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

• O2
• H2O2

ONOO- OH• SOD, Catalase Peroxidase Vit’s C,E thiols

“Oxidative Stress”

Oxidative Stress in human disease

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

Oxidative Stress and Disease

Identification & Distribution of NADPH Oxidase (Nox)

Discovery of NADPH Oxidase 2 (Nox2) in the Vasculature

e-

Fe H+

p47phox p22phox p67phox

Nox2

Cytosol

O2 O2

• NADPH

Pagano et al., AJP, 1995; PNAS, 1997; Hypertension, 2008

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

CNS CNS

CNS

Human Vasculature

Nox2/ Nox1/ Nox4 p22phox p67phox? p40phox p47phox PLC/ PLD c-Src PKC

Epidermal Growth Factor, etc.

Angiotensin II RhoA Tyrosine Kinases

GPCR

Diversity of Nox Modulation

c-abl PKD? ?

Touyz et al.

Rational Design of First-in-Class Nox2 Inhibitor

Development of an In Vivo NOX Inhibitor:

Nox2ds-tat

plasma membrane

Nox2

p22

Nox2ds-tat

p47 p67

tat

Nox2ds

O2

• O2

p22

cytosol

Nox2ds-tat

Nox2ds-tat Inhibits Vascular O2

• . & Delays

Angiotensin II-induced Elevation in Systolic Blood Pressure

Rey et al., Circ Res 2001;89: 408

80 90 100 110 120 130 140 150

• 2

2 4 6 8

Systolic Blood Pressure (mmHg)

Days after Pump Implantation

Sham Ang II + Nox2ds-tat Ang II + Scrmb-tat

* *

2

0.00 0.05 0.10 0.15 0.20

* **

trol b-tat 91ds-tat

Tissue O2

• . Generation

(pmoles/min/mg)

Nox2ds-tat Applied Systemically Reduces Neointimal Hyperplasia

Vehicle Nox2ds-tat Scrmb-tat

Jacobson et al. Circ. Res. 2003 Balloon-Injured Carotid Artery + Nox2ds-tat +Scrmb-tat + Vehicle

 Paracrine Effect of Adventitial Nox2-derived

ROS On Smooth Muscle Hypertrophy

Intima Media Adventitia

Lumen

Large Vessel Cross-Section

 in vivo transfection with virus (3.5 x 108 pfu/ml)

expressing Nox2ds and green fluorescent protein: (Ad-PDGFßrec-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 Mac-3

Liu, Pagano et al. Circ. Res. 2004

E M A A M E = Endothelium M = Media A = Adventitia

Adventitial Targeting of Nox2ds Attenuates Carotid Arterial Hypertrophy

+AngII

• AngII

Medial/SMC Area

Nox2 in Pulmonary Hypertension: Potential Therapy

 5.6 mg Nox2ds-tat

dissolved in 5 mL PBS.

 Nebulizer set to deliver

NOX2ds-tat or vehicle

• ver 20 min into

nebulizing chamber to achieve 1 µM in lung airways. Aerosolized Nox2ds-tat Delivered to Mice Exposed to Hypoxia for Treatment of Right Ventricular (RV) Hypertrophy?

Unpublished data, Pagano, Zuckerbraun, Bauer et al.

0,14 0,16 0,18 0,20 0,22 0,24 0,26 0,28 0,30

Control Hypoxia Hypoxia + Nox2ds- tat

Fulton Index (RV/LV + septum)

Aerosolized Nox2ds-tat Decreases RV Hypertrophy in Hypoxic Mice.

Unpublished data, Pagano, Zuckerbraun, Bauer et al.

