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 (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) 2 Table of Contents

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Pathway © 2014 Amgen Inc. All rights reserved. Not for Reproduction.

4 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: Brown MS, et al. J Lipid Res. 2009;50:S15-S27. Clathrin-coated vesicle Hepatocyte LDL LDLR

5 Recycling of LDLRs Enables Efficient Clearance of LDL-C Particles LDL-C = low-density lipoprotein cholesterol 1. Steinberg D, et al. Proc Natl Acad Sci. 2009;106: Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29: Brown MS, et al. Proc Natl Acad Sci. 1979;76: Increased LDLR surface concentration Lysosomal degradation LDLR recycling

6 PCSK9 = proprotein convertase subtilisin/kexin type 9 1. Qian YW, et al. J Lipid Res. 2007;48: Horton JD, et al. J Lipid Res. 2009;50:S172-S Brown MS, et al. Proc Natl Acad Sci. 1979;76: Steinberg D, et al. Proc Natl Acad Sci. 2009;106: Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29: Zhang DW, et al. J Biol Chem. 2007;282: 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

7 Genetic Variants of PCSK9 Demonstrate Its Importance in Regulating LDL Levels PCSK9 Gain of Function = Less LDLRs 1 PCSK9 Loss of Function = More LDLRs 1 1. Steinberg D, et al. PNAS. 2009;106: Cohen JC, et al. N Engl J Med. 2006;354: Benn M, et al. J Am Coll Cardiol. 2010;55: Mutations in the human PCSK9 gene that lead to a loss of PCSK9 function are found in 1% to 3% of the representative populations 2,3 Lysosomal degradation of LDLR Gain-of-function PCSK9 Loss-of-function PCSK9 Recycling of LDLR

8 Gain-of-Function Mutations in PCSK9 Cause Familial Hypercholesterolemia* † 1. Abifadel M, et al. Hum Gen. 2009;30: Lopez D. Biochem Biophys Acta. 2008;1781: Cameron J, et al. Hum Mol Genet. 2006;15: PCSK9 VariantPopulation Clinical/Biochemical Characteristics D374Y 1 British, Norwegian families, 1 Utah family Tendon xanthomas, severe hypercholesterolemia S127R 1 French, South African, Norwegian families Tendon xanthomas R218S 2 French familiesTendon xanthomas, arcus corneae † For a full list of ADH mutations, please refer to Abifadel reference. Associated with: –High serum LDL-C 1 –In vitro testing in many identified mutations shows decreased levels of LDLRs 3 *Autosomal Dominant Hypercholesterolemia

9 Loss-of-Function Mutations in PCSK9 Are Associated With Decreased LDL-C LOF = loss of function ARIC = 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: Cohen J, et al. Nat Genet. 2005;37: Benn M, et al. J Am Coll Cardiol. 2010;55: Zhao Z, et al. Am Journal of Hum Gen. 2006;79: PCSK9 VariantPopulationLDL-C R46LARIC 1, DHS 2 ↓ 15% 1 Y142X or C679XARIC 1, DHS 2 ↓ 28%–40% 1 R46LCGPS 3 ↓ 11% 3 Heterozygous LOF mutations found in 1% to 3% of representative populations 1,3 Associated with –Lower serum LDL-C 1 PCSK9 null individual identified (compound heterozygote for two inactivating mutations) –No detectable circulating PCSK9 with strikingly low LDL-C (14 mg/dL) 4

10 SREBP = sterol regulatory element-binding protein 1. Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29: Dubuc G, et al. Arterioscler Thromb Vasc Biol. 2004;24: LDLR and PCSK9 Expression Are Both Upregulated When Intracellular Cholesterol Levels Are Low

11 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.

12 Summary LDLR and PCSK9 Expression Are Both Regulated by Intracellular Cholesterol Levels 1,2 Genetic Variants of PCSK9 Support its Role in Regulating LDL Levels 2 –Gain-of-function mutations result in increased LDL-C 3,4 –Loss-of-function mutations are associated with decreased LDL-C 5 1. Dubuc G, et al. Arterioscler Thromb Vasc Biol. 2004;24: Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010: Abifadel M, et al. Hum Mutat. 2009;30: Horton JD, et al. J Lipid Res. 2009;50:S172-S Cohen JC, et al. N Engl J Med. 2006;354: