Strategie teraputiche

Modulazione farmacologica dell'omeostasi del NADmitocondriale: implicazioni terapeutiche

Alberto ChiarugiI° Convegno Nazionale sulle Malattie Mitocondriali

Roma, 21-22 Maggio 2011

4 Nobel nella “storia del NAD”

Destino metabolico del NAD

NAD

De novopathway

Tryptophan

Kynurenine pathway

cADPR

NAADP

NADK

NADP

NADases

PARPARPs PARG

AADPR

Sirtuins Nam

ADPR

MARTs

Rescuepathway

ATP

NMN

NPRT

NMNAT

ATP

ADPRPP

Reazioni metaboliche che distruggono e riformano il NAD

Alcuni ruoli metabolici aggiuntivi del NAD mitocondriale

Organizzazione dei complessi Fe/S

Rigenerazione del glutatione ridotto

Attività della piruvato deidrogenasi

Origine del NAD mitocondriale: un enigma ancora irrisolto

NAD

NAD

NAD support therapy

NAD depletion therapy

….nascono quindi 2 nuovi settori della farmacologia

Occorre però porre attenzione a:

Equilibri omeostaticiFarmacocinetica

direct

indirect

Effetti della deplezione di NAD cellularesulla funzione mitocondriale

C 1 10 100

60

70

80

90

100

110

M, FK 866

C6MT2FibroblastsGliaNeurons

HeLa

NA

D(%

of c

ontr

ol)

Effetti del blocco della resintesi del NAD sulla concentrazioni di NAD

Pittelli M. et al, JBC 2010

ATP

ATP

FK866Nam

NMN

NMNAT

ARTSIRT1-7

CD38PARPs

NAD

Nampt

C FK C FK0

30

60

90

120

150

**

NA

D%

of c

ontr

ol

Ma la deplezione di NAD non avviene nei mitocondri

MitocondriCytosol

C FK100µM/1h FK100nM/24h

Cell autofluorescence (NADH)

0

100

200

300

400 FK866

C PYR PYRADP

AT

P

(arb

itra

ry u

nits)

C PYR PYRADP

******

* *

TMRE Merge

NamptGFP

cytGFP

MitGFP

Nampt

MitGFP

TMRE

TMRE

Nampt is localized in citosol and not in mitochondria of HeLa cells

Pittelli M. et al, JBC 2010

Effetti del NAD esogeno sulla funzione mitocondriale

0 1 2 6 120

50

100

150

200

250

300

350

400

1µM

10µM

100µM

1mM

hrs

1mM 4°C

% o

f int

race

llula

r N

AD

CRL NAD 1mM (6h)

C 0 5 C 0 50

50

100

150

200

250 Mit-Lucif

h

NAD 1mMNAD 1mM

Cyt-Lucif

*

** **

**

Ph

oto

n e

mis

sio

n%

of

con

tro

l

C NAD C NAD0

2

4

6

8

10 mitochondria

nuclei

**

*nmol

NA

D/ m

g pr

ot

Effetti del NAD esogeno sulla funzione mitocondriale

CTRL NAD0

1

2

3

4 **

Oxy

ge

n c

on

sum

ptio

n r

ate

(nm

ol/m

l/min

)

0 2 4 6 8 10

205.0

210.0

215.0

220.0

225.0

230.0

235.0

min

nm

ol o

xyge

n/m

l

Control

NAD 1mM

BA

Effetti del NAD esogeno sul consumo di ossigeno

STP + NAD 1 mMSTPCTRL

Effetti del NAD esogeno sulla sopravvivenza cellulare

Total NAD+ cellular content in HEK293 shNMNAT3 stable cell line(3 exp in duplicate)

shSCRAM

BLED

shNM

NAT30

50

100

150

200

**

NA

D+

(A

BS

/mg

prot

.)Total ATP cellular content in HEK 293 shNMNAT3 stable cell line

(2 exp in triplicate)

shSCRAM

BLED

shNM

NAT30

50

100

150

200

*

ATP

/mg

prot

.

NAD tot NMNAT3-flag (var.2) overexpression 48 hHEK293 (1 exp. in duplicate)

Control

NMNAT3-

flag

0

1

2

3

**

NA

D+

(A

BS

/mg

prot

.)

Total ATP cellular content overexpression NMNAT3-flag 48h HEK 293 (1 exp in triplicate)

Control

NMNAT3

+0

1.0107

2.0107

3.0107

4.0107

ATP

/mg

prot

.

Ruolo dell’enzima mitocondriale NMNAT3

NMN + ATP NAD + PPi

Strategie teraputiche

Potenziali precursori del NAD

Acido Nicotinico

Nicotinamide

Nicotinamide riboside

Nicotinamide adenin mononucleotide

Intermedi della via delle chinurenine

Potenziale terapeutico dei precursori del NAD

Conclusioni

Laura FormentiniFrancesco BlasiMirko Muzzi Giuseppe FaracoAndrea LapucciDaniela BuonvicinoLeonardo Cavone

Elisa LanducciTania ScartabelliElisabetta GeraceAlessio MasiMaria SiliAndrea CozziVincenzo Carlà

