Nox: Glimpses Past and Future Patrick J. Pagano, Ph.D. Dept. of - PowerPoint PPT Presentation

Nox: Glimpses Past and Future Patrick J. Pagano, Ph.D. Dept. of Pharmacology & Chemical Biology Vascular Medicine Institute, Univ. of Pittsburgh October 24, 2011 Ri.Med Symposium, Palermo, Sicilia

Seminar Outline I. Reactive Oxygen Species (ROS) & Oxidative Stress II. Oxidative Stress in Human Disease III. Identification & Distribution of NADPH Oxidases (Nox) IV. Design of First-in-Class Specific Inhibitor of Nox2 V. A Paracrine Role of Adventitial Nox2? VI. Small Molecule Discovery & Rational Design- HTS

SOD, Catalase Peroxidase Vit’s C,E thiols • O 2 - H 2 O 2 ONOO - OH • “Oxidative Stress”

Oxidative Stress in human disease

Oxidative Stress and Disease AIDS Parkinson's Disease Diabetes Inflammatory Bowel Disease Pancreatitis Cataractogenesis Ischemia-Reperfusion Alzheimer's Disease Rheumatoid Arthritis Muscular Dystrophy ARDS Cancer Macular Degeneration Multiple Sclerosis

Identification & Distribution of NADPH Oxidase (Nox)

Discovery of NADPH Oxidase 2 (Nox2) in the Vasculature O 2 - O 2 H + Nox2 Fe p22 phox e- Cytosol p47 phox NADPH p67 phox Pagano et al., AJP, 1995; PNAS, 1997; Hypertension, 2008

CNS CNS CNS Human Vasculature Quinn MT, et al., Clinical Science (2006) 111, 1-20

Diversity of Nox Modulation Angiotensin II Epidermal Growth Factor, etc. Nox2/ c-abl GPCR Nox1/ p22phox Nox4 c-Src p47 phox PLC/ p67 phox ? Tyrosine PLD Kinases p40 phox PKC RhoA ? PKD? Touyz et al.

Rational Design of First-in-Class Nox2 Inhibitor

Development of an In Vivo NOX Inhibitor: Nox2ds-tat O 2 - O 2 Nox2ds tat Nox2ds-tat plasma p22 Nox2 p22 membrane Nox2ds-tat cytosol p47 p67

-. & Delays Nox2ds-tat Inhibits Vascular O 2 Angiotensin II-induced Elevation in Systolic Blood Pressure Sham 150 Ang II + Nox2ds-tat Systolic Blood Pressure * 140 Ang II + Scrmb-tat 0.20 * 130 -. Generation (mmHg) (pmoles/min/mg) * 120 0.15 110 ** 0.10 2 100 Tissue O 2 90 0.05 80 -2 0 2 4 6 8 0.00 b-tat 91ds-tat trol Days after Pump Implantation Rey et al., Circ Res 2001;89: 408

Nox2ds-tat Applied Systemically Reduces Neointimal Hyperplasia Balloon-Injured Carotid Artery + Nox2ds-tat +Scrmb-tat + Vehicle Vehicle Nox2ds-tat Scrmb-tat Jacobson et al. Circ. Res. 2003

 Paracrine Effect of Adventitial Nox2-derived ROS On Smooth Muscle Hypertrophy

Large Vessel Cross-Section Lumen Intima Media Adventitia

 in vivo transfection with virus (3.5 x 10 8 pfu/ml) expressing Nox2ds and green fluorescent protein: (Ad-PDGF ßrec - Nox2ds -- eGFP )  Control virus only expressing GFP (Ad-CMV-eGFP)  Virus applied in 15% pluronic gel

Oxidase Inhibitor Nox2ds is Targeted to the Adventitia AngII + Ad-gp91ds-eGFP eGFP A E M E = Endothelium M = Media A = Adventitia M Mac-3 A Liu, Pagano et al. Circ. Res. 2004

Adventitial Targeting of Nox2ds Attenuates Carotid Arterial Hypertrophy -AngII +AngII Medial/SMC Area

Nox2 in Pulmonary Hypertension: Potential Therapy

Aerosolized Nox2ds-tat Delivered to Mice Exposed to Hypoxia for Treatment of Right Ventricular (RV) Hypertrophy?  5.6 mg Nox2ds-tat dissolved in 5 mL PBS.  Nebulizer set to deliver NOX2ds-tat or vehicle over 20 min into nebulizing chamber to achieve 1 µM in lung airways. Unpublished data, Pagano, Zuckerbraun, Bauer et al.

Aerosolized Nox2ds-tat Decreases RV Hypertrophy in Hypoxic Mice. 0,30 0,28 (RV/LV + septum) 0,26 Fulton Index 0,24 0,22 0,20 0,18 0,16 0,14 Control Hypoxia Hypoxia + Nox2ds- tat Unpublished data, Pagano, Zuckerbraun, Bauer et al.

