3 case studies on how to characterize challenging drug discovery targets — membrane proteins, peptide libraries, and PROTACs

A caveat to a successful pharmaceutical industry in which many effective drugs have already been discovered is the notion that the high hanging fruits left unpicked come in the form of challenging or “undruggable” targets. Among these are proteins and enzymes that exist in complex biological states and are difficult to characterize. Some examples are membrane proteins with sensitive folding patterns, low-expression proteins with inadequate yields for assays, and multi-component complexes. To characterize these challenging targets, researchers must utilize the right technology.

The webinar, Tackle the high-hanging fruits in challenging drug discovery pipelines, explores three case studies in which researchers tackled challenging targets using Dianthus, a fast and high-throughput instrument designed to precisely measure the binding affinities or proteins in a 384-well format via Temperature Related Intensity Change or TRIC.


Case study 1: Fragment screen for the receptor protein STING (Stimulator of Interferon Genes)

The first case study focuses on a fragment screen for the receptor protein STING (Stimulator of Interferon Genes) — a transmembrane dimeric protein with a promising role in cancer immunotherapy. With fragment screening, it is crucial to identify binding partners with high binding affinities to the target protein, as this paves the way for the discovery of therapeutic ligands. Here, Dianthus was used in mass-independent fragment screening for potential STING ligands. After two screening events, fragment binders were identified that featured low micromolar affinities comparable to the binding of cGAMP. Dianthus’s key features that made this possible were its fast measurement speed, low sample consumption, and broad affinity range.


Case study 2: Assess interactions between a membrane-anchored scaffolding protein and ligands from a peptide library

In the second study, Dianthus was used to assess the interactions between a membrane-anchored scaffolding protein and ligands from a peptide library. Due to Dianthus’s flexibility, it was possible to change the experimental strategy from a direct binding assay to a displacement assay. In this new approach, protein interaction is measured when a new unlabeled peptide ligand displaces a labeled peptide from the protein binding site. With a good signal-to-noise ratio and a high amplitude, it was possible to run an unpurified overlapping peptide library and successfully confirm two binding sites.


Case study 3: Characterize PROTAC interactions with a bromodomain protein

Finally, Dianthus’s capabilities were demonstrated in the investigation of multi-component interactions with PROTACs (proteolysis targeting chimeras). PROTACs offer a therapeutic means of tackling undruggable proteins by forming ternary complexes with ubiquitin ligase and the protein of interest. However, equilibrium conditions for the formation of such complexes can be difficult to control. Here, Dianthus is used to characterize PROTAC interactions with a bromodomain protein. To do this, the binary Kds were first determined in separate PROTAC-ligase and PROTAC-target interactions. This was followed by titrating the ligase for the entire PROTAC complex to determine the ternary Kd. In the end, this strategy led to a reliable method of calculating the cooperativity value of a complex, oligomeric system in solution, under controlled equilibrium conditions.


Watch the webinar to get all the details of how Dianthus was used to study challenging molecular interactions that underlie some of the most challenging drug targets.