Presented by: Dave Madge, vice president, research division, WuXi AppTec
Madge described methods that his company applies to identify and evaluate bifunctional molecules that mediate targeted protein degradation (TPD). He pointed out that although much TPD research focuses on proteolysis targeting chimera (PROTAC) compounds, the tools used to identify such molecules also can be used to evaluate the therapeutic potential of other bifunctional molecules. Of particular interest are those that bring “housekeeping proteins” to disease-causing targets. More broadly, researchers are seeking out molecules that influence protein– protein interactions, immune responses, stabilization of protein complexes, and modulation of other biological processes.
WuXi AppTec leverages several technologies to facilitate in vitro ligand discovery. Affinity-selection mass spectrometry (ASMS) and DNA-encoded libraries (DELs) are useful because they do not require specification of a target-protein binding site or functional effect. ASMS provides rapid and accurate plate-based assessment of molecules. DELs help to ensure broad coverage of the chemical space. Other methods for ligand identification include high-throughput screening (HTS) and fragment-based drug discovery (FBDD).
After identifying potential target and housekeeping ligands, biochemists must develop a molecule that enables simultaneous binding of proteins to those ligand receptors. Madge notes that research in this stage of bifunctional molecule development is advancing rapidly as scientists consider more ligand types for a broader range of ligases. Inventive linking chemistries now play a large role in determining a bifunctional compound’s absorption, distribution, metabolism, and excretion (ADME) profile. WuXi AppTec continues to expand its collection of ligand–linker chemistries for rapid development of proof-of-concept TPD molecules. The company also can synthesize multikilogram quantities of PROTAC molecules for such purposes.
Madge highlighted that the complexity of bifunctionals can raise significant challenges for compound purification and analysis. Such molecules often require multistep purification because they exhibit high polarity, low solubility, and poor stability in the presence of common chromatography modifiers. Experience, systematic method development, and extensive application of triple-quadrupole MS and ultraperformance liquid chromatography (UPLC) have helped WuXi AppTec to identify appropriate analytical protocols for bifunctionals.
WuXi AppTec is augmenting its capabilities for biophysical characterization to support validation of a bifunctional’s mechanism of action (MoA) and mapping of its protein–ligand interactions. Madge explained that characterization tools are important not only for determining how a linker molecule interacts singly with a ligase and target protein, but also for evaluating formation of the ternary complex that enables the molecule’s degradative function. Surface plasmon resonance (SPR) can determine the strength and longevity of a protein– ligand interaction during ternary complex formation. Microscale thermophoresis by temperature-related intensity change (MST-TRIC) quantifies a degrader molecule’s affinity for a ternary complex. And X-ray crystallography can visualize such complexes at high resolution, enabling rational ligand optimization for one or both sides of a bifunctional molecule. These methods also help biochemists to enhance bifunctional stability and permeability into dense tissue bases.
Several tools support in vitro studies of bifunctional activity. Time-resolved fluorescence-resonance energy transfer (TR-FRET) requires careful preparation and optimization, Madge noted, but it is valuable for early screening of candidate ligands. Cell-engagement assays using platforms such as the NanoBRET system (Promega) help to evaluate degradermolecule permeability. AlphaLISA assays (Perkin Elmer) nicely complement biophysical techniques in visualizing ternary-complex formation. To demonstrate in-cell target degradation, WuXi AppTec has adapted the HiBiT protein-tag system to enable end-point analysis of the extent and kinetics of a protein’s degradation. That platform provides a rapid and efficient solution for nondruggable targets in difficult-tohandle primary immune cells. Meanwhile, quantitative immunofluorescent assays (or “in-cell Western blots”) can confirm a degrader’s functional effects quickly and easily.
WuXi AppTec is extending its collection of E3 ligases to help improve bifunctional selectivity and potency. The company also is bolstering its computational capabilities to advance linker and ligand discovery further.