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Drug discovery > Lecture 2.2 > Flashcards

Lecture 2.2 Flashcards

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1
Q

Target discovery is comprised of 3 steps:

A
  1. provision of disease models
  2. target identification
  3. target validation
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2
Q

What are the 2 approaches to target discovery?

A
  1. ‘molecular’ approach
  2. ‘systems’ approach
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3
Q

What is the ‘molecular approach’ to target discovery?

A

uses techniques such as genomics, proteomics, genetic association and reverse genetics

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4
Q

What is the ‘systems approach’ to target discovery?

A

uses clinical and in vivo studies to identify potential targets.

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5
Q

Briefly explain how target validation is done?

A

modulation of gene expression and/or protein function in both cell and animal models is used to confirm the role of the target

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6
Q

What are some techniques for target identification?

A
  1. radioligand binding
  2. DNA microarray
  3. in silico methods
  4. correlative technologies (proteomics,
    genomics, genetic association)
  5. phenotype-driven target identification
    –> forward genetics and reverse genetics
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7
Q

Briefly explain the technique of ‘radioligand binding’

A

Bind the potential receptors with radioligands so that the targets can be picked out from a
pool of other receptors

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8
Q

What are the steps involved with radioligand binding?

A
  1. ligands with high specificity for particular
    targets are labeled with radioisotopes
  2. tissue (containing target of interest) mixed
    with radioligands
  3. bound receptors separated, cloned and their
    nucleotide sequence decoded
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9
Q

Briefly explain the purpose of ‘DNA microarray’

A

By measuring the expression profile, identify the target genes that cause disease

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10
Q

What are the steps involved with DNA microarray?

A
  1. array of genes (ssDNA) printed on solid
    support of membrane
  2. mRNA from healthy + diseased used to
    generate cDNAs
  3. cDNA labeled with fluorescent tags
  4. incubated with microarray support
  5. genes in samples pair up with
    complementary counterparts
  6. fluorescence indicates level of expression

–> disease-related genes revealed

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11
Q

Briefly explain an in silico method for target identification.

A

ESTs of interest compared with those in database for known protein structures

–> further aided by database on 3D protein structures, AA sequence in sample

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12
Q

What are Expressed Sequence Tags (ESTs)?

A

Short nucleotide sequences of cDNA that code for the expression of particular proteins

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13
Q

What are some correlative technologies used for target identification?

A

techniques to measure differential expression of mRNA or protein

for RNA (genomics) –> PCR

for protein (proteomics) –> gel electrophoresis + mass spec

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14
Q

How are genetic association studies used to identify targets?

A

Researchers compare the frequency of genetic variants (such as SNP) between individuals with a particular trait or disease (cases) and individuals without the trait or disease (controls).

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15
Q

What is ‘forward genetics’ in phenotype-driven target identification?

A

random modulation of the phenotype using in vitro biological and chemical library screen or in vivo chemical mutagenesis and the subsequent identification of the gene (phenotype to gene)

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16
Q

What is ‘reverse genetics’ in phenotype-driven target identification?

A

gene manipulation and examination of the phenotype (target gene is known beforehand)

–>determine their functional roles in disease processes and assess their potential as therapeutic targets

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17
Q

Genomics includes developing and utilizing tools for the prediction and detection of:

A
  1. genes
  2. sequence similarity
  3. motif/domain similarity
  4. gene expression variants
    (measurement of mRNA levels through
    microarray analysis)
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18
Q

What are the limitations of genomics?

A

mRNA expression levels do not reliably predict protein expression levels

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19
Q

Why can’t we use mRNA expression levels to predict protein expression levels?

A
  1. different half-lives in cells
  2. PTMs after translation
  3. cannot predict localisation of proteins
  4. cannot provide info on protein interactions
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20
Q

How to overcome the limitation of genomics studies?

A

study the function, structure and interactions of proteins themselves at a system-wide scale

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21
Q

What is functional genomics?

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A

systematic analysis of gene activity in healthy versus diseased organisms/ organs/ tissues/ cells.

22
Q

According to functional genomics, disease mechanisms can be broadly classified into:

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A
  1. defects in distinct genes - genetic disorder (down
    syndrome)
  2. infection by bacteria, fungi, viruses (AIDS, HIV-
    infection, COVID-19)
  3. immune-autoimmune diseases (SLE)
  4. multicausal disease (functional dyspepsia)
  5. trauma and acute disease based on injury or organ
    failure
23
Q

What environmental factos may raise the risk of developing a particular disease?