Nox2ds-tat Implicates Nox2 in Signaling and Disease: Park et al. Nox2-derived radicals contribute to neurovascular and behavioral dysfunction in mice overexpressing the amyloid precursor protein. PNAS 2008. Walch et al. Pro-atherogenic effect of interleukin- 4 in endothelial cells…2006. Miller et al. NADPH oxidase activity and function are profoundly greater in cerebral versus systemic arteries. 2005 Yang et al. Insulin-stimulated NAD(P)H oxidase activity increases migration of cultured vascular smooth muscle cells. 2005 Al-Shabrawey et al. Inhibition of NAD(P)H oxidase activity blocks vascular endothelial growth factor overexpression and neovascularization during ischemic retinopathy. 2005 Harfouche et al. Roles of reactive oxygen species in angiopoietin-1/tie-2 receptor signaling. 2005 Keller et al. Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species. 2004 Fürst et al. Atrial natriuretic peptide induces mitogen-activated protein kinase phosphatase-1 in human endothelial cells via Rac1 and Nox2-activation. 2005 Jung et al. gp91phox-containing NADPH oxidase mediates endothelial dysfunction in renovascular hypertension. 2004 Sohn et al. Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine. 2003 Krötz et al. NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment. 2002

- Nox2ds Specifically Attenuates Nox2-derived O 2 Nox2ds Dose Response Cytochrome C reduction Scrmb Nox2ds IC 50 : o.77 µM 1.7 Nox2ds Rate of Superoxide Generation (nmol O2-/min/10 7 cell equivalent) 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1 -0.1 0 0 0.1 0.3 1 3 10 0 μ 0 μ 0.1 μ 0.3 μ 1 μ 3 μ 10 μ [Nox2ds or Scrmb], µM Gabor Csányi no LIDS LiDS *No effect on Nox1 and Nox4 Oxidase Systems FRBM 2011 , Csányi,G. et al.

Development & Validation of a High-throughput Assay of Nox Small Molecule Inhibitors (384-well Format) Strategy: Assay: .- O 2 L-012 + 5µM PMA COS-Nox2 SOD Optimization: H 2 O 2 -Cell seeding -PMA dose response - Time course of reaction - DMSO tolerance -Automation Validation: - LOPAC screening. Planned Screening: -220,000 small molecule library Eugenia Cifuentes

225,000 compounds HTS No X Strategy Inhibition @ 1 - 10 µM ( > 50%) Yes Non-specific O 2 - scavenging Yes X (Xanthine Oxidase assay) No Yes X Cos cell Cytotoxicity ( CytoTox-Glo) No Yes X Mitochondrial Function (OCR and H2O2 production) No No X Confirmation of hits at various concentrations Yes Yes Nox 1, 4, 5 inhibitory activity Pan-Nox Inhibitors? No Improvement of physicochemical properties by analoguing. Confirmed Nox2 inhibitors (~200 compounds) Secondary testing in whole animals. Cifuentes & Pagano , 2011

Five Percent of Molecules from NIH Clinical Collection Passed Initial screens; Being Tested for Isoform Specificity Screening # of Assays compounds % of total 480 100 Total tested 77 16 Nox2 Assay ( ≥ 50 % inhibition) 71 15 Cytotoxicity Assay (≤50% dead cells ) Xanthine Oxidase Assay 30 6 ( ≤ 50 % inhibition) Mitochondrial function 23 5 (≤ 50% inhibition)

Glimpses Past and Future -AngII +AngII Vehicle Ad-CMV-eGFP Ad-Nox2ds-eGFP HTS First HTS for Nox2 Inhibitors inhibitor New Inhibitors Nox2ds Nox1ds Nox4 Sham Ang II+Losartan Ang II 122kD 85kD gp91phox/ Nox2 42kD Cifuentes and Pagano, AJP 2000 Peptido- mimetics/ Rational Design - SOD + SOD Nox Initial Discovery In Vasculature Therapeutics

Acknowledgments University of Pittsburgh Department of Pharmacology and Chemical Biology Vascular Medicine Institute Eugenia Cifuentes, Ph.D. Gabor Csanyi , Ph.D Imad Al Ghouleh , Ph.D Loreto Egaňa , M.S. Giovanna Frazziano, Ph.D. Andres Rodriguez, Ph.D. Eric Kelley, Ph.D Daniel Ranayhossaini *Funding Sources: NIH NHLBI Drug Discovery Institute American Heart Association ITXM Hemophelia Ctr. Of Elizabeth Sharlow, Ph.D. Western Pennsylvania David Close Stephanie Leimgruber

Leading Causes of Death in the Western World Center for Disease Control, National Vital Statistics Report, 2007 -More than 150, 000 Americans killed by Cardiovascular Disease (CVD) in 2005 were < 65 years of age. Circulation. 2009;119:e21-e181

Hypertension-Related Cardiovascular Disease (CVD)  Persistent hypertension is a major risk factor for stroke, heart attacks, heart failure and arterial aneurysm, and is a leading cause of renal failure.  Even moderate elevation of arterial blood pressure leads to shortened life expectancy. Guyton & Hall (2005). Textbook of Medical Physiology (7th Ed. ed.).

Experimental Procedure Ad-PDGF-Nox2ds-eGFP AdCMV-eGFP Day -2 0 7

NADPH Oxidase (Nox Subunits & Activators) 2 O 2 2 • O 2 - e- (10 nmol/min/10 6 neutrophils) e- e- H + Flavocytochrome e- Fe b558 Nox2 e- Fe e- FAD NADP + e- H + e- p22 phox Rac2 p47 phox p67 phox p40 phox Cytosol NADPH = electron

NADPH Oxidase 2 Inhibitor: Nox2ds (a.k.a. gp91ds) C-S-T-R-I(V)-R-R-Q-L p22 phox Nox2 FAD FAD NADPH NADPH Taylor et al. JBC, 281, pp. 37045 – 37056

Assay Development: Stable and Robust Nox2 Assay Detecting Superoxide 35000 Max 30000 -. Generation 25000 (RLU) unstimulated 20000 15000 O 2 Stimulated with PMA 10000 PMA, 5 m M Min Stimulated with PMA in 5000 the presence of SOD 0 0 20 40 60 80 100 120 140 Time (min) Cifuentes et al., unpublished data