1. Is there a rational strategy for biomarkers that predict disease outcomes and translate into humans? Update on the rapidly advancing field of causal pathway measurements Marc K. Hellerstein, M.D., Ph.D. Co-Founder and Chief of SAB, KineMed, Inc., Emeryville, CA Professor (D.H. Calloway Chair), University of California, Berkeley; Professor of Medicine, UCSF

5. Attrition should not be a surprise: Fundamental unpredictability of complex networks Drug/Nutrient Predicted Effect Unexpected Effect

9. Causal Pathways of Disease Link Molecular Targets to Outcomes of Complex Systems Agent Molecular target Biochemical pathway Clinical outcome (Microscopic) (High throughput) (No functional significance) (Low throughput) (Macroscopic) (Functional significance)

10. Fibrogenesis as Causal Pathway: Complex Regulation, Simple Final Process WBC & Lymph M  HBV Target IFN  viral replication Cell Death Toxicants Target Steatosis Tissue Inflammation IL-6 Target TNF  IFN  Hepatocyte TNFR Fibroblast nucleus matrix metalloproteinases (MMPs) procollagenase AA peptides Lysyl oxidase Crosslinked collagen Transcription Procollagen Translation GlycOH AA hydroxyl Collagen mRNA acetaldehyde oxidative stress T-cell Target systemic & dietary protein degradation Extracellular Space Organ failure & Death PK3 NK PGE 2 M  Target CTGFR TGF  R IL-10 Target WBC & Lymph TGF-  CTGF Target Target STK Target MMP inhibitors Collagen buildup Fibrosis Collagen

20. Dynamic Proteomics 2 H 2 O-labelled cells plasma or tissues Enrich protein of interest (e.g., IP) SDS-PAGE Reduce, carboxymethylate Trypsinise LC/ESI-MS/MS analysis (Thermo OrbiTrap) Calculate fraction new protein ( f ) De Riva et al., in prep. Analysis of tryptic peptides from 2H2O-labelled proteins

21. LC/MS/MS analysis of tryptic peptide Relative abundance LC Chromatogram SIM: m/z ≈ 876.45 (M+H) 2+ MS1: mass spectrum of intact peptide m/z Retention time m/z MS2: fragmentation for sequencing A. De Riva et al., in prep.

22. Mass Shift In Plasma Albumin Peptides Fractional synthesis calculated from the change in %m0 Mass isotopomer fractional abundances %M1 %M2 %M3 %M4 %M0 m/z=1245 m/z=1487 m/z=826 Published half-life of albumin (17 d) Day 0 6 12 26 41

23. Mass Shift In Plasma IgA Peptides natural 100% labeled Mass isotopomer fractional abundances %M1 %M2 %M3 %M4 %M0 Day 0 6 12 26 41 h m/z=918 m/z=1172 Published half-life of IgA (6 d) Fractional synthesis calculated from the change in %m0 Predicted values CONFIDENTIAL PF BH KL JP 8-02.10

24. RELATIVE ABUNDANCES [ % ] UNLABELED PEPTIDE ISOTOPOMERS 2 H-PEPTIDE

25. F=0% ISOTOPOMERS RELATIVE ABUNDANCES [ % ]

26. F=25% ISOTOPOMERS RELATIVE ABUNDANCES [ % ]

27. F=50% ISOTOPOMERS RELATIVE ABUNDANCES [ % ]

28. F=75% ISOTOPOMERS RELATIVE ABUNDANCES [ % ]

29. ISOTOPOMERS RELATIVE ABUNDANCES [ % ] F=100%

30. Basic Principles of MIDA Precursor Pool Polymer/Product Isotope Pattern M2 M3 M0 M1 (M+0) (M+1) (M+2) (M+3) M0 M1 M2 M3 M4 p=1.1% Excesses (M+0) (M+1) (M+2) (M+3) p=5%

33. Axonal transport: essential to neuronal health Proteins made here Proteins transported here Proteins used here Secreted Proteins

34. Clinical Translational Tool: CSF-based secreted biomarkers of MT-mediated fast axonal transport in ALS MT-mediated Fast axonal transport Healthy Neuron Degenerating Neuron Introduction of label Introduction of label Nucleus Time to Appearance in the CSF Nucleus 2 H – newly synthesized cargo molecules MTs Hyperdynamic MTs Time of CSF appearance 2 H – labeled secreted cargo molecules

