Assay Interference by Chemical Reactivity

Real and virtual high-throughput screens (HTS) are crucial components of the modern drug discovery process. Assays that determine the biological activity of compounds found by both of these approaches may be subject to interference by a variety of processes, one of which is non-specific chemical reactivity. Within this mode of assay interference, test compounds can chemically react with assay reagent(s) or biological molecule(s), confounding the assay readout by producing apparent biological activity. Failure to identify and triage (remove from consideration) these compounds can result in wasted resources and project dead-ends. This chapter contains practical strategies to mitigate the impact of assay interference by compound chemical reactivity. Two general approaches are described to identify and triage reactive compounds in the context of drug discovery and development: knowledge-based and experimental-based methods. Knowledge-based strategies covered in this chapter include substructure filters, literature and database searches, and the consulting of experienced medicinal chemists. Experimental-based strategies covered in this chapter include mechanistic experiments, thiol-based probes and multiple types of counter-screens. Employing a well-designed and comprehensive screening tree that incorporates these strategies should reduce the likelihood of pursuing reactive assay artifacts and intractable hits from HTS campaigns.

[1]  A. Stoppani,et al.  Redox cycling of beta-lapachone and structural analogues in microsomal and cytosol liver preparations. , 2004, Methods in enzymology.

[2]  J. Denu,et al.  Site-Specific Reactivity of Nonenzymatic Lysine Acetylation , 2015, ACS chemical biology.

[3]  Olivier Sperandio,et al.  FAF-Drugs3: a web server for compound property calculation and chemical library design , 2015, Nucleic Acids Res..

[4]  D. Ehmann,et al.  Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor , 2012, Proceedings of the National Academy of Sciences.

[5]  C. Crews,et al.  From epoxomicin to carfilzomib: chemistry, biology, and medical outcomes. , 2013, Natural product reports.

[6]  J. Baell.  Observations on screening-based research and some concerning trends in the literature. , 2010, Future medicinal chemistry.

[7]  S. Thrall,et al.  A Simple Assay for Detection of Small-Molecule Redox Activity , 2007, Journal of biomolecular screening.

[8]  A. Becker,et al.  Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases. , 2013, Journal of medicinal chemistry.

[9]  P. Sachdev,et al.  Inhibition of indoleamine 2,3 dioxygenase activity by H2O2. , 2006, Archives of biochemistry and biophysics.

[10]  Ming Li,et al.  Small‐Molecule APOBEC3G DNA Cytosine Deaminase Inhibitors Based on a 4‐Amino‐1,2,4‐triazole‐3‐thiol Scaffold , 2013, ChemMedChem.

[11]  R. Hartmann,et al.  A detective story in drug discovery: elucidation of a screening artifact reveals polymeric carboxylic acids as potent inhibitors of RNA polymerase. , 2013, Chemistry.

[12]  Ian A. Watson,et al.  Rules for identifying potentially reactive or promiscuous compounds. , 2012, Journal of medicinal chemistry.

[13]  Erica M. Stec,et al.  A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production. , 2008, Assay and drug development technologies.

[14]  Jayme L. Dahlin,et al.  The essential roles of chemistry in high-throughput screening triage. , 2014, Future medicinal chemistry.

[15]  H. R. Evans,et al.  Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme. , 2004, Biochemistry.

[16]  W. Patrick Walters,et al.  A guide to drug discovery: Designing screens: how to make your hits a hit , 2003, Nature Reviews Drug Discovery.

[17]  Adam Yasgar,et al.  Evaluation of micro-parallel liquid chromatography as a method for HTS-coupled actives verification. , 2007, Assay and drug development technologies.

[18]  D. Epps,et al.  A competitive fluorescence assay to measure the reactivity of compounds. , 2001, Analytical biochemistry.

[19]  Meenal Patel,et al.  PTC124 targets genetic disorders caused by nonsense mutations , 2007, Nature.

[20]  Jonathan B Baell,et al.  Screening-based translation of public research encounters painful problems. , 2015, ACS medicinal chemistry letters.

[21]  Robert A Copeland,et al.  Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. , 2005, Methods of biochemical analysis.

