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PAINS (Pan-Assay INterference compounds)

  • What are PAINS?
  • Pan-Assay INterference compounds
  • Frequent hitters, False positives, Non-specifics
  • Use multiple, different sites on enzyme

Widespread in biochemical HI enzyme activity assays

  • “Painsare unpleasant feelings often caused by intense or damaging stimuli

Often not recognised as non-specific ⇒ Confusion & wasted time

Chemical Con Artists

Adapted from Baell & Walters Nature 513, 481–483

Many mechanisms

  • Compound and/or its impurities
  • e.g. heavy metals, reactive contaminants
  • Technology interference
  • Fluorescence quench
  • Form molecular aggregates
  • Redox
  • Generate reactive species
  • Reactive
  • Promote denaturation by binding unfolded protein

Identification Guidelines

  • IC50 > 3µM
  • Steep concentration-inhibition curve (super stoichiometric)
  • Non-competitive
  • Irreversible
  • Time-dependent
  • Active in unrelated screens
  • Sensitive to detergent (aggregators)
  • No comparable activity in orthogonal assay
  • Assay with different physical basis: e.g. binding v. enzyme activity
  • A known or predicted PAIN structure
  • Flat SAR

Avoidance

  • Don’t remove from screening set
  • Some PAINS are potent drugs and hits
  • Chelators
  • To remove heavy metal impurities
  • Detergent
  • To disrupt aggregates
  • Check with DLS
  • Reductant
  • To mop up oxidants
  • Ensure enzyme is stable
  • FTSA to ID optimal conditions for stability (buffer, pH, ionic strength etc.)
  • Expect low hit rate for known non-specifics

Lysine-Specific Demethylase 1 Inhibitor

  • Epigenetic target
  • Primary screen
  • H2O2 detection: peroxidase-coupled to resorufin fluorescence
  • 2 hit series identified from HTS
  • Some SAR…………

 

Hit Identification

 

Deconstruct LSD1 H2O2 Assay

  • Peroxidase interferes with compound inhibition of LSD1
    • Primary HTS as parallel LSD1/Peroxidase assay
    • Co-addition to enzyme/substrate and product detection reagents
  • Re-formatted as serial 2-step assay for comparison

 

A Generic PAIN-Free Screening Plan?

  • Key elements
    • PAIN primary assay
    • Binding ≡ Biochemical
    • Structure with activity
    • SAR
    • MOI
  • Multidisciplinary
    • Bioscience
    • Biophysics
    • Chemistry

Position Statement

  • Aim to identify and avoid PAINS in…..
    • Early drug discovery
    • Publications on new hits
  • Keep PAINS in screening sets and flagged
    • Use prospective tools to identify PAIN risk
  • Develop a robust PAIN assay cascade specific to target
    • Primary screen (to avoid PAINs)
    • Orthogonal screen (enzyme binding and activity)
  • SAR
    • Confirm structure and activity
    • Early synthetic chemistry at risk to increase SAR confidence
  • MOI
    • Once hit series structure/activity confirmed
  • Share the PAIN
    • PAIN management best practice & learning

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