Developing Inhibitors of the p47phox-p22phox Protein-Protein Interaction by Fragment-Based Drug Discovery.

NADPH oxidase isoform 2 is an enzyme complex, which generates reactive oxygen species (ROS) and contributes to oxidative stress. The p47phox-p22phox interaction is critical for activation of the catalytical NOX2 domain, and p47phox is a potential target for therapeutic intervention. By screening 2,500 fragments using fluorescence polarization (FP) and a thermal shift assay (TSA) and validation by surface plasmon resonance (SPR) we found eight hits towards the tandem SH3 domain of p47phox (p47phoxSH3A-B) with KD values of 400-600 μM. Structural studies revealed that fragments 1 and 2 bound two separate binding sites in the elongated conformation of p47phoxSH3A-B and these competed with p22phox for binding to p47phoxSH3A-B. Chemical optimization led to a dimeric compound with the ability to potently inhibit the p47phoxSH3A-B-p22phox interaction (Ki of 20 μM). Thereby, we reveal a new way of targeting p47phox and present the first report of druglike molecules able to bind p47phox and inhibit its interaction with p22phox.

[1]  K. Krause,et al.  Pharmacological characterization of the seven human NOX isoforms and their inhibitors , 2019, Redox biology.

[2]  L. Pardo,et al.  Design of a True Bivalent Ligand with Picomolar Binding Affinity for a G Protein-Coupled Receptor Homodimer. , 2018, Journal of medicinal chemistry.

[3]  David Lagorce,et al.  FAF‐Drugs4: free ADME‐tox filtering computations for chemical biology and early stages drug discovery , 2017, Bioinform..

[4]  Richard E. Gillilan,et al.  BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis , 2017, Journal of applied crystallography.

[5]  M. Rosales-Hernández,et al.  Aromatic Regions Govern the Recognition of NADPH Oxidase Inhibitors as Diapocynin and its Analogues , 2017, Archiv der Pharmazie.

[6]  J. Tam,et al.  Comment on “A small-molecule antivirulence agent for treating Clostridium difficile infection” , 2016, Science Translational Medicine.

[7]  Uzay E Emir,et al.  Effect of the putative lithium mimetic ebselen on brain myo-inositol, sleep and emotional processing in humans , 2016, Neuropsychopharmacology.

[8]  Ju-Young Kim,et al.  Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo , 2016, International journal of biological sciences.

[9]  Megan C. Garland,et al.  A small-molecule antivirulence agent for treating Clostridium difficile infection , 2015, Science Translational Medicine.

[10]  Timo Krotzky,et al.  One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists , 2015, ChemMedChem.

[11]  J. Irwin,et al.  An Aggregation Advisor for Ligand Discovery. , 2015, Journal of medicinal chemistry.

[12]  J. Lambeth,et al.  NOX2 As a Target for Drug Development: Indications, Possible Complications, and Progress. , 2015, Antioxidants & redox signaling.

[13]  John P. Overington,et al.  The promise and peril of chemical probes. , 2015, Nature chemical biology.

[14]  Donald P. Huddler,et al.  CHAPTER 2:Getting the Most Value from Your Screens: Advances in Hardware, Software, and Methodologies to Enhance Surface Plasmon Resonance Based Fragment Screening and Hit-to-Lead Support , 2015 .

[15]  R. Tomar,et al.  Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways , 2014, Molecular Biology Reports.

[16]  J. Lambeth,et al.  High-throughput Assays for Superoxide and Hydrogen Peroxide , 2014, The Journal of Biological Chemistry.

[17]  M. Williamson.  Using chemical shift perturbation to characterise ligand binding. , 2013, Progress in nuclear magnetic resonance spectroscopy.

[18]  M. Rosales-Hernández,et al.  Ethers and esters derived from apocynin avoid the interaction between p47phox and p22phox subunits of NADPH oxidase: evaluation in vitro and in silico , 2013, Bioscience reports.

[19]  N. Yuldasheva,et al.  Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction , 2013, Diabetes.

[20]  Olan Dolezal,et al.  Parallel Screening of Low Molecular Weight Fragment Libraries , 2013, Journal of biomolecular screening.

[21]  K. Block,et al.  Aiding and abetting roles of NOX oxidases in cellular transformation , 2012, Nature Reviews Cancer.

[22]  J. Snyder,et al.  Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits. , 2012, Chemistry & biology.

[23]  Steen H. Hansen,et al.  BayesApp: a web site for indirect transformation of small-angle scattering data , 2012 .

[24]  J. Quinn.  Modeling Taylor dispersion injections: determination of kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing. , 2012, Analytical biochemistry.

[25]  J. Quinn.  Evaluation of Taylor dispersion injections: determining kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing. , 2012, Analytical biochemistry.

[26]  R. Giffard,et al.  Significance of marrow‐derived nicotinamide adenine dinucleotide phosphate oxidase in experimental ischemic stroke , 2011, Annals of neurology.

[27]  C. Sobey,et al.  Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets , 2011, Nature Reviews Drug Discovery.

[28]  L. Olsen,et al.  Design and synthesis of highly potent and plasma-stable dimeric inhibitors of the PSD-95-NMDA receptor interaction. , 2009, Angewandte Chemie.

[29]  Detlef Snakenborg,et al.  BioXTAS RAW, a software program for high‐throughput automated small‐angle X‐ray scattering data reduction and preliminary analysis , 2009 .

[30]  M. Gougerot-Pocidalo,et al.  p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases , 2009, Experimental & Molecular Medicine.

[31]  Myron D. Ginsberg,et al.  Neuroprotection for ischemic stroke: Past, present and future , 2008, Neuropharmacology.

