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Jeremy K. Nicholson, PhDProfessor and Head of Biological
ChemistryImperial College
University of London
STATISTICAL SPECTROSCOPY AND GLOBAL SYSTEMS BIOLOGY APPROACHES IN
DISEASE MODELING
NEDMDG SYMPOSIUMWorcester, Mass. June 8th,
2006
Summary• The quest for new drugs - ‘Top-down’ Systems Biology, gene-environment interactions and the Personalized Healthcare Paradigm.
• Generating and modeling system level metabolic data in experimental disease states.– Pharmaco-metabonomics and predictive models
– Biomarker recovery via statistical spectroscopy
•NMR, UPLC-MS, UPLC-MS/NMR, Proteo-metabonomics
• Characterizing genetic,dietary and microbial contributions to Human Metabolic Phenotypes: Molecular epidemiology and “The Health of Nations”.
Can Systems Biology save the Pharmaceutical
Industry?
High technology has generally not helped the rate of practical drug
discovery!
PHARMACEUTICAL PRODUCTIVITY DECLINE: REAL COST PER DRUG HAS INCREASED 10 FOLD IN 30 YEARS!THIS IS AN UNSUSTAINABLE BUSINESS MODEL!
ONLY 11% OF DRUGS IN CLINICALTRIALS MAKE IT TO MARKET!
Lipinski Rules: Compound Bioavailability is poor if…
> 5 H-bond donors
CLog P > 5
Sum {N + O} > 10
Mass > 500 DALTONS
23,710
3,051
HUMAN GENOME23,710
DRUGGABLE GENOME3,000
DISEASEMODIFYING
GENES3,000
DRUGTARGETS 1000
LIBRARY (NME)
PHARMACOLOGY/TOXICOLOGY
ADME/ANIMALS
Ph1 Ph2 Ph3
DISCOVERY DEVELOPMENT
MARKET
CLINICAL TRIALS
INVESTIGATIONAL NEW DRUG
FATALWithdrawal
from market
OBLIGATELead Selectionand Optimization
CRITICAL$$$ failure!
Product Rescue?
EARLYChemistry and in vitro screens
© Imperial College, 2006
PHARMACEUTICAL PIPELINE ATTRITION
Quasi-DarwinianSelectionPreclinical Efficacy
Models & Biomarkers
Preclinical Safety Biomarkers and Mechanisms
Screening Studies: Transcriptomic, Proteomic and
Metabolomic/Metabonomic
Clinical Efficacy Biomarkers
Clinical Safety Biomarkers
Pre
-Le
ad
Pri
orit
iza
tion Response Analysis: Genotyping,
Proteomics, Pharmaco-genomics.Pharmaco-metabonomics
Sys
tem
s B
iolo
gy A
pplic
atio
n
CLINICALDIAGNOSTICMARKERS
NEW DRUGTARGETS
Genomics ProteomicsMetabonomics
Microbiome Xeno-metabolome
Global Systems Biology
Personalized Healthcare
© Imperial College,2006
COMPREHENSIVE PHENOTYPE
Theranostics& patient
stratification
Optimized drugefficacy &
minimized toxicity
OptimizedNutrition
Multivariate Descriptions of Metabolism
•MetaboLome Definition: The quantitative analyisis or description of all low molecular weight metabolites in specified cellular, tissue or biofluid compartments. (Metabolomics: Numbers, chemical classes, structures, concentrations: < 1KDa)
•MetaboNome Definition: The sums, products & interactions of all the individual compartments/metabolomes (including extra-genomic sources) dispersed in a complex organism…The ‘Global’ System.
METABONOMICS“Quantitative measurement of multivariate
metabolic responses of multicellular systems to pathophysiological stimuli or
genetic modification”
(AIMS TO MODEL GLOBAL METABOLICREGULATION OF COMPLEX SYSTEMS INCLUDING DYNAMIC
INTERACTIONS & COMPARTMENTALIZATION OF COMPONENTS)METABOLOMICS (various definitions)
e.g. “measurement of metabolite concentrations& fluxes in cell systems”.
OR “measurement & modelling of all metabolites & pathways in a system”
Extra-cellular metabolite pool (biofluids)
External secretions /excretion
Cellular transcriptome
Cellular proteome
Intracellular metabolome
METABONOMICS OFCOMPLEX SYSTEMS
Multiple cell linesC1,C2…C8 etc.
Intervention
with
specific
target
C1 C2
C3
C4
C5C6
C7
C8
multiple
targets?
Extra-cellular metabolite poolTissue profilesExcretion signaturesMolecular compartmentsReaction profilemeasurement & modeling
BIOLOGICAL FLUID TYPES(primary secretory and connective roles)
Key Diagnostic Fluids: Plasma, Urine.
