Multiomics and Biomarkers in Drug Discovery and Personalised Medicine

Multiomics and Biomarkers in Drug Discovery and Personalised Medicine

Intended Learning Outcomes

• Appreciate the contribution of multiomics technologies in biomarker discovery, drug development, and personalised medicine.

• Understand the principles of genomics, proteomics, and metabolomics, and appreciate their current applications in drug and biomarker discovery, development, and validation.

• Understand the principles of advanced bio-analytic methodologies, in particular biological mass spectrometry and its applications.

• Understand the challenges and bottlenecks in developing and validating biomarkers, including sample biobanking and experimental design.

Personalised and Precision Medicine

• Personalised medicine: A medical model using molecular profiling technologies for tailoring the right therapeutic strategy for the right person at the right time, determining the predisposition to disease at the population level, and delivering timely and stratified prevention.
  Definition source: ‘OMICS in Personalised Medicine, EU Workshop, 2010

• Precision medicine: An emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person.

getting away from ‘one size fits all’

Molecular Profiling Technologies: Defining Multiomics

• Disciplines whose names end in the suffix 'omics'.

• In biological sciences, an 'omics' discipline aims at the collective characterization and quantification of pools of biological molecules (e.g., proteins, sugars, lipids, genes) that translate into the structure, function, and dynamics of an organism or organisms (e.g., genomics, proteomics, lipidomics).

• Multiomics is an integrative approach that combines experimental data generated by different omics techniques with computational approaches.

• investigating more than one molecule - gethering info from hunfreds and thousands of molecules/ species

The Main Omics Technologies

• Genomics: Sequencing and analysing an organism’s genome, the complete set of DNA.

• Transcriptomics: The study of RNA molecules arising from the expression of genes.

• Proteomics: Large scale analysis and study of proteins in an organism.

• Metabolomics: Study of the complete collection of metabolites within cells, body fluids, tissues or organisms; various sub-disciplines as the metabolome is vast and includes diverse species e.g. sugars, lipids, amino acids etc.

Technology Platforms Supporting Omics Applications

• Omics applications are facilitated by large technology platforms, supported by high throughput assays and automation.

• Sequencing platforms used in genomics and transcriptomics.

• Mass spectrometry is a key technology for proteomics and metabolomics; NMR also used.

• Large data sets are managed by computational approaches; application of bioinformatics; support for computational biology, systems biology, systems medicine.

What is a Genome

• The entirety of DNA in an organism; 30,000 - 40,000 genes.

• Publication of the complete human genome in 2004.

• It includes genes that encode proteins.

• Non-coding DNA is 95-98% of human genome.

  • It regulates the activity of genes.

  • Other unknown functions.

Basic Genetic Research

• Many differences in drug response attributed to variations in the genes that metabolise drugs or determine cellular sensitivity to drugs.

• 100,000 people die per year and about 2,000,000 suffer serious reactions to medications generally considered safe.

• Cancer is the first area to have benefited from the genomic revolution and has supported the concept of personalised (precision) medicine.

Medical and Pharmaceutical Applications of Genetic Research

• Disease With Genetic Component

  • Identify Responsible Genetic Variation

  • Diagnostics

    • Genetic tests

    • Protein or enzyme tests

  • Gene Therapy

    • Repair or replace malfunctioning gene

  • Understand Basic Biological Defect

    • Which proteins and enzymes are involved?

    • What is their normal function?

    • How do they malfunction?

  • Pharmacogenetics

    • Prescribe most effective drug

    • Avoid serious side effects

  • Preventive Medicine

    • Identify healthy people at risk for future disease

    • Optimize follow-up

    • Recommend lifestyle changes

  • Drug Therapy

    • Develop drugs that target the specific biological malfunction

From Cancer Genomics to Personalised Medicine

• Knowledge: drug targets & biomarkers

• Drug and biomarker discovery and development

• Genomics-informed clinical trials

• Regulatory and commercial challenges

• Patient consents

• Sample acquisition

• Clinical annotation

• Study design

• Functions & mechanism of action

• Analysis

• Cancer genomics

Personalised Medicine Bottlenecks

• Understanding of the genetic, molecular and cellular mechanisms underlying common diseases is currently limited.

• Omics platforms for nucleic acids (genomics) ready for clinical applications, but other omics need further development.

• Need for large scale studies to identify and validate disease biomarkers and signatures.

