MoA Profiling Service

Understanding precisely how a drug candidate engages its target, modulates cellular signalling networks, and affects the wider proteome is fundamental to successful drug discovery. Traditional single-target assays provide limited mechanistic depth, often missing compensatory pathway activation, off-target pharmacology, or polypharmacology that may determine preclinical advancement outcomes. Our MoA Profiling service deploys three complementary mass spectrometry-based platforms — phosphoproteomics and kinase activity profiling, thermal proteome profiling (TPP), and interaction proteomics (AP-MS) — to deliver a comprehensive, systems-level view of drug mechanism from direct target engagement through downstream pathway effects.

• Phosphoproteomics & Kinase Activity Profiling: Quantitative phosphoproteomics with TiO2/IMAC enrichment identifies thousands of regulated phosphorylation sites upon drug treatment. Kinase-substrate enrichment analysis and kinase activity inference algorithms translate phosphosite-level changes into pathway-level mechanistic understanding, revealing on-target pathway modulation, compensatory signalling, and resistance mechanisms. SILAC-based or label-free quantification with FDR-controlled statistics ensures high-confidence phosphosite assignment.
• Thermal Proteome Profiling (TPP): Multiplexed TMT-based TPP detects drug-induced changes in protein thermal stability across the proteome, providing direct biophysical evidence of target engagement in cellular context without requiring chemical modification of the drug. Ten-temperature gradient profiling with melt-curve fitting and ΔTm quantification for 6,000–8,000 proteins identifies both the intended target and additional proteins stabilised or destabilised by drug treatment.
• Interaction Proteomics (AP-MS): Affinity purification coupled with LC-MS/MS identifies drug-induced changes in protein-protein interaction networks. Antibody-based or epitope-tag pull-down of the target protein followed by quantitative proteomics distinguishes specific interactors from background, revealing drug-dependent complex assembly, disassembly, or recruitment that informs mechanism and potential toxicity.

Understanding MoA Profiling in Drug Discovery

Mechanism of action studies address a fundamental question in drug discovery: how does a compound produce its biological effect? The answer is rarely limited to a single target interaction. A drug may bind its intended target with high affinity while also engaging related family members, modulating downstream signalling pathways, inducing adaptive cellular responses, or re-wiring protein interaction networks. Comprehensive MoA profiling provides the experimental evidence needed to distinguish intended pharmacology from off-target effects, identify the most therapeutically relevant pathways, and guide medicinal chemistry optimisation from a mechanism-informed perspective.

Mass spectrometry has become the platform of choice for systems-level MoA profiling because it delivers unbiased, proteome-wide readouts across multiple dimensions of drug action — from direct target binding to pathway-level signalling responses. For researchers beginning with broader target deconvolution investigations, our Chemoproteomics platform provides complementary workflows for activity-based protein profiling, while the dedicated Target Validation Proteomics service offers deeper integration of orthogonal validation approaches for confirmed targets.

Platform Selection: Choosing the Right MoA Profiling Strategy

The choice of profiling platform depends primarily on the drug modality and the specific mechanism-of-action questions that need to be addressed. Each platform captures a distinct dimension of drug action, and the most informative MoA studies often combine two or more approaches to build a complete mechanistic picture.

Phosphoproteomics and kinase activity profiling is the method of choice for kinase inhibitors, targeted signalling modulators, and any compound where understanding pathway-level effects is critical. By quantifying thousands of phosphorylation sites and inferring kinase activity changes, this approach reveals not only whether the intended pathway is engaged but also whether compensatory or resistance pathways are activated — information that can directly inform combination therapy strategies.

Thermal Proteome Profiling is the most broadly applicable approach, compatible with any drug modality without requiring chemical modification. TPP provides direct biophysical evidence of protein binding in cellular context, making it the ideal first-line approach for target engagement confirmation and for identifying unexpected off-target interactions that may contribute to pharmacology or toxicity. TPP data also complements phosphoproteomics findings by distinguishing direct targets from downstream signalling effects.

Interaction proteomics provides a unique third dimension by revealing how drug treatment alters the interaction network of the target protein and its pathway neighbours, identifying changes in complex composition, post-translational modification-dependent interactions, and recruitment of downstream effectors that may be critical for therapeutic efficacy. For comprehensive MoA programmes requiring all three dimensions, our integrated workflow combines enrichment strategies from our Chemoproteomics and Target Validation Proteomics platforms into a unified experimental design.