[22]  Rajarshi Guha,et al.  KNIME Workflow to Assess PAINS Filters in SMARTS Format. Comparison of RDKit and Indigo Cheminformatics Libraries , 2011, Molecular informatics.

[23]  Scott B Ficarro,et al.  A structure-guided approach to creating covalent FGFR inhibitors. , 2010, Chemistry & biology.

[24]  J. Poncet,et al.  Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor α , 2006, The Journal of Steroid Biochemistry and Molecular Biology.

[25]  J. Cooke,et al.  A Novel and Potent Inhibitor of Dimethylarginine Dimethylaminohydrolase: A Modulator of Cardiovascular Nitric Oxide , 2014, The Journal of Pharmacology and Experimental Therapeutics.

[26]  R. Copeland,et al.  Impact of enzyme concentration and residence time on apparent activity recovery in jump dilution analysis. , 2011, Analytical biochemistry.

[27]  Li Di,et al.  Stability Challenges in Drug Discovery , 2009, Chemistry & biodiversity.

[28]  J. Bajorath,et al.  What is the Likelihood of an Active Compound to Be Promiscuous? Systematic Assessment of Compound Promiscuity on the Basis of PubChem Confirmatory Bioassay Data , 2013, The AAPS Journal.

[29]  Robert Mah,et al.  Drug discovery considerations in the development of covalent inhibitors. , 2014, Bioorganic & medicinal chemistry letters.

[30]  Jason W. Lancaster,et al.  Telaprevir: a hepatitis C NS3/4A protease inhibitor. , 2012, Clinical therapeutics.

[31]  Michael A. Dyer,et al.  On the Mechanism of Action of SJ-172550 in Inhibiting the Interaction of MDM4 and p53 , 2012, PloS one.

[32]  Andrew I Su,et al.  HierS: hierarchical scaffold clustering using topological chemical graphs. , 2005, Journal of medicinal chemistry.

[33]  S. Sieber,et al.  Electrophilic natural products and their biological targets. , 2012, Natural product reports.

[34]  Jean-François Truchon,et al.  A generally applicable method for assessing the electrophilicity and reactivity of diverse nitrile-containing compounds. , 2007, Bioorganic & medicinal chemistry letters.

[35]  Michelle R. Arkin,et al.  Mining a Cathepsin Inhibitor Library for New Antiparasitic Drug Leads , 2011, PLoS neglected tropical diseases.

[36]  A. Duranti,et al.  Identification of a bioactive impurity in a commercial sample of 6-methyl-2-p-tolylaminobenzo[d][1,3]oxazin-4-one (URB754). , 2007, Annali di chimica.

[37]  R. Forbes,et al.  Impact of metal-induced degradation on the determination of pharmaceutical compound purity and a strategy for mitigation. , 2011, Journal of pharmaceutical and biomedical analysis.

[38]  Stanislav Gobec,et al.  False positives in the early stages of drug discovery. , 2010, Current medicinal chemistry.

[39]  James Inglese,et al.  Mitigating risk in academic preclinical drug discovery , 2015, Nature Reviews Drug Discovery.

[40]  Adrian Whitty,et al.  The resurgence of covalent drugs , 2011, Nature Reviews Drug Discovery.

[41]  Jayme L. Dahlin,et al.  PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS , 2015, Journal of medicinal chemistry.

[42]  James Inglese,et al.  Apparent activity in high-throughput screening: origins of compound-dependent assay interference. , 2010, Current opinion in chemical biology.

[43]  J. Baell,et al.  New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. , 2010, Journal of medicinal chemistry.

[44]  B. Cravatt,et al.  Disparate proteome reactivity profiles of carbon electrophiles. , 2008, Nature chemical biology.

[45]  Kate S. Carroll,et al.  Orchestrating redox signaling networks through regulatory cysteine switches. , 2010, ACS chemical biology.

[46]  Jayme L. Dahlin,et al.  A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation , 2013, PloS one.

[47]  Yanli Wang,et al.  A survey of across-target bioactivity results of small molecules in PubChem , 2009, Bioinform..