• Bio-banking is very expensive; we need:

  • Gender- and age-stratified collections of healthy individuals.

  • Gender- and age-stratified disease related collections.

  • Population-wide collections with health status, life-style, environmental exposure and diet information.

• Training: clinical bio-informaticians; researchers; clinical community, funders and regulators and health care provides.

The UK Biobank Resource

• The UK Biobank is a prospective cohort study that has collected genetic and phenotypic data from some 500,000 people aged 40–69 from across the United Kingdom.

• The participants have undergone health measurements, provided blood, urine and saliva samples, given detailed information about themselves and agreed to have their health followed.

Biomarkers

• What is a biomarker: “A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” (NIH, 2001).

• Characteristics of an ideal biomarker

  • Safe and easy to measure

  • Cost efficient to follow up

  • Modifiable with treatment (eg cholesterol)

  • Consistent across gender and ethnic groups

  • Objective measurement

Proteomics

• Proteomics is defined as “large-scale study of proteins, particularly their structures and functions”, usually using mass spectrometry (to distinguish from antibody-based measurements of single proteins).

• Clinical Proteomics is a field of proteomics research focussing on primary samples (tissue, biofluid) i.e. taken direct from a patient.

• Translational proteomics is the process of taking putative biomarkers towards clinical use.

• Proteins express the genetic potential of an organism – active biological agents in cells.

• They are involved in almost all cellular processes and fulfil a multitude of functions.

  • Functions: enzyme catalysis, transport, mechanical support, organelle constituents, storage reserves, metabolic control, protection mechanisms, toxins, osmotic pressure.

• Why proteins

  • Whole genome sequence: complete but, does not show how proteins function or what biological processes occur.

  • Gain insight into alternative splicing (i.e. multiple protein isoforms encoded by the same gene).

  • Study post-translational modifications - proteins chemically modified or regulated after synthesis.

Why Study the Proteome

• Gain insight into alternative splicing (i.e. multiple protein isoforms encoded by the same gene).

• Study post-translational modifications - proteins chemically modified or regulated after synthesis.

• Sometimes proteins need post translational modification to become active.

Mass Spectrometry and Omics

• Mass spectrometry offers a versatile, sensitive, selective method for the qualitative and quantitative analysis of various classes of metabolites of biological importance (e.g. proteins, peptides, lipids, sugars).

• Various modes of ionisation make it suitable for different matrixes.

• It can be coupled to chromatographic separation to support quantitative analyses.

• It can be automated and support high throughput analyses.

• It generates large data sets that can be processed via computational approaches.

Basic Components of a Mass Spectrometer

• Inlet

• Source

• Analyzer

• Ion Detector

• Data System

• Vacuum Pumps

take the outcome of mess spec and link it to the structure of protein.

Ionisation – Analysers - Detectors

• Ionisation (formation of ions)

  • Matrix assisted laser desorption ionisation (MALDI)

  • Electrospray ionisation (ESI)

  • Fast atom bombardment (FAB)

  • Atmospheric pressure chemical ionisation (APCI)

  • Electron ionisation (EI)

  • Chemical ionisation (CI)

• electrospray is good if the metabolite of interest is soluble.

• Mass analysers (separation of ions according to their mass)

  • Time of flight (TOF)

  • Ion Trap (IT)

  • Quadrupole (Q)

• Detectors

  • Photomultipliers and array detectors

MALDI

Surrounds metabolite and makes it more friendly for mass spec through radiation with a laser. Whole thing is lifted over surface, ionised and goes into mass spectrometer.

Is useful for molecules that are on a surface and are not dissolved in solvent that can be used for electrospray.

• Matrices:

  • Organic matrices: cinnamic or benzoic acid derivatives

  • Liquid crystalline matrices

  • Inorganic matrices, such as graphite

MALDI-ToF

Time of flight

• ToF: Ions are accelerated to have the same kinetic energy. The time taken to travel a fixed distance is used to find their mass.

• land on detector according to how heavy they are.

Quantitative Proteomics

• Gel based

  • Electrophoresis

    • 2-DE, 1-DE

  • In-gel digestion

  • HPLC/MS/MS

• HPLC based

  • Label

  • Digest

  • Label

  • Digest

  • 2D-HPLC/MS/MS

• Database search and protein quantification

many clinical applications, proteomics are used a lot in cancer discovery.

