David Sinclair, Ph.D. - Dose-Responsedose-response.org/.../2014/05/Sinclair_David_2011.pdf ·...
Transcript of David Sinclair, Ph.D. - Dose-Responsedose-response.org/.../2014/05/Sinclair_David_2011.pdf ·...
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David Sinclair, Ph.D.
Departments of Pathology and Genetics
Harvard Medical SchoolBoston
Disclosure statement: DAS is a consultant to Sirtris and Genocea.
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EnvironmentalCues
Causes of Disease& Aging
Longevity RegulationPathways
Small Molecules
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ERCs
SIR2 SIR2homologs
LOWGLUCOSE
LOWAA
PNC1
AGING
TOR,Msn2/4
Anderson et al., Nature (2004)Lamming et al., Science (2005)Medvedik et al., PLoS Biol. (2007)
NAD+
NAM
HEATSHOCK
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2% glucose 0.5% glucose 0.1% glucose low amino acids low nitrogen
Anderson et al., Nature (2003)
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yeast Sir2
SIRT1
SIRT2
SIRT3
SIRT4SIRT5
core domain
SIRT6
SIRT7
Humans possess 7 NAD+-dependent deacetylasesa.k.a. “sirtuins”
humans
Save the date: Sirtuin session tomorrow 5 PM
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Sirtuins
NAM
NANAMN
NaAD+
SIRTsPARPs
NMN
Yeast,Flies,
nematodesProteobacteria,
Mammals
Nampt(PBEF)
Pnc1
The NAD+ salvage pathway
NAD+ NAD+ NAM
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ANT
CypDAc 166
PTP
NAD+
Mitochondrial matrix
SIRT3
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ANT
CypD
Ac166
PTP
Mitochondrial matrix
SIRT3
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ANT
CypD
Ac166
PTP
Mitochondrial matrix
SIRT3
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ANT
DepolarizationReduced ATPMitochondrial destruction
Leakage of solutes out of mitochondria
Neurodegeneration, Cardiac hypertrophy
CypD
Ac166
PTP
Mitochondrial matrix
SIRT3
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Haigis and Sinclair, Annual Reviews, 2010
Circadian clock
DietExercise
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Quercetin (4.6x)
Fisetin (6.6x)
Piceatannol (9.0x)
Butein (8.5x)Resveratrol (13.0x)
Isoliquiritigenin (7.6x)
Representatives from three classes of plant-derived STACs
Stilbenes Chalcones Flavones
Howitz et al., Nature, Aug (2003)
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Gruber et al., 2007 Valenzano et al., 2006Jarolim et al., 2004
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Calorie Restriction (EOD)
High CalorieDiet (HC)
Lean Diet(SD)
ActivatorActivator
Rafa deCabo
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Standarddiet
High calorie diet+ resveratrol
15 month old mice
High caloriediet
**photo by Doug Hansen
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Oil Red O fat stain
LIVER
Fixed whole liver
H&E
Standard diet(SD)
High calorie(HC)
High calorie+ resv (HCR)
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Lagouge et al., Cell (2006)
Baur et al., Nature (2006)
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Last mouse Resveratrol fed every other day
36 months of age
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Howitz and Sinclair, Cell (2008)
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Generation of inducible SIRT1‐knockout mice
E1 E2 E3 E5 E6 E7 E8 E9E4
NEO
Cre‐ERT2
E1 E2 E3 E5 E6 E7 E8 E9
E1 E2 E3 E5 E6 E7 E8 E9
E4
Cre‐ERT2(Cytoplasmic)
Cre‐ERT2(Nuclear)
Tamoxifen
Cre‐ERT2
Cre‐ERT2
E4
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0
10000
20000
30000
40000
50000
60000
70000
StD WT StD T1KO HFD WT HFD T1KO
HFD Low RSV WT
HFD Low RSV
T1KO
HFD High RSV WT
HFD High RSV
T1KO
Rhod
amine 123 Fluo
rescen
ce(RFU
s)
WT KO WT KO WT KO WT KOStandard
Diet High Fat
DietHigh Fat
Diet + resveratrol
Mitochondrial membrane potential
Low dose High Dose
Low dose = 100 mg/kg/dHigh dose = 400 mg/kg/d
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0
0.5
1
1.5
2
2.5
3
ND1 ND2 ND3 ND4 ND4l ND5 ND6 Cytb COX1 COX2 ATP6 ATP8
mR
NA
(rel
ativ
e ex
pres
sion
)+ resveratrol (wildtype)+ resveratrol (SIRT1 knockout)
Mitochondrially-encoded ETC genes
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Baur et al., Nature (2006)
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Haigis and Sinclair, Annu Rev Pathol., 2010
LKB1
Mito poison?Lower ATP SIRT1-independent
SIRT1 activating?Higher ATPSIRT1-dependent
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0
5
10
15
20
25
30
35
40
45
50
Standard Diet HFD HFD + Low Resveratrol
HFD + High Resveratrol
ATP
(pmol/m
g protein)
Resveratrol increases ATP in a SIRT1-dependent manner in vivo
Gastrocnemius
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p –AMPK (Thr 172)
Total AMPK
SIRT1 KOWT WT SIRT
1 KOWT
SIRT1
KO
WTSIRT1 KO
Standard diet
HFD HFD + High RSV
HFD + Low RSV
0
0.5
1
1.5
2
2.5
Standard diet
HFD HFD + Low RSV
HFD + High RSV
p-A
MP
K/A
MP
K
WTSIRT1 KO
Is AMPK activation SIRT1-dependent?
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1st screen of ~18,000 molecules: found 18(best in class - resveratrol)
Discovery and Development of SIRT1 activators (STACs)
Screened 1,000,000 molecules: 6 novel chemical scaffoldsSynthesis of 4000+ STACs
Potency increase 10,000-foldEfficacy in rodent models of type II diabetes and neuroD
2003-4
2005-9
Human safety and efficacy studies with synthetic STACsHuman data to be publicly released June, 2011 (ADA meeting)
2010-11
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Milne et al., Nature (2007)
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SIRT1
Ac
SIRT1 SIRT1
W
SIRT1
Amino methyl coumarin (AMC) Tryptophan (W)
Ac
Dai et al., JBC (2010)
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Supported by an NIH/NIA-Sirtris CRADA to Rafa deCabo
SRT1720
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Joseph Baur, Rafa deCabo, Robin Minor
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Muscle Disorders
SarcopeniaDisuse Atrophy
Ophthalmic Diseases
AMDDry eye
Others
Inflammatory BowelSepsis
Metabolic Disease
Type 2 DiabetesOthers
Respiratory
COPD
SRT2104 and SRT2979
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A. Camins et al., Biochimica et Biophysica Acta (2010)
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Dr. Rafa de CaboAlejandro Martín-Montalvo(NIA/NIH)
Joe BaurBeamon Agarwal(U. Pennsylvania)
Yana CenAnthony Sauve(Cornell University, NY)
Sinclair Lab:Ana GomesAngela HafnerAlvin LingBasil HubbardBrian NorthJun LiLuis RajmanNathan PriceSean Armour
Carlos PalmieraAnabela Rolo(U. Coimbra, Portugal)
Guilio Pasinetti(Mt. Sinai, NY)
Marc DonathMarianne Böni-Schnetzler(University Hospital Basil)
Jim EllisVipin Suri and team(Sirtris)
Funding: Thanks to NIA/NIH, The Glenn Foundation for Medical Research, The Ellison Medical Foundation.