Specialized Functions: Cerebrospinal, thyroid. Saliva (sub-lingual, parotid, sub-maxillary), Gastric, Bile, Pancreatic.Amniotic, Follicular, Milk, Seminal Vesicle, Prostatic, Epidydimal, Seminal.
Pathological Fluids: Ascites, Cystic, Blister.
Artificial Fluids: Bronchiolar lavage fluid, peritoneal dialysates, hemodialysates, fecal water, rectal dialysates, cell extracts and cell supernatants.
PHYSICAL-BIOCHEMICAL FEATURESOF URINE AND PLASMA
URINE: Variable pH, ionic strength, osmolarity.
High dielectric constant. Extreme dynamic concentration range
(>1011). Thousands of molecules < 1KDa, (polarity?).
Metal complexes and supramolecular aggregates.
Many small proteins, high enzyme activities in pathological states-dynamically reactive matrix.
PLASMA: Relatively constant pH, ionic strength, osmolarity.
Lower bulk dielectric constant. High dynamic concentration range
(>105). Hundreds of of molecules < 1KDa and
>1KDa. Metal complexes and supramolecular
complexes. Multi-compartment -multi diffusional-
matrix Many large proteins and protein
complexes.
timeaveraged
snapshot
Mass Spectrometry
Linked chromatography/MS
TLC/MS
CE-MS
GC-MS
HPLC-MS
UPLC-MSn
LC-ICPMS-MS
NMR Spectroscopy
Single pulse 1H, 13C, 31P
2+ D methods COSY TOCSY HMQC HMBC etc
LC-NMR, CE-NMR, CEC-NMR
PFG Diffusion analysis DOSY etc…
HR-MAS (cells + tissues)PFG-MAS(cryoprobes/robotic FI etc)
Standard Analytical Information: Identity, Structure, Quantity (BOTH)
Physical Biochemical Information: Interactions, Compartments (NMR)
Analytical Approaches in Metabonomics and Metabolomics
Biofluids and extractsBiofluids, extracts, cells/tissues
Single quadTriple quad
TOF-MSQTOF-MS
Ion trapLinear ion trap
FTMS
LC- NMR -MSn
Many Ionization MethodsCHEMOMETRIC MODELLING(pattern recognition for
classification, diagnostics & biomarker analysis)
STATISTICAL SPECTROSCOPY(Linking multiple spectra & spectral types for structure elucidation/pathway analysis)
CHEMOMETRIC TOOLS FOR INFORMATION RECOVERY FROM
MULTIVARIATE DATA
UNSUPERVISED
• Principal Components Analysis (PCA)
• Hierarchichal Cluster Analysis (HCA)
• Logical blocking-PCA• Non-linear Mapping (NLM)
• Supergravity Association Mapping (SAM)….etc.
SUPERVISED
• Partial Least Squares (PLS) & PLS-DA
• O-PLS & O2-PLS• Soft Independent Modeling of Class Analogy (SIMCA)
• Rule Induction• Bayes Nets/Machine Learning
• Genetic Algorithms• Neural Networks• CLOUDS…etc…© Imperial College, 2006
900 MHz 1H NMR Spectrum of Untreated Human Urine
Contains LatentBiomarker information on:
GenotypePhysiological stateNutritional state
Gut microbes‘Biological’ Age
Presence of Disease
Translatable BiomarkersDiagnosticPrognosticToxicityEfficacy
A B C D E F
Secretory Metabolomes
HOST GENOME
Cellular transcriptomes
Cellular proteomes
1o Intracellular metabolomes
Extracellular metabolite pool
Primary andco-metabolome interactions
in mammalian systems(Nicholson et al Nature, Microbiology, 2005, 3, 2-8)
Humans: > 500 functionally distinct NORMAL cell types/ca.10 trillion
parenchymal cells
GUT ‘MICROBIOME’
Species transcriptomes
Species proteomes
Species metabolomes
Enteron
Co-metabolome enters via hepatic portal + mesenteric veins
1 2 3 4 5 6
microbial and dietary2o metabolites
Biliary secretions enterduodenum fromcommon bile duct
ENTEROHEPATICCIRCULATION
Humans: > 1000 Species.> 100 trillion cells
Microbial-mammalian
metabolic axis:
COMBINATORIAL METABOLISM!
