IC50 Methodology

This page describes the curve fitting algorithm, quality control metrics, and classification system used to calculate and evaluate IC50 values.

Dose-Response Curves

Dose-response experiments measure the cellular response (typically luminescence) to varying concentrations of a compound. The relationship between concentration and response typically follows a sigmoidal (S-shaped) curve.

The application supports two assay types:

  • Reporter Assay — Measures transcriptional activity
  • CTG (CellTiter-Glo) — Measures cell viability

IC50 Calculation

IC50 (half maximal inhibitory concentration) is the concentration of a compound that produces 50% inhibition of the maximum response. It is a key metric for compound potency.

The application provides two sources of IC50 values:

  1. CRO IC50 — Provided directly from the CRO experiment data
  2. Calculated IC50 — Derived by fitting a dose-response curve to the raw measurement data

Curve Fitting Models

The system fits two competing sigmoid models and selects the best one using AICc (corrected Akaike Information Criterion).

4-Parameter Logistic (4PL) Model

y = bottom + (top - bottom) / (1 + (x / IC50)^hill)

Where:

  • y = response (luminescence)
  • x = concentration
  • bottom = minimum response (lower asymptote)
  • top = maximum response (upper asymptote)
  • IC50 = concentration at 50% response
  • hill = Hill slope (steepness), constrained to [0.1, 5.0]

3-Parameter Logistic (3PL) Model (bottom = 0)

y = top / (1 + (x / IC50)^hill)

This model assumes complete inhibition (bottom fixed at zero). It is useful for compounds that fully suppress the response and is selected when it fits as well as or better than 4PL by AICc.

Model Selection via AICc

For each curve, the system:

  1. Fits both 4PL and 3PL models using robust optimization (soft_l1 loss)
  2. Computes AICc for each model (penalizes complexity while rewarding fit)
  3. Selects the model with the lower AICc
  4. Compares the selected sigmoid model against a null (flat) model

The sigmoid model is accepted only if: delta_AICc = AICc(sigmoid) - AICc(null) ≤ -10 — ensuring the sigmoid is substantially better than a flat line.

Quality Control Metrics

Metric Formula Pass Threshold Interpretation
Efficacy (baseline - min_response) / baseline ≥ 0.20 (20%) Fraction of response reduction from baseline
SNR (max_response - min_response) / pooled_SD ≥ 3.0 Signal strength relative to measurement noise
Monotonicity Spearman correlation of log(dose) vs response rho < 0 and p < 0.05 Statistically significant decreasing trend
Model test AICc(sigmoid) - AICc(null) ≤ -10 Sigmoid model is substantially better than flat line

IC50 Censoring

IC50 values are flagged based on whether they fall within the tested concentration range:

Status Condition Interpretation
None IC50 within tested range IC50 is reliable
Left-censored IC50 < minimum tested concentration Compound is very potent; IC50 is an upper bound
Right-censored IC50 > maximum tested concentration Compound shows weak/no activity; IC50 is a lower bound

Classification System

Based on the QC metrics, each curve is automatically classified:

Classification Criteria Meaning
ACTIVE Passes all 4 QC tests (model, monotonicity, efficacy, SNR) High-quality dose-response with reliable IC50
PARTIAL Passes model and monotonicity, but fails efficacy OR SNR Detectable response but weaker signal or incomplete inhibition
INACTIVE Fails monotonicity OR model comparison No significant dose-response detected
UNCERTAIN Edge cases not fitting other categories Requires manual review

CRO IC50 Classification

For CRO-provided IC50 values, classification is based on the value prefix:

  • IC50 with > prefix (e.g., ">30000") → INACTIVE (right-censored, compound exceeded max tested concentration)
  • IC50 without > prefix → ACTIVE

Percent Inhibition

Percent inhibition is calculated differently for CRO and calculated values:

Source Formula
Calculated ((baseline - min_response) / baseline) × 100
CRO ((baseline - max_conc_response) / baseline) × 100
Note: The CRO uses the response at the maximum concentration, while the calculated method uses the minimum response across all concentrations. This can lead to differences between CRO and calculated percent inhibition values.

Interpretation:

  • 100% = complete inhibition (response reduced to zero)
  • 50% = half-maximal inhibition
  • 0% = no inhibition
  • Negative values = activation/stimulation