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

e-

Fe

H+

p22phox

Nox4

Cytosol

e-

NOXO1

p22phox

NOXA1

Nox1

Cytosol

Cytosol

Nox5

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 0 2 4 6 8

Syst

olic

Blo

od

Pre

ssu

re

(mm

Hg)

Days after Pump Implantation

Sham

Ang II + Nox2ds-tat

Ang II + Scrmb-tat

* *

O2

- (n

mo

l o

f O

2-

/min

/m

g o

f ti

ssu

e)

0.00

0.05

0.10

0.15

0.20

*

**

Con

trol

AII

AII+sc

ram

b-ta

t

AII+gp

91ds

-tat

Tis

sue

O2-.

Ge

ne

rati

on

(p

mo

les/

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

over 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

Fu

lto

n In

de

x

(RV

/LV

+ s

ep

tum

)

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

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-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 μM 0 μM 0.1 μM 0.3 μM 1 μM 3 μM 10 μM

no LIDS LiDS

Rat

e o

f Su

pe

roxi

de

Ge

ne

rati

on

(nm

ol

O2

-/m

in/1

07

cell

eq

uiv

ale

nt)

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

Vehicle

+AngII

Ad-CMV-eGFP

Ad-Nox2ds-eGFP

-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

0

5000

10000

15000

20000

25000

30000

35000

0 20 40 60 80 100 120 140

O2

-. G

en

era

tio

n

(RLU

)

Time (min)

unstimulated

Stimulated with PMA

Stimulated with PMA inthe presence of SOD

PMA, 5mM

Assay Development: Stable and Robust Nox2 Assay Detecting Superoxide

Max

Min

Cifuentes et al., unpublished data