Activity Assays
Activity Assays for Drug Discovery
WuXi Biology and Testing offers a comprehensive suite of biochemical activity assays to determine whether a test compound inhibits, activates, or otherwise modulates its target's catalytic or biological function. Leveraging purified proteins, recombinant enzymes, and reconstituted cell-free systems, our teams generate quantitative potency (IC50/EC50) selectivity, and mechanistic data across diverse target classes, including kinases, proteases, phosphatases, deubiquitinases, ATPases, and E3 ligases.
Our platforms combine luminescent, fluorescent, and energy-transfer technologies (including ADP-Glo, Kinase-Glo, PhosphoSens, LANCE, TR-FRET/HTRF, Caliper, Z'-LYTE, Luminescence assay, Fluorometric assay, etc.) to support hit identification, lead optimization, selectivity profiling, and mechanism-of-action studies. Activity assays can be integrated with biophysical characterization and cell-based studies to provide a complete understanding of target modulation across multiple modalities including small molecules, peptides, oligonucleotides, biologics, conjugates, and bifunctional molecules..
Biochemical Assay Capabilities at a Glance Across Modalities
Targets With Established Biochemical Activity Assay Conditions
Biochemical Assays Available
Typical Turnaround for Ready-to-Go Assays
Enzymatic and Biochemical Activity Assays
Biochemical activity assays directly quantify target activity in controlled systems, measuring catalytic turnover, substrate conversion, cofactor consumption, or product formation. Unlike binding assays, which establish target engagement, activity assays determine whether a molecule alters the biological activity of its target.
Our biochemical platforms include:
- ADP-Glo and Kinase-Glo: luminescent ATP/ADP assays for ATP-dependent enzymes. ADP-Glo reports ADP production; Kinase-Glo measures residual ATP. High sensitivity and wide dynamic range.
- LANCE, and TR-FRET/HTRF assays: homogeneous time-resolved FRET assays using labeled reagents to detect phosphorylation, cleavage, or product formation with low background and tolerance to compound fluorescence.
- Caliper, Z'-LYTE, and ADP-Quest: orthogonal technologies for SAR and selectivity. Caliper separates substrate/product directly; Z’-LYTE is an endpoint FRET kinase assay; ADP-Quest detects ADP via enzyme coupling.
- Protease, deubiquitinase, phosphatase, and ATPase assays: class-specific fluorogenic, chromogenic, or luminescent formats for sensitive kinetic readouts.
Biochemical Activity Assays Across Drug Modalities
Functional and biochemical readouts vary depending on the therapeutic modality and mechanism of action. Our teams select and optimize assay formats that best capture target modulation and pharmacological activity:
- Small molecules: enzymatic inhibition and selectivity assays to determine potency (IC₅₀, EC₅₀, Kᵢ), mechanism of inhibition (e.g., competitive, allosteric, irreversible), and kinetic parameters.
- Biologics and antibodies: neutralization and biochemical assays that measure blockade of ligand–receptor interactions, inhibition of enzymatic activity, or modulation of signaling pathways.
- Antibody–drug conjugates (ADC, PDC, AOC, etc.): enzymatic cleavage and payload-release and biochemical activity assays that evaluate linker stability, payload integrity, and target-dependent activity.
- Oligonucleotides: assays assessing nuclease resistance, target RNA cleavage efficiency, target knockdown, and sequence-specific activity.
Bifunctional molecules (PROTAC, molecular glue, etc.): In vitro ubiquitination and protein degradation assays quantifying degradation potency (DC₅₀) and maximal degradation (Dmax).
Building Confidence with Orthogonal Biochemical Data
A single biochemical readout rarely provides a complete picture of target modulation. Robust drug discovery programs integrate complementary assay formats to confirm potency, mechanism, and selectivity.
- Primary activity assay: establishes potency (IC₅₀, EC₅₀) and maximal modulation in a purified biochemical system.
- Orthogonal biochemical assay: confirms activity using an alternative detection principle to rule out false positives and assay artifacts.
- Biophysical characterization: SPR, MST, ITC, nanoDSF, and Native MS confirm target engagement and binding kinetics.
- Cellular confirmation: functional cell-based assays translate biochemical findings into physiologically relevant systems.
- Structural characterization: X-ray crystallography and Cryo-EM reveal binding modes to drive structure-based optimization.
This layered strategy differentiates genuine modulators from assay interference (e.g., PAINS), aggregation effects, and detection artifacts.
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Frequently Asked Questions
An activity assay measures the functional consequence of a compound's interaction with its target whether it inhibits or activates an enzyme's catalytic output, blocks or triggers receptor signaling, or otherwise changes what the target does, as a compensation to a binding assay, which measures only whether the interaction occurs.
Binding assay confirms physical engagement, proving a compound directly interacts with its target. An activity assay goes further, measuring the pharmacological outcome of that engagement — such as enzyme inhibition, receptor activation, or downstream signaling. Most discovery programs run both: binding assays to confirm target engagement, and activity assays to confirm this interaction drives biological modulation..
ADP-Glo, Kinase-Glo, PhosphoSens, LANCE, TR-FRET/HTRF, Caliper, Z'-LYTE, Luminescence assay, Fluorometric assay, etc. for IC₅₀ determination. The right format depends on the target class, substrate, and throughput needs.
Yes. Activity data is most valuable when evaluated alongside biochemical binding assays, biophysical confirmation, or cell-based functional readouts to establish a comprehensive picture of how a candidate engages and modulates its target, and to confirm results aren't driven by platform-specific artifacts or false positives.
Our platform supports multiple targets such as kinases, proteases, deubiquitinases, phosphatases, receptors, across different modalities, with well-validated Ready-to-Go activity assays to enable rapid project initiation.
For targets with pre-validated Ready-to-Go assays, programs can typically begin generating screening data within about two weeks.
Novel or more complex targets may require customized assay development, which our team defines at the start of the project.