35. Neuregulin-1, Chromogranin B and sAPPa exhibit altered CSF kinetics in symptomatic SOD1G93A mice ***p< 0.001 *p< 0.05 **p< 0.01 T max in CSF: 3d 2 H- labeled Chr-B (EM1) Time (day post-labeling) 2.0 0 2 4 6 8 10 12 0.0 0.5 1.0 1.5 * *** *** ** SOD1G93A Wild type 13 weeks ♀/♂ Body water 2 H- enrichment 2 H- labeled NeuR-1 (EM1) 0 2 4 6 8 10 12 0.0 0.5 1.0 1.5 2.0 Time (day post-labeling) SOD1G93A Wild type 13 weeks ♀/♂ 0 2 4 6 8 10 12 SOD1G93A Wild type 13 weeks ♀/♂ 0.0 1.0 2.0 3.0 4.0 Time (day post-labeling) ** *** ** * *** 2 H- labeled sAPP  (EM1) Time (day post-labeling) 0 2 4 6 8 10 12 0.0 1.0 2.0 3.0 4.0 5.0 6.0 *** * *** *** SOD1G93A Wild type 13 weeks ♀/♂

36. Pulse-chase Labeling – Healthy Control Patients 2 H-Enrichments

37. Pulse-chase Labeling – ALS Patient 0 10 20 30 40 50 0.0 0.5 1.0 1.5 2.0 Control subject #7033 and ALS subject #6082 2H-labeled Chr-B (EM1%) 0 10 20 30 40 50 0.0 0.5 1.0 1.5 2.0 2H-labeled sAPP  (EM1%) Time (day) Time (day) Chromogranin-B sAPP  CTRL 7033 ALS 6082 CTRL 7033 ALS 6082

38. Pulse-chase Labeling - Parkinson’s Disease Patient 2 H-Body Water 2 H-Enrichments Control subject #7033 and PD subject #6080 0 10 20 30 40 50 0.0 0.5 1.0 1.5 CTRL 7033 PD 6080 Time (day) 2 H-labeled Cargo (Fraction of Peak)

39. Sensitive clinical biomarker: kinetically distinct, disease-specific defects in neuronal transport? ALS- Defect #1: Gross delay representing global defect in bulk transport 0 10 20 30 40 50 0.0 0.5 1.0 1.5 2.0 2H-labeled Chr-B (EM1%) Time (day) PD - Defect #2: Transport is irregular uneven & less uniform CHASE 0 10 20 30 40 50 0.0 0.5 1.0 1.5 2.0 2H-labeled Chr-B (EM1%) Time (day) PULSE CTRL 7033 ALS 6082 CTRL 7033 PD 6080

40. Peptide Isotopomer Analysis of Control Subject #6079 9-16-10 BH JC KL Cystatin-C peptide m/z=613.81, z=2 ALDFAVGEYNK

41. 2 H-Enrichment in Pulse-Chase Subject #6079 (QTOF Analysis) #6079 Fraction Labeled

42. Proteome wide measure of protein turnover 1716 Unique proteins (total) 45 222 113 >10 peptides 71 244 165 5-10 peptides 111 343 353 2-4 peptides 107 313 379 1 peptide 334 1,122 1,010 Total proteins analyzed 1,968 7.226 4,619 Peptides utilized in protein analysis 3,340 11,643 10,389 Detected peptides (t0) Blood Liver Brain

43. Rapid POC in Humans Skin Keratin and Keratinocyte Dynamics in Psoriasis

44. Epidermal Keratin Turnover: Fsn Mice Glucocorticoid treatment has no effect on high turnover rate of keratin in fsn mice 0 0.02 0.04 0.06 0.08 0.1 0.12 0 5 10 15 20 25 Days on D2O EM1 FSN treated FSN untreated C57Bl/6

45. Kinetics of Human Skin Keratin (Tape Strips) Lindwall, JinvestDerm 2006 0.00 0.01 0.02 0.03 0.04 0.05 0 20 40 60 80 100 Day EM1 (Ala)

46. Skin Turnover in Psoriatic Patients e

48. Clotting Rate in Vivo : Turnover (Consumption) of Coagulation Factors in Cascade

49. Complement Pathway: Turnover (Consumption)

51. Label Pathways for Measuring DNA Synthesis G6P R5P PRPP Purine and Pyrimidine bases Precursors NDP dNTP DNA dN 2 H 2 O GNG Deoxy-ribonucleoside salvage 2 H 2 O 2 H 2 O Base Salvage DNPS 2 H 2 O Glycogen (RR) Glucose DNNS 3 H-dT, BrdU

52. Application: Patient Subgroup Selection in CLL Messmer et al., J Clin Invest 2005 GMH, 02/10/2008 Fast Turnover/Bad Disease Fraction labeled 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 50 100 150 200 250 WBCs (10 3 /mm 3 ) 100 10 Days Peak f = 68.1% f %/day = peak f days of labeling = 0.81% new cells/day (birth rate)

