies rely on two antibodies to either detect distinct proteins (e.g. for molecular glue assays in which we are detecting a protein:protein interaction) or distinct epitopes of the same protein (e.g. for PROTAC assays in which we are quantitating levels of a specific protein in cell lysates).
When binding to the protein(s), the antibodies bring together the readout partners to generate a signal: In AlphaLISA, these partners are two beads; in HTRF these are fluorescence tagged antibodies; and in Lumit these are antibodies labeled with SmBiT and LgBiT tags.