Application of Affinity Selection Mass Spectrometry: Molecular Glues Drug Discovery
Introduction:
New modalities, such as molecular glues, require robust screening strategies to identify and develop the most promising candidates efficiently. In this article, we spotlight Affinity Selection Mass Spectrometry (ASMS), a versatile and cost-effective high-throughput screening technique used in drug discovery.
ASMS: A Powerful High-throughput Screening Technology
Since the mid-1990s, ASMS has emerged as a powerful high-throughput screening (HTS) tool used in small-molecule drug discovery to identify novel ligands targeting therapeutic proteins. It leverages the binding affinity of small molecules to the active sites on target proteins and separates bound complexes from unbound small molecules via size-exclusion chromatography. The bound complexes are subsequently identified using reverse-phase liquid chromatography and mass spectrometry (Figure 1).
Unlike traditional high-throughput screening (HTS) methods, ASMS allows for the simultaneous evaluation of multiple compounds. Furthermore, ASMS is not constrained by the requirement for specific biological assays to identify active compounds; it can be applied to any soluble target, including single proteins, protein complexes, and RNA. Leveraging high-resolution mass spectrometry, ASMS achieves detection rates of 0.5% to 1.5% per screening, significantly reducing the workload for subsequent chemical synthesis and improving overall drug discovery efficiency. Additionally, its unbiased screening approach enables the optimization and validation of multiple candidate molecules. The use of positive controls and multi-condition screening strategies minimizes false positives, thereby increasing both reliability and success rates.
Figure 1: ASMS Screening Workflow
ASMS Facilitates the Discovery of Molecular Glues
Molecular glues are a class of small molecules that stabilize protein-protein interactions by binding at the interface between an E3 ubiquitin ligase and a target protein, precisely promoting targeted ubiquitination and subsequent degradation.
Unlike PROTACs (Proteolysis Targeting Chimeras), which consist of two distinct ligand domains connected by a variable linker (Figure 2), molecular glues have simpler, lower-molecular-weight structures that enhance drug-like properties, particularly for previously undruggable targets. As a novel approach to targeted protein degradation, molecular glues offer a unique mechanism of action, making them a promising strategy in small-molecule drug discovery.
Figure 2: Mechanism of Action of Ubiquitin-Proteasome System (UPS) in Protein Degradation [1]
Recent studies have highlighted the potential of CDK12-targeting molecular glues in cancer therapy. For example, the molecular glue HQ461 has been shown to facilitate interactions between CDK12 and the DDB1-CUL4-RBX1 E3 ubiquitin ligase complex. This interaction results in the ubiquitination and subsequent degradation of cyclin K (CCNK), impairing CDK12 function, downregulating DNA damage response genes, and inducing tumor cells’ death (Figure 3).
Figure 3: Mechanism of Action of HQ461[2]
Additionally, CR8 was identified as a molecular glue that binds to the CDK12-cyclin K complex through a solvent-exposed pyridine group in its structure. This binding promotes interaction between CDK12 and DDB1, acting as a molecular bridge that stabilizes a large protein-protein interface. This mechanism bypasses the traditional requirement for an E3 ubiquitin ligase substrate receptor, leveraging the E3 ligase directly to mediate the ubiquitin-dependent degradation of cyclin K. Both examples demonstrate how molecular glues selectively target CDK12 for degradation by inducing specific protein complexes.
Although the potential of CDK12-targeting molecular glues has been recognized in both research and clinical settings, there are still limited methods for systematically identifying and designing these molecules. To address this, researchers employed ASMS, processing samples in batches of 400 to enhance detection efficiency and minimize protein consumption. From WuXi AppTec’s library of 2,000 small molecules, two compounds were identified that promote CDK12 degradation at concentrations as low as 0.5 µM/cmpd. ASMS confirmed significant binding of these compounds to the CDK12/CCNK/DDB1 complex (Figure 4). The highly automated ALIS-ASMS system, requiring minimal sample pretreatment, reduced human error and ensured objective results.
Subsequent crossover studies validated these findings using Surface Plasmon Resonance (SPR), which measured ternary binding affinities of 3 µM and 22 µM, and HTRF assays which confirmed that both small molecules significantly promote the formation of the CDK12/CCNK/DDB1 complex (Figure 4). These results underscore the strong potential of the ASMS screening platform for molecular glue discovery, offering efficient and integrated techniques to meet diverse research and development needs.
Figure 4: ASMS for Molecular Glue Discovery Targeting the CDK12/CCNK/DDB1 Complex
ASMS Platform at WuXi Biology
Established in 2018, the ASMS platform at WuXi Biology is backed by a library of over 270,000 small molecules and offers customized compound screening services. The platform utilizes automated workflows, high-resolution mass spectrometry, efficient data processing software, and standardized protocols. For a single target, it can screen up to 200,000 compounds within 3-4 weeks, with comprehensive validation through functional crossover studies. The platform provides dissociation constant (KD) values to accurately assess small molecule-protein binding affinity, ensuring precise ligand-binding measurements.
References
[1] ZHANG Xiao-Yuan,ZHANG Yan-Yan,SUN Xiao-Kang,ZHANG Lin-Jun,CHEN Mian,LIU Fei. Research Progress of Targeted Protein Degradation Technology[J]. Progress In Biochemistry and Biophysics, 2022, 0(1)
[2] Lv, Lu, et al. “Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation.” Elife 9 (2020): e59994.
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