Liquid chromatog approaches used to analyse different proteins

If have very large proteins, resolved using gel

Gel Based Proteomics: 2D Gel Separation of Proteins

Proteins are separated in the gel.

LC-MS/MS

Liquid chromatography can be used to separate them in a similar wau- use the ability of the small molecules to be dissolved in and separated on a column.

• A quadrupole is a mass analyzer that separates ions based on the stability of their flight trajectories through an oscillating electric field in the quadrupole (consisting of four cylindrical rods).

Importance of Biological/Clinical Sample

How Suitable is Plasma?

• Twenty-two proteins constitute 99% of the protein content of plasma.

• Huge conc of albumin in blood so can mask a lot. Very difficult t isolate protein biomarkers from these.

Proteomics Biomarker Discovery Projects Follow a Similar Pipeline

• DISCOVERY

  • Few samples

  • Many molecules

  • Complex data analysis

  • Define list of ‘putative’ markers

• VALIDATION

  • More samples

  • Fewer molecules

  • Complex data analysis

  • Refine list of ‘putative’ markers

• ASSAY DEVELOPMENT

  • Test samples

  • Target molecules

  • Complex design and testing

  • Build a robust and well-controlled test

• CLINICAL VALIDATION

  • Many samples

  • Target molecules

  • Apply targeted, robust assay

  • Define utility of marker(s) in a ‘real- life’ setting

Proteomics in Biomedical and Clinical Research

• A growing sector; challenges remain.

• Shows promise for use in both discovery of novel biomarkers, and in translation it the clinical laboratory.

• However, it lags behind genomics (could be up to 10 years).

• Methods now are capable of identifying putative biomarkers.

• Methods for translation of multi-protein panels into routine clinical use under development.

• Requires knowledge of disease, a good/specific question and good experimental design/analysis: experimental design and choice of sample are of importance.

• Proteomics in the NHS: few validated diagnostics using protein mass spectrometry and proteomics.

Metabolomics

• The metabolome

  • The total quantitative collection of low molecular weight organic and inorganic species (metabolites) present in a biological organism.

  • Involved in endogenous metabolism (biosynthesis or catabolism).

  • Uptaken from the external environment (drugs, food nutrients, growth medium components).

  • Involved in exogenous metabolism (drug metabolism).

  • Symbiotic relationships (gut microflora)

  • Important for health of mammals.

What are Metabolites

• Small molecular weight (MW) organic and inorganic species generally molecular weight less than 1500Da.

• Not proteins or peptides - there are a few exceptions (e.g. glutathione is a tetrapeptide)

• Polar metabolites: amino acids, carbohydrates, organic acids, inorganic salts.

• Non-polar metabolites: fatty acids, bile salts, steroids, glycerophosphotidylcholines etc

• A wide diversity of physical and chemical properties: molecular weight, reactivity, concentration, hydrophobicity.

• Metabolomics is the process of identifying and quantifying all small molecule metabolites of an organism in a specified biological state.

• The metabolites of an organism represent a chemical “fingerprint” of the organism in a well-defined state as defined by specific circumstances.

• Metabolomics can provide an overview of the metabolic status and global biochemical events associated with a cellular or biological system.

PHENOTYPE

everything together is phenotype- genome, transcriptome, proteome , metabolome

This is what metabolites would like to be able to feed back.

METABOLOME

• More than 6500 endogenous and exogenous metabolites

• Complex, changes as we age, identifying biomarkers here is difficult.

The Metabolome is Complex

• Wide dynamic concentration range (femptomoles to millimoles).

• Large number of metabolites (>7,000 in the human metabolome).

• Spatial, temporal and behavioural variability.

• Identifying metabolites a major bottleneck.

Why Study the Metabolome?

• Final downstream product of gene transcription and is therefore closest to the phenotype of the biological system studied.

• Highly sensitive to genetic or environmental perturbations – allows detection of external factors (markers of nutrition, pharmacology, microbiome etc).

• Dynamic in nature; changes in concentrations are measured in sub- second/seconds timescales depending on area of metabolism and context e.g. physical perturbation to heart produces many metabolic changes in serum within one minute of the perturbation.

• Metabolic profiling is much cheaper and very much more high-throughput, making it feasible to examine large numbers of samples.

Global Metabolic Profiling: Untargeted Analysis

• Shotgun analysis of many metabolites – largely based on concentration.

• Provides a snapshot of global metabolism.

• Mass spec allow us to take an extract from a cell.