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A

diet, toxic exposure, trauma, stress

24
Q

Functional genomics explores gene function through the analysis of:

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A
  1. Regulatory pathways
  2. biochemical pathways
  3. protein-protein interactions
  4. effects of gene knockouts, gene up-regulation, gain-of-
    function
  5. results of functional complementation of knockouts
25
What is proteomics?
the study and manipulation of the protein make-up of a cell and the set of enabling techniques associated with the study.
26
Why is the study of proteins important in drug discovery?
proteins in a living organism are the molecular targets of most drugs on the market or being developed
27
Proteomics studies aim to reveal information on:
◼ Protein structure and function ◼ Protein expression levels ◼ Post-translational modifications ◼ Subcellular localization ◼ Protein-protein interactions ◼ Protein-nucleotide interactions (e.g., to identify and study transcription factors) ◼ Protein-lipid interactions (membrane-associated proteins in signal transduction pathway)
28
Why do we study protein-nucleotide interactions?
to identify and study transcription factors
29
Why do we study protein-lipid interactions?
membrane-associated proteins in signal transduction pathway
30
What do the major functional categories of proteomics include?
1. fractionation and purification 2. identification 3. quantitative analysis 4. characterization (sequence homologies, PTMs, functional analysis,structural analysis)
31
Why does the number of proteins far exceed the number of genes?
Due to splice variants and post-translational modifications, the number of proteins far exceeds the number of genes.
32
Why is it important to study proteins on the protemic scale instead of studying them in isolation?
proteomic scale yields more insights --> study of protein-protein interaction enables the mapping and functional elucidation of an entire metabolic or signaling pathway --> provides information on relevant constituent proteins and their interconnections
33
What 2 categories can the fractionation and purification techniques be divided into?
- low-scale techniques - high throughput applications
34
Name some low-scale techniques for protein fractionation and purification.
1. gel and column based fractionation 2. differential centrifugation/ centrifugation through density gradients 3. immunoprecipitation 4. affinity chromatography
35
Name some 'high throughput applications' techniques for protein fractionation and purification.
1. 2D PAGE --> 1st dimension by isolectric point, 2nd by mass 2. HPLC
36
What are the limitations of 2D PAGE?
1. does not replicate well 2. cannot detect sample at low [C] 3. problem with hydrophobic protein 4. poor separation for proteins with similar mass and pI
37
What is the principle of HPLC?
Packing materials in a column selectively capture the protein of interest while the rest of the sample solution is washed through.
38
What are some detectors used in HPLC?
Refractive index, UV, fluorescent, electrochemical, MS, NMR, ELSD
39
What are the advantages of HPLC?
1. faster 2. reproducibility 3. sensitivity to low [C] 4. separation based on more attributes than just mass and pI: ionisation, molecular size, specific affinity to an antibody, hydrophobicity
40
What attributes could the separation of proteins in HPLC be based on?
ionisation, molecular size, specific affinity to an antibody, hydrophobicity
41
What are the identification techniques of proteins?
1. For known proteins, mass and pI observed in 2D-PAGE --> look up database 2. For novel protein, AA can be determined by: --> edman sequencing --> MS analysis for high-throughput application
42
What are some quantitation methods of proteins?
1. UV absorption 2. gel-based methods using staining reagents (fluorescent or light emitting dyes) 3. Mass spectrometry
43
Name a method of seperating out glycosylated proteins
Affinity purification coupled with MS analysis --> Lectin binds the sugar part of a glycoprotein, thus can be used to separate out glycosylated proteins
44
What are some ways to 'characterize' proteins?
1. Search for sequences that share an evolutionary relationship 2. Determine the presence or absence of particular post- translational modifications (PTMs) such as glycosylation, phosphorylation, proteolysis 3. functioanl analysis to evaluate interactions with ligans, lipids and other proteins
45
What are some high-throughput methods for protein- protein interaction?
1. phage display 2. protein chips 3. yeast two-hybrid 4. Tandem affinity purification
46
What are some techniques for structrual analysis of proteins?
1. laboratory structure determination - NMR - X-ray crystallography 2. Computational structure determination
47
What are the pros and cons of using NMR or X-ray crystallography in determinng protein structure?
pro: images down to fine resolution con: snapshot of a protein and may miss other configurations
48
What is the con of using computational structure determination method for proteins?
Not as accurate as laboratory methods
49
How do computational structure determination methods predict protein structure using the AA sequence?
1. homology modelling 2. Ab initio fold algorithms (fold from the beginning) 2. secondary strcutural prediction
50
Overall, what is the main purpose of protemics in drug discovery?
1. Identify novel proteins as new drug targets (using 4 functional areas) 2. Perform initial functional characterization of a novel protein --> Computational sequence homology analysis 3. Screen a compound with known therapeutic effect against a large number of proteins to pinpoint a possible target.
51
What is the purpose of computational sequence homology analysis in drug discovery?
help putatively assign a novel protein to a known pathway or network where its homolog participates in

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