COOH
OH
HO OH
COOH
H
HO
C
OH
HO OH
ON
COOHH
C
H
HO
ON
COOHH
C
OH
HO OH
ON
H
SO3H
C
H
HO
ON
H
SO3H
1. Biosynthesis: cholic acid
deoxycholic acid
glycocholic acid glycodeoxyocholic acid taurodeoxyocholic acidtaurocholic acid
COOH
OH
HO OH
4. Regeneration ofcholic acid
2. Phase II glycine ConjugationPhase II taurine Conjugation 7. Phase II glycine ConjugationPhase II taurine Conjugation
MAMMALIAN LIVER
MAMMALIAN GUT
3. Secretion into bile
amino acid deconjugationby gut microbiota
5. 7- 7 -dehydroxylation by gut microbiota
6. reabsorption into bloodvia hepatic portal system
enterohepaticcirculation
8. Secretion into bile
9. Deconjugation further reactions
MICROBIAL-MAMMALIAN CO-METABOLISM OF CHOLIC ACID
De-conjugated bile acids areless efficient at emulsifying fats
PHARMACO-METABONOMICS
A new paradigm for personalized predictive
drug metabolism and toxicology.
Definition: The prediction of the quantitative outcome of an intervention based on a pre-treatment metabolic model: Applications in drug metabolism, xenobiotic toxicity, drug efficacy…etc.
Age Hormonal status
Genetic Factors P450 Polymorphisms
SNP variations
Understanding drug interaction responses in relation to individual metabolic variation: Gene-environment interactions determine the pre-dose
starting phenotype.
Gut Microbiome
Nutrition
Individual (dot) location is theresultant of the influencevectors in m-space.
Are there locations that are more risk averseFor particular interventions?Prognostic biomarker clusters?
Conditional Metabolic Phenotype
Host Genetic ConstitutionInterspecies variations
& individual SNP variations
Specific drug metabolizing enzyme complement
(CYP450) polymorphisms
Tissue-specific CYP450induction state
(e.g., in liver & gut)
Individual Gut Microbiomemicrobial species variation
& ACTIVITY
Nutritional status& dietary composition
Metabolic Fateand Toxicity
of Drug
Global System Interactions Affecting Drug Metabolism & Toxicity
Nicholson, JK et alNature, Biotechnology
22 (10) 1268-1274. (2004)
STATISTICAL SPECTROSCOPY
“The application of multivariate statistical methods to extract
latent structural or connectivity information in multiple spectral
data sets from samples or experiments collected serially or
in parallel.”
1. STATISTICAL SEARCH SPACE REDUCTION FOR BIOMARKER
IDENTIFICATION IN SERIAL UPLC-MS DATA SETS
Crockford et al Analytical Chemistry (2006), in press
PARTIAL LEAST SQUARES DISCRIMINANT ANALYSIS (PLS-DA) SORTS FEATURES ACCORDING TO IMPORTANCE
FOR CLASS SEPARATION.
SPECTRAL LOADINGS BACK-PROJECTED DIRECTLY TO LC-MS CHROMATOGRAM TO IDENTIFY RETENTION TIMES &
MASSES OF CANDIDATE BIOMARKERS.
356 features
O-PLS-DA STATISTICAL SEARCH SPACE REDUCTION
© IMPERIAL COLLEGE 2006
Hydrazine dosed (10) vs control (10):Statistically significant peaks (r > 0.6)Back-projected into UPLC-MS time domain
51 features
r > 0.8
O-PLS-DA STATISTICAL SEARCH SPACE REDUCTION
STRONG CANDIDATEBIOMARKERS
© Imperial College, 2006
RECONSTRUCTION OF LATENT BIOMARKER INFORMATION FROM LARGE SPECTROSCOPIC SETS BY STATISTICAL
TOTAL CORRELATION SPECTROSCOPY (STOCSY) Cloarec et al Analytical Chemistry, 77 (5) 1282-
1289, 2005.
2D STOCSY: Plot / correlation matrix for all samples, color code by r2. Gives both self-molecular correlations (assignment) and also pathway and compartment correlations.
Calculate correlation matrix (C) between all computer points(/) for all 1D spectra in all datasets to be compared:
X1 and X2 are the auto-scaled experimental matrices of n x v1 and n x v2
n = number of spectra in each classv1 and v2 = number of variables in each matrix (32K)
NMR Control Rat urine
Hydrazine treated Rat
Hydrazine treated Rat
UPLC-MS Control Rat urine
SequentialNMR & UPLC-MSspectra can beobtained on eachsample forstatistical integration
SHYParallel NMR& MS dataCollection
© Imperial College, 2006
Direct Pathway Connection Co-variance (Am/z- B)
Direct Structure Assignment Co-varianceX-Ym/z (parent)-Zm/z (fragment)
MSdata
NMRdata
Ym/z X
BAm/z
Zm/z
SHY CONNECTIONS IN PARALLELNMR-MS SPECTROSCOPIC SETS
•
MSdomain
m/z
NMR domain
Statistical HeterospectroscopY (SHY): Expansion- shows NMR to parent ion, fragment pattern & pathway correlates.