[32]  G. Whitesides,et al.  Dependence of effective molarity on linker length for an intramolecular protein-ligand system. , 2007, Journal of the American Chemical Society.

[33]  K. Itoh,et al.  Ebselen, a seleno-organic antioxidant, as an electrophile. , 2006, Chemical research in toxicology.

[34]  Brian K Shoichet,et al.  A detergent-based assay for the detection of promiscuous inhibitors , 2006, Nature Protocols.

[35]  G. Donnan,et al.  1,026 Experimental treatments in acute stroke , 2006, Annals of neurology.

[36]  Kenji Ogura,et al.  NMR Solution Structure of the Tandem Src Homology 3 Domains of p47phox Complexed with a p22phox-derived Proline-rich Peptide* , 2006, Journal of Biological Chemistry.

[37]  Christopher W Murray,et al.  Fragment-based lead discovery: leads by design. , 2005, Drug discovery today.

[38]  G. Booker,et al.  Synthesis of 5-, 6- and 7-substituted-2-aminoquinolines as SH3 domain ligands. , 2005, Organic & biomolecular chemistry.

[39]  K. Rittinger,et al.  Activation and assembly of the NADPH oxidase: a structural perspective. , 2005, The Biochemical journal.

[40]  Daniel Fritz,et al.  Identification and specificity studies of small-molecule ligands for SH3 protein domains. , 2004, Journal of medicinal chemistry.

[41]  Xueliang Fang,et al.  Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. , 2004, Analytical biochemistry.

[42]  M. Horiuchi,et al.  Solution Structure of the Tandem Src Homology 3 Domains of p47phox in an Autoinhibited Form* , 2004, Journal of Biological Chemistry.

[43]  Randy Hoffman,et al.  Overcoming compound interference in fluorescence polarization-based kinase assays using far-red tracers. , 2004, Assay and drug development technologies.

[44]  Kenji Ogura,et al.  A molecular mechanism for autoinhibition of the tandem SH3 domains of p47phox, the regulatory subunit of the phagocyte NADPH oxidase , 2004, Genes to cells : devoted to molecular & cellular mechanisms.

[45]  M. Congreve,et al.  A 'rule of three' for fragment-based lead discovery? , 2003, Drug discovery today.

[46]  B. Shoichet,et al.  A specific mechanism of nonspecific inhibition. , 2003, Journal of medicinal chemistry.

[47]  Kenji Ogura,et al.  Crystallization and preliminary crystallographic analysis of the autoinhibited form of the tandem SH3 domain of p47phox , 2003 .

[48]  C. Chung,et al.  Effect of detergent on "promiscuous" inhibitors. , 2003, Journal of medicinal chemistry.

[49]  Katrin Rittinger,et al.  Molecular Basis of Phosphorylation-Induced Activation of the NADPH Oxidase , 2003, Cell.

[50]  Robert W. Bryant,et al.  Evaluation of Fluorescent Compound Interference in 4 Fluorescence Polarization Assays: 2 Kinases, 1 Protease, and 1 Phosphatase , 2003, Journal of biomolecular screening.

[51]  D. Earnshaw,et al.  Real Experiences of uHTS: A Prototypic 1536-Well Fluorescence Anisotropy-Based uHTS Screen and Application of Well-Level Quality Control Procedures , 2001, Journal of biomolecular screening.

[52]  J. Owicki,et al.  Fluorescence Polarization and Anisotropy in High Throughput Screening: Perspectives and Primer , 2000, Journal of biomolecular screening.

[53]  M. Parnham,et al.  Ebselen: prospective therapy for cerebral ischaemia , 2000, Expert opinion on investigational drugs.

[54]  Tetsuro Ago,et al.  Mechanism for Phosphorylation-induced Activation of the Phagocyte NADPH Oxidase Protein p47 phox , 1999, The Journal of Biological Chemistry.

[55]  U. Haupts,et al.  Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice. , 1999, Drug discovery today.

[56]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[57]  P. Newburger,et al.  Activation of the Leukocyte NADPH Oxidase by Phorbol Ester Requires the Phosphorylation of p47 PHOX on Serine 303 or 304* , 1998, The Journal of Biological Chemistry.

[58]  Yoshiyuki Sakaki,et al.  Assembly and Activation of the Phagocyte NADPH Oxidase , 1996, The Journal of Biological Chemistry.

[59]  S. Chanock,et al.  The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis. , 1995, The Journal of clinical investigation.

[60]  N. Vermeulen,et al.  Mechanism of the reaction of ebselen with endogenous thiols: dihydrolipoate is a better cofactor than glutathione in the peroxidase activity of ebselen. , 1990, Molecular pharmacology.

[61]  A. Bach.  Targeting Oxidative Stress in Stroke , 2017 .

[62]  T. Hazbun,et al.  Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells. , 2017, Biochimica et biophysica acta. General subjects.

[63]  Roderick E Hubbard,et al.  Experiences in fragment-based lead discovery. , 2011, Methods in enzymology.

[64]  K. Krause,et al.  The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. , 2007, Physiological reviews.

[65]  Geoffrey A. Donnan,et al.  Experimental Treatments in Acute Stroke , 2006 .

[66]  K. Ogura,et al.  Crystallization and preliminary crystallographic analysis of the autoinhibited form of the tandem SH3 domain of p47(phox). , 2003, Acta crystallographica. Section D, Biological crystallography.

[67]  Jeen-Woo Park,et al.  PHOSPHORYLATION OF Ser-359 OR Ser-370 PRECEDES PHOSPHORYLATION AT OTHER SITES AND IS REQUIRED FOR ACTIVITY* , 1998 .