[48]  P. Cary,et al.  Structure of the C-terminal domain of human La protein reveals a novel RNA recognition motif coupled to a helical nuclear retention element. , 2003, Structure.

[49]  E. Weerapana,et al.  Investigating the proteome reactivity and selectivity of aryl halides. , 2014, Journal of the American Chemical Society.

[50]  Sridhar Ramaswamy,et al.  Targeting transcription regulation in cancer with a covalent CDK7 inhibitor , 2014, Nature.

[51]  J. Kos,et al.  Redox-Based Inactivation of Cysteine Cathepsins by Compounds Containing the 4-Aminophenol Moiety , 2011, PloS one.

[52]  Christopher P Austin,et al.  Quantitative analyses of aggregation, autofluorescence, and reactivity artifacts in a screen for inhibitors of a thiol protease. , 2010, Journal of medicinal chemistry.

[53]  Ruben Abagyan,et al.  Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor. , 2010, Chemistry & biology.

[54]  Patrick R. Griffin,et al.  PubChem promiscuity: a web resource for gathering compound promiscuity data from PubChem , 2012, Bioinform..

[55]  Renaldo Mendoza,et al.  ALARM NMR: a rapid and robust experimental method to detect reactive false positives in biochemical screens. , 2005, Journal of the American Chemical Society.

[56]  Jean M. Severin,et al.  Toxicological evaluation of thiol-reactive compounds identified using a la assay to detect reactive molecules by nuclear magnetic resonance. , 2007, Chemical research in toxicology.

[57]  Peter Wipf,et al.  Profiling the NIH Small Molecule Repository for compounds that generate H2O2 by redox cycling in reducing environments. , 2010, Assay and drug development technologies.

[58]  Ewgenij Proschak,et al.  Approved Drugs Containing Thiols as Inhibitors of Metallo-β-lactamases: Strategy To Combat Multidrug-Resistant Bacteria. , 2015, Journal of medicinal chemistry.

[59]  A. Kalgutkar,et al.  Drug discovery for a new generation of covalent drugs , 2012, Expert opinion on drug discovery.

[60]  Andrew C. Good,et al.  An Empirical Process for the Design of High-Throughput Screening Deck Filters , 2006, J. Chem. Inf. Model..

[61]  Anton Simeonov,et al.  Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124 , 2010, Proceedings of the National Academy of Sciences.

[62]  Cristian Bologa,et al.  Exploring the biological promiscuity of high-throughput screening hits through DFT calculations. , 2014, Bioorganic & medicinal chemistry.

[63]  Adam Yasgar,et al.  Thiol-Containing Fluorescent Probe High-Throughput Identification of Promiscuous Inhibitors from Screening Libraries with the Use of a , 2013 .

[64]  R. Fletterick,et al.  Discovery of Small Molecule Inhibitors of the Interaction of the Thyroid Hormone Receptor with Transcriptional Coregulators* , 2005, Journal of Biological Chemistry.

[65]  C. Winterbourn,et al.  Conjugation of Glutathione to Oxidized Tyrosine Residues in Peptides and Proteins* , 2012, Journal of Biological Chemistry.

[66]  D. Auld,et al.  Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression , 2009, Proceedings of the National Academy of Sciences.

[67]  Peter Wipf,et al.  Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents. , 2008, Assay and drug development technologies.

[68]  S. Olesen,et al.  The fungal product terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) . , 2010, Biochemistry.

[69]  G. S. Walker,et al.  Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. , 2014, Journal of medicinal chemistry.

[70]  C. Klein,et al.  Target Binding Properties and Cellular Activity of Afatinib (BIBW 2992), an Irreversible ErbB Family Blocker , 2012, Journal of Pharmacology and Experimental Therapeutics.

[71]  Caroline Engeloch,et al.  Stability of Screening Compounds in Wet DMSO , 2008, Journal of biomolecular screening.

[72]  Timothy L. Foley,et al.  4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), a Potent Inhibitor of Bacterial Phosphopantetheinyl Transferase That Attenuates Secondary Metabolism and Thwarts Bacterial Growth , 2014, Journal of medicinal chemistry.