Ringraziamenti

Fondazione Giuseppe Tomasello

Roberta Felici

PARP-1 and necrosis(the suicide hypothesis)

glucose

GAP

ATPADP

1,3-DPG

pyruvate

NADNADH

ATP

DNAdamage

NAD

NADH

NA+PRPP

NMN

NAD

NMNAT

PARP1

ADP

H+

H+

III

IIIIV

ATP

PRT

ATP

CELL NECROSIS

NAD e NAMPT e degenerazione assonale

CRL NAD 1mM (6h)

NAD e mitocondri concetto di saturazione

NADH(autofluorescenza cellulare)

A key role of PARP-1 in epigenetic chromatin remodeling

Homeostatic role

NAD

III

IIIIV

nPARP1

mPARP1

ROS

I II III IV

ANT

nPARP1

Detrimental role

Poly(ADP-ribose) polymerase-1 (PARP-1) in nucleus-mitochondrial cross-talk under homeostatic or pathogenetic conditions

1

2

3

4

5

6

BER

I IIIIV

1 - nPARP-1 regulates nuclear respiratory chain gene expression2 - nPARP-1 regulates mitochondrial DNA repair gene expression3 - mPARP-1 regulates mitochondrial respiratory chain gene expression4 - mPARP-1 assists mitochondrial genome repair

5 - mitochondrial ROS hyperactivate nuclear PARP-1 and prompt energy failure6 – mitochondrial ROS hyperactivate nuclear PARP-1 and alter epigenetic regulation

ATP

Ca2+

Ca2+

Ca2+

IP3

PLC

GPR

0 15 30 45 600

102030405060708090

100110

NAD

ATP

MNNG 100µM

min hrs2 4 86

% o

f co

ntro

l

MNNG

0 1h

Wash

Working model

10 -7 10 -6 10 -5 10 -4 10 -3 10 -2

0

25

50

75

100 PHEBZD

NA

D d

eple

tion

% o

f in

hibi

tion

10 -7 10 -6 10 -5 10 -4 10 -3 10 -2

0

25

50

75

100 PHEBZD

AT

P d

eple

tion

% o

f in

hibi

tion

NH 2

O N H

O

Phenanthridinone (PHE)IC50 = 3µM

Benzamide (BZD)IC50 = 30µM

The DNA alkylating agent Methyl-Nitro-Nitrosoguanidine (MNNG) induces PARP-1 The DNA alkylating agent Methyl-Nitro-Nitrosoguanidine (MNNG) induces PARP-1

hyperactivation and a long-lasting depletion of NAD and ATP in HeLa cellshyperactivation and a long-lasting depletion of NAD and ATP in HeLa cells

5’ 10’ 15’C

Cel

l cou

nts

TMRE fluorescence intensity

MNNG 2h

MNNG 3h

MNNG 1h

MNNG 4h

MNNGControl

0 1 2 3 4 5 60

20

40

60

80

100

120

140

160

MNNG

*

******

hrs

TM

RE

fluo

resc

ence

(% o

f con

trol)

Mitochondrial membrane potential () in HeLa cells undergoing PARP-1 hyperactivation

Poly(ADP-ribose) metabolism

NAD

NA

PAR

ATP

ATP

NADNMNAT

NMN

PARP

ADPRPARG

NAD rescue pathway

PPi

5-P ribose

ATP

ADP-ribosepyrophosphorylase

ATPmitochondria

ATPglycolysis

and catabolism

PRT

5-P ribose

AMP

ADP-ribosepyrophosphatases

(NUDIX hydrolases)

5-P ribose

AMP

Phosphodiesterases ?

From McLennan A.G., Cell. Mol. Life Sci. 63 (2006) 123–143

Summary of the human Nudix genes and hydrolases

ADP

AMP

ADPAMP

= 6.3±0.9

Control

ADP

AMP

ADPAMP

= 0.35±0.05

PARP-1 hyperactivation

Inverted ADP/AMP ratio underlies PARP-1-dependent energy failure

AMP reduces ADP uptake and ATP output from isolated mitochondria

ADP

ADP

ATP

ATP

INTERMEMBRANESPACE

MATRIX

The Adenine NucleotideTranslocator

Amino acid residues involved in the orientation and molecular constraintof ADP during its journey down to the ANT cavity

- Non fix anti conformation- NH in C6- No sobstistution in C2 - 2 or 3 PO4 groups

Transported Nucleotides

Ser227

Gly224

Arg279

Glu29

Lys22

Arg137

Arg235

Lys32

Ser227

Gly224

Arg279

Glu29

Lys22

Arg137

Arg235

Lys32

Ser227

Gly224

Arg279

Glu29

Lys22

Arg137

Arg235

Lys32

Ser227

Gly224

Arg279

Glu29

Lys22

Arg137

Arg235

Lys32ADP AMP

0

25

50

75

100

125

CRL ATR

CP

MA

(% o

f con

trol

)

0 1 10 100 1000

[AMP] M

ADP AMP

Bioactive Conformation (ΔE) = +7.60 kcal/mole Bioactive Conformation (ΔE) = +3.25 kcal/mole

Conformational Energy Evaluation of ADP or AMP bound to ANT

PARP NAD PAR AMP

ADP

ATPADP

glycolysis

metabolism

AK

ATP

NADrescue

Conclusion: The Nudix Hypothesis

ANT

AKAK

AK

AK

EVOLUTIONARY IMPLICATIONSPARP-1-dependent energy failure helps disclosing some of the ancestral strategies adopted

by eukaryotic cells to preserve bioenergetic exchanges with the protomitochondrion