CYP Inhibition | IC50 Shift

About the Assay

The IC₅₀ Shift assay is designed to identify and characterise time‑dependent inhibition (TDI) of drug‑metabolising CYP450 enzymes using human liver microsomes. TDI represents an important mechanism of drug–drug interactions, arising when an inhibitor becomes more potent following pre‑incubation with NADPH‑activated metabolic systems. This behaviour can indicate quasi‑irreversible or irreversible enzyme modification, resulting in prolonged inhibition and increased risk of clinical exposure changes. The assay covers seven CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) across eight probe substrates, each selected according to FDA guidance for in vitro inhibition studies. By comparing inhibitor potency with and without a pre‑incubation step, the assay differentiates reversible inhibition from TDI.

Protocol Summary

Seven test compound concentrations are incubated with human liver microsomes with or without a 30-minute pre-incubation in the presence and absence of NADPH. Following this, an incubation is performed in the presence of the isoform-specific probe substrate at 37 °C shaking at 700 rpm. Selective CYP TDIs are screened alongside the test compounds as positive controls.  Reactions are terminated by sampling into cold MeCN before being centrifuged at 3,000 rpm for 30 minutes at 4 °C. Formation of the metabolite is monitored by LC-MS/MS.

Formation of metabolite is measured in each sample and a percent activity calculated, 100% is defined as the metabolite formed in the incubation containing vehicle with no test compound. Non-linear regression is used to fit a four-parameter logistic curve to a plot of percent activity against concentration of test compounds (where test concentration is plotted on a log scale). An IC₅₀ value is obtained, this is the concentration of test compound at which 50% inhibition is achieved. The fold-shift (increase in potency) is calculated by taking the IC₅₀ of the 30-minute pre-incubation without NADPH and dividing by the IC₅₀ of the 30-minute pre-incubation with NADPH. A fold-shift of ≥2 indicates TDI. Positive control compounds with known TDI against each isoform are included

Validation Results

Validation demonstrated that the IC50 Shift assay is a reliable, consistent and well‑controlled platform for identifying TDIs across seven CYP isoforms. The validation strategy assessed intra‑ and inter‑assay variability by repeating studies across three independent occasions with multiple replicates. IC₅₀ determinations were consistent across all isoforms, demonstrating strong reproducibility and confirming that the assay can reliably detect both reversible inhibitors and TDIs.

Table 1 summarises the validated assay conditions for each isoform, including substrates, inhibitors, concentration ranges and protein levels for each isoform. These optimised conditions reflect the outcomes of prior kinetic characterisation and ensure accurate determination of IC₅₀ shifts. Collectively, the results confirm that the IC₅₀ Shift assay provides a sensitive and dependable means of screening for CYP‑mediated TDI, supporting risk assessment in early drug discovery and development.