Biochemical Secondary Assays for Hit Qualification and Mechanistic Validation
Completion of primary screening is a key milestone, but it marks only the beginning of a rigorous validation process designed to confirm on-target activity, assess selectivity, and elucidate the mechanism of action. We develop secondary assays with the throughput and quality parameters required to handle large compound libraries to effectively prioritize your hit compounds.
Hit validation assays
Orthogonal validation: We confirm on-target activity using spectroscopically divergent detection technologies. If a compound is active in both the primary and orthogonal assays, its on-target effect is corroborated by definition.
Diverse technology suite: Our platforms include luminescence, Fluorescence Intensity (FI), TR-FRET, Fluorescence Polarization (FP), and alpha technology to ensure results are not detection-technology dependent.
Concentration-response & reproducibility: We ensure hit reliability through rigorous potency determination and reproducibility testing, advancing only the most robust scaffolds.
Interference and artifact detection assays
False positive identification: We design assays to flag compounds that interfere with spectroscopic properties or readout systems.
Target-omission methodology: By replacing the target enzyme with the reaction product, we identify compounds affecting the detection system rather than the biological target.
PPI artifact detection: For protein-protein interaction (PPI) projects, we utilize molecules that mimic a “100% assembled” complex to filter out non-specific interferents. Axxam maintains a curated set of reference PAINS compounds to validate assays for PAINS-like behaviors.
Off-target and selectivity panels
Family & paralogue panels: We evaluate selectivity across entire classes (e.g., Kinases, Proteases, and HDACs) to ensure your compound hits the right target within a protein family.
Species cross-reactivity: We utilize orthologous counterparts from different species to anticipate results for upcoming pharmacological studies.
Target variants: We screen against disease-relevant mutants, splice variants, or recombinant versions with different regulatory domains.
Secondary pharmacology
We assess the selectivity and specificity of hit compounds by identifying off-target activities that may cause adverse reactions or side effects. This evaluation screens compounds against unintended proteins beyond the primary target, helping to predict and mitigate safety risks early in discovery.
A panel of ready-to-use biochemical assays is available at Axxam to support secondary pharmacology studies, including assays for cardiovascular, CNS, metabolic, and immune response targets.
Mechanism of action studies
Mechanistic insights: By using alternative substrates and interaction partners, we expand the description of compound selectivity and physiological roles.
Functional enzymology for kinetic & covalent analysis: We offer deep-dive characterization for advanced Mechanism-of-Action (MOA) studies, including:
- Competitive vs. non-competitive and allosteric modulation analysis, including substrate concentration-dependent IC50.
- Kinetic profiling: Measurement of association/dissociation rates, catalytic efficiency, and time-dependent IC50 for reversible inhibitors.
- Residence time & irreversible inhibitors: Determination of τ and kinetic parameters (kinact/Ki) for covalent or slowly dissociating compounds, including access to specialized libraries, i.e., Axxam’s proprietary covalent collection.