53. Application: Patient Subgroup Selection in CLL Messmer et al., J Clin Invest 2005 GMH, 02/10/2008 Slow Turnover/Stable Disease WBCs (10 3 /mm 3 ) Fraction labeled 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 50 100 150 200 250 100 10 Days Peak f = 15.2% f %/day = peak f days of labeling = 0.18% new cells/day (birth rate)

54. KineMed Test Distinguishes between Patients with Stable vs Progressive Disease KineMarker 

56. Micro-dissection of Normal & Tumor Tissues GMH, 02/10/2008 Tissue stained for SRPK1 (increased expression in tumors compared to normal epithelia) Benign epithelial Tumor (Grade 3) 1) Make a series of ~ six 5-10 micron thick tissue slides 2) Determine benign and tumoral areas to be excised from first and last stained slides 3) Micro-dissect mapped areas from slides in the middle

57. Micro-dissection of Normal & Tumor Tissues GMH, 02/10/2008 Determine tumor areas to excise

58. Micro-dissection of Normal & Tumor Tissues GMH, 02/10/2008 Micro-dissection of tumoral cells Into microfuge tube Derivatize & analyze

59. Human Breast cancer: DCIS vs normal tissue

60. Purification of PEC from Semen: GMH, 02/10/2008 Semen sample Remove enriched PEC fraction Viscolytic Enzyme Percoll Gradient Stain prostate-specific membrane antigen (PSMA-cell surface) & prostate-specific alkaline phosphatase (PAP-intracellular) SORT via flow cytometry Cytospin & Determine purity (via anti-CK staining) Pass through 50 μ m filter (removes squamous epithelial cells) sperm PEC +ve sort -ve sort fraction PSMA PAP

61. Proliferation Rates of ProstateTissue Microdissected Cells and Seminal Fluid PEC: Radical Prostatectomy Patients

62. Proliferation Rates of ProstateTissue Microdissected Cells and Seminal Fluid PEC

63. Correlation Between Proliferation Rates of Prostate Tissue Microdissected Cells and Seminal Fluid PEC PEC Tissue

67. HDLc in Hypoalpha Patients ABCA1

68. Cholesterol Efflux in Hypoalpha Patients ABCA1

70. Effect of ALP-1 on in-vivo esterification rate in rabbit P < 0.05 P > 0.05 +93 +133 +20% +47%

71. Relation of RCT Fluxes to Reduction in Atherosclerosis with rrLCAT Treatment Vehicle rrLCAT *** Vehicle rrLCAT 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Efflux Ra (mg/kg/hr) 0 5 10 15 20 25 30 Neutral Sterol Excretion (mg/day) 0.00% 20.00% 40.00% 60.00% Surface area with lesions (%) * Abdominal Aorta 60 - 40 - 20 - 0 0.00% 20.00% 40.00% 60.00% Surface area with lesions (%) * Abdominal Aorta 60 - 40 - 20 - 0 Vehicle rrLCAT Efflux Esterification Fecal Sterol Output 0 10 20 30 40 50 60 Rate of CE clearance (mg/day/rabbit) * Vehicle rrLCAT

72. Torcetrapib inhibits absolute rate of esterification 8 cmpA and 7 cmpB Torcetrapib inhibits rate of esterification * Rate of esterification Torcetrapib 0 5 10 15 Rate of esterification (mg/dl/6h) Vehicle

78. Measurable Processes in Complex Diseases: AD/ Neurodegeneration APP Synthesis A  1-42 production Tau/MT Dysfunction Plaque accum. Neuronal Function Axonal Transport Neuro Transmitters Synaptic plasticity Plaque turnover Complement  -secr 1° NI Stimuli Neuro-inflammation Cytokine Synth Microglia Caspases, apoptosis Cell death

79. Measurable Processes in Complex Diseases: Neoplasia (Prostate Cancer) Androgens Growth factors Mutagens etc PEC proliferation Apoptosis Cellular pathways Clonal Selection PIN Prolif Angiogenesis ECM destruction Invasion Lymphangio-genesis Metastases Inflammation Cytokine Synth

80. Measurable Processes in Complex Diseases: Atherogenesis Diet Activity Genes Etc. TG ApoB particles DNL Obesity VLDL TG turnover FFA Release Adipose dynamics Macrophage proliferation HDL LDL RCT Complement Activation Cytokine Synth Coagulation Cascade Vessel Wall APGP Synth Inflamm-asomes C-VD Events