N-acetyl-lysine
Co
rrel
atio
n/a
nti
corr
elat
ion
co
effi
cien
ts
MOLECULAR EPIDEMIOLOGY
DATA DRIVEN TOP-DOWN SYSTEM METABOLIC MODELING
Can genetic, dietary, microbial, and environmental influences in large
scale population studies be deconvolved?
J. Stamler (PI)
P. Elliot (PI)
M. Daviglus
H. Kesteloot
H. Ueshima
B. Zhou
Q. Chan
M. D Iorio
E. Maibaum
S. Bruce
C. Teague
R.L. Loo
L. Smith
The INTERMAP study has been supported by Grant 5-RO1-HL50490-09, 5-RO1 HL65461-04 and 5 RO1 HL71950-02 from the US National Heart, Lung, and
Blood Institutes, National Institutes of Health, Bethesda, MD, USA; by the Chicago Health Research Foundation; and by national agencies in Japan, People’s Republic of
China and the United Kingdom.
INTERSALT Study was supported by the Council on Epidemiology and Prevention of the International
Society and Federation of Cardiology; World Health Organisation; International Society of Hypertension;
Wellcome Trust; National Heart, Lung, and Blood Institute, US; Heart Foundations of Canada, Great
Britain, Japan and the Netherlands; Chicago Health Research Foundation; Parastatal Insurance, Company,
Belgium; and by many national agencies supporting local studies.
Acknowledgements:
Examples from the INTERMAP and INTERSALT studies.
•
CHINESE
COMBINATION OF GENETIC ENVIRONMENTAL& NUTRITIONAL FACTORS© Imperial College, 2006
JAPANESE
FUNDAMENTAL METABOTYPE DIFFERENCESPCA-DA of population data (disease outliers removed)
AMERICAN
Concluding RemarksMetabonomics is a powerful top-down systems biology tool for investigating drug toxicity, disease processes, phenotypic variation & differential gene function in vivo.
NOVEL OUTPUTS:Metabolic biomarker information on system regulation
& failure.Deeper understanding of DISEASE MECHANISMS.Models that incorporate genetic & environmental
interactions.
Omics data must be considered in an extensive biological framework with robust statistical interrogation and integration to visualize system activity
Analyzing modulations of the MICROBE-MAMMAL-METABOLIC AXIS will be crucial for understanding genotype-phenotype interactions and variation in toxicity and efficacy of drugs in man.
Top down metabolic modeling is likely to prove to be a powerful tool in the pursuit of Personalized Healthcare Solutions and understanding the Health of Nations.
The Metabonomics Engine @ IC & Collaborators
Academic: Dr Elaine Holmes, Prof John C. Lindon, Dr H. Keun Dr T.Ebbels, Dr J. Bundy, Prof James Scott, Prof Tim Aitman, Prof Paul Elliot, Dr H. Tang, Dr G. Tranter, Dr S. Mitchell. (Imperial)Post Doctoral Group: Drs, O. Cloarec, M. Dumas, A. Craig, A. Maher, B. Beckwith-Hall, E. A. Clayton, R. Barton, J., Y. Wang, E. Meibaum, I. Douarte, S. Bruce. T. Tseng, C.Stella, M. Coen. J. Sidhu, E.Skiordi, M. Bollard, ………..etcGraduate Students: T. Athersuch, I. Yap, R. C. Bailey, C. Teague, D. Parker, A. Tregay. J. Pearce, J. Bowen, S. Lowdell, L.Smith, A. Cooray, N.Jones, G. McLaughlin, D. O’Connor, R.Liu, M.Ratalainen, K. Veselkov, F.P. Martin. …etcCollaborators: Dr Rob Plum, John Shockcor (WATERS), Prof Ian D. Wilson and Dr T.Orton (AZ ), Prof J. Everett, Drs M. Reily and D. Robertson, (Pfizer), Prof Jose Ordovas (Tufts University), Prof Burt Singer (Princeton University), Drs M. Spraul (Bruker), Dr Sunil Kochhar (Nestle), Frans van D’Ouderra,J. Powell, M. Faughan et al (Unilever).Dr D. Gaugier (Oxford University), Prof D.Withers (UCL).FUNDING: NIH, The Wellcome Trust, BBSRC, MRC, EPSRC, NERC, The Royal Society, Roche Foundation, Servier, Lilly, P&G, Pfizer, AstraZeneca, Nestle, Unilever, Novo Nordisk, Roche Foundation, BMS, Hi-Q, Metabometrix, METAGRAD, WATERS CORPORATION.