Dealing with PAINs in a drug discovery CRO D Swift, N Bland, R Lane, D Laughton, R Satchell, L Birch, S Pollack, J Unitt Sygnature Discovery Ltd, BioCity, Pennyfoot Street, Nottingham NG1 1GF, United Kingdom d.swift@sygnaturediscovery.com Abstract ID:                        146480 Overview: Pan-Assay INterference compounds (PAINs) is a term used to describe a broad range of compounds that interfere with biological screening assays by acting through a range of mechanisms (see box “what are PAINS”). As such, PAINs are frequent hitters in screens, producing often unknowingly false positives that could waste much effort in drug discovery research, although almost 4% of FDA approved drugs are known promiscuous aggregators, indicating that PAIN activity does not preclude a compound as a potential therapeutic. At Sygnature Discovery, rather than removing known PAINs from screening libraries, which could miss legitimate hits, we have implemented a target-specific systematic strategy to avoid, identify and characterize false positives during the hit identification and validation stages of a drug discovery campaign. We illustrate these approaches with two case studies which highlight two common PAIN mechanisms: detection technology interference and redox activity due to compound impurities. What are PAINs? Frequent hitters, False positives, Non-specifics Many mechanisms Impurities e.g. heavy metals, reactive contaminants Multiple binding sites on enzyme Protein destabilisers Form molecular aggregates Technological interference (e.g. fluorescence quench) Redox: Generate reactive species Reactive Widespread in biochemical enzyme activity assays PAINs are often not recognized as non-specific  Confusion & wasted time Rhodanines A family of compounds that are frequently found to be non-specific hits in high throughput screens Identifying PAINs: IC50 > 3µM Steep concentration-inhibition curve (super stoichiometric) Non-competitive Irreversible Flat SAR Time-dependent Sensitive to detergent (aggregators) Active in unrelated screens-metal ion chelators No comparable activity in orthogonal assay Assay with different physical basis: e.g. binding v. activity A known or predicted PAIN structure Case Study 2: Dealing with PAINs in a high throughput screen (HTS) 244,000 diverse compounds were screened at 10 µM against a metabolic enzyme (with an active site thiol) target as part of a drug discovery program. Conditions in the HTS were tailored to minimize the potential for false positives. Counter-screening for PAIN identification: In addition, a set of counter-screens was introduced to identify PAINS. A significant source of false positives were compounds that showed redox activity to due contamination. Redox activity can be detected by the reduction of resazurin in the presence of a reductant such as the cysteine in our assay buffer. Results of HTS and follow-up: Of the 244,000 compounds 150 of these had IC50 values of ≤ 60 µM against the main target. These 150 actives were then analysed in a series of counter-screens, including the redox assay described above, with the following outcomes: 89 compounds with PAIN structural flag; 122 with no PAIN counter-screen liabilities for follow-up. Conclusions: A clear counter-screening cascade following the HTS allowed for an efficient removal of PAINs and triaging of actives to take forward as validated hits. Eflornithine PAIN-like characteristics (irreversible with IC50>20µM) Used to treat African trypanosomiasis Assay condition/screen Purpose L-cysteine in buffer Quench thiol-reactive compounds [Tween] 0.01% Minimise effects of aggregators Case Study 1: Lysine Specific Demethylase 1 (LSD1) Epigenetic target Primary screen H2O2 peroxidase-coupled to resorufin fluorescence 2 hit series identified from HTS Some SAR Hit to Lead: Orthogonal screen: HCHO Production HCHO inhibition  Counter-screen: HRP+H2O2 No HCHO inhibition  An Amplex Red technology counter-screen excluded series 1 but not series 2 An additional orthogonal assay based on HCHO product identified series 2 as a PAIN. Counter-screen Purpose Outcome Resazurin redox assay Identify redox-active PAINS 15% with redox activity ≤ 50 µM ADP-GloTM assay w/o enzyme Identify detection step interferers 3% with ADP-GloTM activity ≤ 50 µM Unrelated Cys enzyme assay Flag for nonspecific thiol-reactive compounds 27% with IC50 ≤ 10 µM 6 fold increase [Tween] Identify aggregators 1% with potency shift of ≥ 10-fold Dealing with PAINs as a CRO: The case studies described above demonstrate the importance of recognising PAINs early on during a drug discovery campaign. Case study 1- stopped the project early Saving client time and importantly costs associated with pursuing nuisance compounds. Case study 2 – PAIN management Multiple classes of PAINS identified, efficiently triaging compounds for hit to lead optimisation Orthogonal screens Employ FTSA and SPR as complementary techniques, not only to measure binding affinity, but to identify small molecules with undesirable mechanisms. Profiled common types of PAIN in both technologies and these data are a valuable reference when screening novel compounds. Summary: Aim to identify and flag PAINS using: Publications on new hits Use prospective tools to identify PAIN risk But keep PAINS in screening sets 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 chemistry at risk to increase SAR confidence Mechanism of Inhibition Once hit series structure/activity confirmed Share the PAIN Publicize PAIN management best practice Primary screen Orthogonal screens: biophysics Chemical filtering Confirm structure and activity Clear SAR MOI Studies Hit Series Minimise effects: Detergent, chelator Resynthesis repurification Rescreen Flag potential PAINS Not super stoichiometric No technological interference Inhibition = binding Identify non-specifics Chemical clustering Reversible/irreversible Competitive/non-competitive Detergent sensitive Medicinal chemist input Compound binding Kinetics by SPR Compound binding by FTSA