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

• f 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-tat Implicates Nox2 in Signaling and Disease:

Nox2ds Specifically Attenuates Nox2-derived O2

• FRBM 2011 , Csányi,G. et al.
• 0.1

0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7

0 μ 0 μ 0.1 μ 0.3 μ 1 μ 3 μ 10 μ

Rate of Superoxide Generation

(nmol O2-/min/107 cell equivalent) Scrmb Nox2ds Nox2ds

0 0 0.1 0.3 1 3 10 [Nox2ds or Scrmb], µM no LIDS LiDS

Cytochrome C reduction Gabor Csányi

IC50: o.77 µM

Nox2ds Dose Response *No effect on Nox1 and Nox4 Oxidase Systems

Development & Validation of a High-throughput Assay of Nox Small Molecule Inhibitors (384-well Format)

Strategy:

Assay: Optimization:

• Cell seeding
• PMA dose response
• Time course of reaction
• DMSO tolerance
• Automation

Validation:

• LOPAC screening.

Planned Screening:

• 220,000 small molecule library

5µM PMA

O2

.-

H2O2

COS-Nox2 L-012

SOD

+

Eugenia Cifuentes

225,000 compounds

Inhibition @ 1 - 10 µM ( > 50%) Non-specific O2- scavenging (Xanthine Oxidase assay) Confirmed Nox2 inhibitors (~200 compounds) Nox 1, 4, 5 inhibitory activity Improvement of physicochemical properties by analoguing.

No

X

Confirmation of hits at various concentrations Cos cell Cytotoxicity ( CytoTox-Glo) Yes

Pan-Nox Inhibitors?

Yes No No No Yes

Secondary testing in whole animals.

HTS Strategy

Yes

X

Yes

X

Cifuentes & Pagano , 2011

Mitochondrial Function (OCR and H2O2 production) Yes

X

No

X

No

Screening Assays

# of compounds % of total Total tested 480 100 Nox2 Assay (≥ 50% inhibition) 77 16 Cytotoxicity Assay (≤50% dead cells ) 71 15 Xanthine Oxidase Assay (≤ 50% inhibition) 30 6 Mitochondrial function (≤ 50% inhibition)

23 5

Five Percent of Molecules from NIH Clinical Collection Passed Initial screens; Being Tested for Isoform Specificity

• SOD

+ SOD Sham Ang II Ang II+Losartan 122kD 85kD 42kD Cifuentes and Pagano, AJP 2000 gp91phox/ Nox2

• AngII

HTS for Nox2 Inhibitors

Peptido- mimetics/ Rational Design

New Inhibitors Nox1ds

Nox Therapeutics

First inhibitor Nox2ds

Glimpses Past and Future

Nox4

Initial Discovery In Vasculature

HTS

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

Drug Discovery Institute

Elizabeth Sharlow, Ph.D. David Close Stephanie Leimgruber *Funding Sources: NIH NHLBI American Heart Association ITXM Hemophelia Ctr. Of Western Pennsylvania

Center for Disease Control, National Vital Statistics Report, 2007

Leading Causes of Death in the Western World

• More than 150, 000 Americans killed by Cardiovascular Disease (CVD) in

2005 were < 65 years of age.

• Circulation. 2009;119:e21-e181

• 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.).

Hypertension-Related Cardiovascular Disease (CVD)

Experimental Procedure

Day -2 0 7

Ad-PDGF-Nox2ds-eGFP AdCMV-eGFP

NADPH Oxidase (Nox Subunits & Activators)

e-

Fe Fe 2 O2 2 •O2

• (10 nmol/min/106 neutrophils)

e- e-

H+

Cytosol p47phox p22phox p67phox Flavocytochrome b558

NADPH

NADP+ e- H+ FAD

e- e- e- e-

Nox2

p40phox

= electron

Rac2

Taylor et al. JBC, 281, pp. 37045–37056

NADPH Oxidase 2 Inhibitor: Nox2ds (a.k.a. gp91ds)

Nox2 p22phox

C-S-T-R-I(V)-R-R-Q-L

FAD FAD NADPH NADPH

5000 10000 15000 20000 25000 30000 35000 20 40 60 80 100 120 140

O2

• . Generation

(RLU) Time (min)

unstimulated Stimulated with PMA Stimulated with PMA in the presence of SOD

PMA, 5mM

Assay Development: Stable and Robust Nox2 Assay Detecting Superoxide

Max

Min

Cifuentes et al., unpublished data