New Insights in the Understanding of Cholesterol Metabolism:The Role of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

in the Regulation of Low-Density Lipoprotein Cholesterol (LDL-C) and LDL

Receptors (LDLRs)

USA-145-100024(1) © 2014 Amgen Inc. All rights reserved. Not for Reproduction.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Pathway

Genetic Variants of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

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Table of Contents

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Pathway

© 2014 Amgen Inc. All rights reserved. Not for Reproduction.

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Hepatic LDLRs Play a Central Role in Cholesterol Homeostasis

LDL = low-density lipoprotein; LDLR = low-density lipoprotein receptor

1. Steinberg D, et al. Proc Natl Acad Sci. 2009;106:9546-9547. 2. Brown MS, et al. J Lipid Res. 2009;50:S15-S27.

Clathrin-coated vesicleHepatocyte

LDL

LDLR

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Recycling of LDLRs Enables Efficient Clearance of LDL-C Particles

LDL-C = low-density lipoprotein cholesterol1. Steinberg D, et al. Proc Natl Acad Sci. 2009;106:9546-9547. 2. Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29:431-438. 3. Brown MS, et al. Proc Natl Acad Sci. 1979;76:3330-3337.

Increased LDLR surface concentration

Lysosomal degradation

LDLR recycling

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PCSK9 = proprotein convertase subtilisin/kexin type 9

1. Qian YW, et al. J Lipid Res. 2007;48:1488-1498. 2. Horton JD, et al. J Lipid Res. 2009;50:S172-S177.3. Brown MS, et al. Proc Natl Acad Sci. 1979;76:3330-3337. 4. Steinberg D, et al. Proc Natl Acad Sci. 2009;106:9546-9547. 5. Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29:431-438. 6. Zhang DW, et al. J Biol Chem. 2007;282:18602-18612.

PCSK9 Regulates the Surface Expression of LDLRs by Targeting for Lysosomal Degradation

LDLR/PCSK9 routed to lysosome

Lysosomal degradation

PCSK9 secretion

Decreased LDLR surface concentration

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Genetic Variants of PCSK9 Demonstrate Its Importance in Regulating LDL Levels

PCSK9 Gain of Function = Less LDLRs1 PCSK9 Loss of Function = More LDLRs1

1. Steinberg D, et al. PNAS. 2009;106:9546-9547. 2. Cohen JC, et al. N Engl J Med. 2006;354:1264-1272. 3. Benn M, et al. J Am Coll Cardiol. 2010;55:2833-2842.

Mutations in the human PCSK9 gene that lead to a loss of PCSK9 function are found in 1% to 3% of the representative populations2,3

Lysosomal degradation of LDLR

Gain-of-function PCSK9 Loss-of-function PCSK9

Recycling of LDLR

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Gain-of-Function Mutations in PCSK9 Cause Familial Hypercholesterolemia*†

1. Abifadel M, et al. Hum Gen. 2009;30:520-529. 2. Lopez D. Biochem Biophys Acta. 2008;1781:184-191.

3. Cameron J, et al. Hum Mol Genet. 2006;15:1551-1558.

PCSK9 Variant PopulationClinical/Biochemical

Characteristics

D374Y1British, Norwegian

families, 1 Utah family

Tendon xanthomas, severe hypercholesterolemia

S127R1 French, South African, Norwegian families Tendon xanthomas

R218S2 French families Tendon xanthomas, arcus corneae

†For a full list of ADH mutations, please refer to Abifadel reference.

• Associated with:– High serum LDL-C1

– In vitro testing in many identified mutations shows decreased levels of LDLRs3

*Autosomal Dominant Hypercholesterolemia

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Loss-of-Function Mutations in PCSK9 Are Associated With Decreased LDL-C

LOF = loss of functionARIC = Atherosclerosis Risk in Communities (N ~ 4,000); DHS = Dallas Heart Study (N = 3,553); CGPS = Copenhagen General Population Study (N = 26,013)

1. Cohen JC, et al. N Engl J Med. 2006;354:1264-1272. 2. Cohen J, et al. Nat Genet. 2005;37:161-165

3. Benn M, et al. J Am Coll Cardiol. 2010;55:2833-2842. 4. Zhao Z, et al. Am Journal of Hum Gen. 2006;79:514-534.

PCSK9 Variant Population LDL-C

R46L ARIC1, DHS2 ↓ 15%1

Y142X or C679X ARIC1, DHS2 ↓ 28%–40%1

R46L CGPS3 ↓ 11%3

• Heterozygous LOF mutations found in 1% to 3% of representative populations1,3

• Associated with

– Lower serum LDL-C1

• PCSK9 null individual identified (compound heterozygote for two inactivating mutations)

– No detectable circulating PCSK9 with strikingly low LDL-C (14 mg/dL)4

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SREBP = sterol regulatory element-binding protein

1. Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29:431-438. 2. Dubuc G, et al. Arterioscler Thromb Vasc Biol. 2004;24:1454-1459.

LDLR and PCSK9 Expression Are Both Upregulated When Intracellular Cholesterol Levels Are Low

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Expression of PCSK9 Depends on Intracellular Cholesterol Levels

- Cholesterol Depletion*

- Statins↑ SREBP-2 ↑ PCSK9

- Dietary Cholesterol

- Cellular Cholesterol↓ SREBP-2 ↓ PCSK9

*Intracelullar Cholesterol Depletion

1. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23.

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Summary• LDLR and PCSK9 Expression Are Both Regulated by Intracellular

Cholesterol Levels1,2

• Genetic Variants of PCSK9 Support its Role in Regulating LDL Levels2

– Gain-of-function mutations result in increased LDL-C3,4

– Loss-of-function mutations are associated with decreased LDL-C5

1. Dubuc G, et al. Arterioscler Thromb Vasc Biol. 2004;24:1454-1459. 2. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23. 3. Abifadel M, et al. Hum Mutat. 2009;30:520-529. 4. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 5. Cohen JC, et al. N Engl J Med. 2006;354:1264-1272.