Molecular Glue Discovery: Current and Future Approaches

Abstract

The intracellular interactions of biomolecules can be maneuvered to redirect signaling, reprogram the cell cycle, or decrease infectivity using only a few dozen atoms. Such "molecular glues," which can drive both novel and known interactions between protein partners, represent an enticing therapeutic strategy. Here, we review the methods and approaches that have led to the identification of small-molecule molecular glues. We first classify current FDA-approved molecular glues to facilitate the selection of discovery methods. We then survey two broad discovery method strategies, where we highlight the importance of factors such as experimental conditions, software packages, and genetic tools for success. We hope that this curation of methodologies for directed discovery will inspire diverse research efforts targeting a multitude of human diseases.

Figure 1.

Cartoon depicting a molecular glue classification system based on its mechanism of action. Type I molecules (left) induce a non-native protein protein interaction (PPI) to “shield” a protein of interest from carrying out its function. Type II molecular glues (middle) inhibit a protein’s function by redirecting an endogenously formed PPI. Type III molecular glues (right) induce a non-native PPI to produce a novel activity (ubiquitination, transcription, etc.).

Figure 2.

Type III molecular glue discovery enabled by time-resolved-FRET (TR-FRET). (A) The oncogenic SMAD4 mutation R361H eliminates its interaction with SMAD3, so Tang et al. sought identify molecular glues that reactivate mutant SMAD4’s interaction with SMAD3. (B) Cells overexpressing epitope-tagged SMAD3 and SMAD4 were lysed and then subjected to screening for molecular glues using an antibody-based TR-FRET approach.

Figure 3.

Molecular glue discovery via fluorescence polarization (FP). (A) Protein binding decreases tumbling velocity of a fluorophore tagged biomolecule, increasing the FP signal. (B) The high BCL6 protein concentration relative to the BCOR peptide increased the chance of discovering a BCL6 molecular glue because molecular glue homodimerizers scale exponentially with protein concentration. (C) Relatively small changes in molecular glue chemical structure resulted in protein oligomerization versus dimerization.

Figure 4.

Computationally identified molecular glue inhibits MERS-CoV replication. A mutant of the MERS-CoV nucleocapsid resulted in low-affinity dimerization and enabled protein crystallization. Docking at the dimerization site identified a molecular glue that oligomerized nucleocapsid proteins in cellulo resulting in MERS-CoV inhibition (PDB 6KL2 and 6KL5).

Figure 5.

Recommended future approaches to discovering type I molecular glues. Triggering homodimerization or oligomerization with molecular glues can effectively inhibit or even degrade a target protein. Ubiquitin and LC3B are abundant and evolutionarily conserved proteins that are already validated as effective molecular glue targets to inhibit protein activity. Moreover, these proteins can be used in nearly any method described herein including NMR and X-ray crystallography.

Figure 6.

A public database drives machine learning (ML) based molecular glue discovery. First, a publicly available molecular glue database (top) should be assembled that consists of interaction detection method (IDM) screening results, validated molecular glues and their target PPIs, and all PPI structures. Next, the database can fuel ML approaches to predict non-native PPI small-molecule binding pockets and a subset of chemical structures that are more likely to bind PPI interfaces (middle). Together, these combined advances will generate novel molecular glues (bottom) and discover chemical and biophysical principles that underlie molecular glue activity.