• send it down mass spectrometer w/o any separatin and measure ions.

• can see initially and see changes- however, hard to put these changes into the context of biological pathways from this.

Targeted Metabolomics

• Analysis of specific metabolites.

• These could be endogenous molecules of interest, drugs (or their metabolites) or nutrient markers.

• Quantitative analysis

• good way to understand how drugs work.

Methods for Global Metabolomics

• LCMS

• GCMS
  Separate & Detect, Feature Finding, Alignment & Statistics, Identify Pathways

• Different platforms used.

Tandem Mass Spectrometer Metabolomics Platforms

• LC, GC

• Ion Source ESI, APCI

• MS1 m/z filter Quadrupole

• Collision Cell

• MS2 m/z filter QQQ ToF (QTOF) Ion Trap (QTrap)

• detector

ESI- electrospray ionisation bc deaking with small molecules.

Liquid Chromatography

• Low organic (polar) solvent: Upon injection, most molecules bind to and ‘focus’ on the head of the column.

• High organic content: Most molecules will now be in the mobile phase and the column ‘cleaned’

• Increase solvent content: As organic solvent increases (some) molecules dissociate from the stationary phase and flow through the column.

• The ramping of organic solvent concentration (e.g. 20% to 80%) over a fixed times causes metabolites to be separated by their polarity or their hydrophobicity.

• Different stationary phases can be used to alter binding and separation of specific compounds.

• can separate molecules depending on their affinity.

Mass Spectrometry for Targeted Metabolomics

• Selected Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM) requires a defined mass pair e.g. mass of metabolite and one of its fragments (chosen based on its fragmentation pattern).

• If you couple liquid chromatography to mass soec, specifically designed for small molecules. Allows isolation of moleucle want to study- fragment it, find the fragment its related to and analyse it.

Gas Chromatography

• Low Temperature: Most molecules, most of the time, are in the stationary phase, and not moving

• High Temperature: Most molecules, most of the time, move down the column with the carrier gas flow

• Intermediate Temperature: At a certain temp., molecules of a given metabolite spend some time stationary, and some moving with the carrier gas

• The “intermediate” behaviour occurs at low temperatures for volatile compounds, higher temperatures for less volatile components.

• The temperature of the column oven is ramped (say 50ᴼ to 300ᴼ over 20 min) to give an analysis over a wide range of volatility.

• chromatograms to separate columns

Gas Chromatography-MS

• Because the mass spectrometer is fast (20 spectra per second), it can recover good spectra even if the components aren’t fully separated

• each molecule can be identified with computers linking these properties to databases.

Metabolomics Bottlenecks

• Huge diversity of chemical structures and large differences in abundance.

• There is no single technology available to analyze the entire metabolome.

• Complementary approaches have to be established for extraction, detection, quantification, and identification of as many metabolites as possible.

• Extract the information from vast numbers of data produced by high throughput methodologies: need for statistical and computational approaches; multi variant data analysis.

• Hundreds to thousands of metabolites -chemometric data analyses can reveal statistically meaningful correlations between the independent variables and the metabolic profile.

  • Clustering techniques and data visualisation.

  • Principal Component Analysis (PCA), dendrograms, hierarchical clustering.

• We dont know what normal levle is fro a lot of the molecules due to huge diversity of structures.

• Need lots of support from bioinformatics.

Strategies of Drug Target Discovery Based on Omics

Disease, Clinical samples, Cell Model, Animal Model, Disease tissue --> OMICS (Genomics, Transcriptomics, Proteomics, Metabolomics, Lipidomics) --> Bio-Informatics (Pathway Analysis, Target Identification, Gene Disease Database, Data Mining) --> Functional Analysis (RNAi Analysis, Transfection, Rev-Docking, Network) --> Potential Targets (Biomarker Discovery, Candidate Drug Target Database) --> Target Validation (Expression Modulation in cell models, Modulation in animal models)

can generate targets for intervention.

Role of Multiomics in Pharmaceutical Research and Drug Development

Pathway, Target, Drug, Disease --> Interactomics, Bioinformatics, Genomics, Proteomics, Metabolomics, Chemoproteomics, Pharmacometabolomics, PhytoChemomics, Pharmacognomics, Reverse Docking, Systems Biology, Network Pathway & Biology --> Mechanism --> Target Discovery, Drug Development, Drug Assessment, Personalized Medicine

Application to humans , from bioinformatics to personalised medicine.