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PY366 Drug discovery > molecular biology > Flashcards

molecular biology Flashcards

1
Q

what is the central dogma

A
  • DNA makes RNA makes protein
  • DNA makes RNA through transcription
  • RNA makes protein through translation
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2
Q

what is molecular biology

A

the study of biological molecules, their reactions and interactions

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3
Q

what is a genome

A

total genetic content of a cell/organism
- the complete DNA sequence of a cell/organism

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4
Q

what is a transcriptome

A

all mRNA molecules currently present in a cell/organism

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5
Q

what is a proteome

A

total set of proteins currently present in a cell/organism

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6
Q

how are genomes studied

A

by transcriptomic and proteomic profiling

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7
Q

how do genomes vary

A

largely the same between cell types within an individual

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8
Q

how will transcriptomes vary

A

will vary between cells, and with environmental conditions

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9
Q

what can be used for the study of mRNA abundance

A
  • RNA sequencing
  • DNA micro assays
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10
Q

what can be used for the study of protein abundance

A

traditionally Ab based: ELISA assays
- needs antibodies and lacks highly parallel methods and universal external standard
- mass spectrometry

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11
Q

how will proteomes vary

A

will vary between types of cells and with environmental condition

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12
Q

what is important in proteomes

A

post translational modifications
- proteolytic cleavage
- covalent modification

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13
Q

what is mass spectrometry

A

method to accurately measure mass of proteins and protein fragments

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14
Q

what does mass spectrometry require

A

proteins/peptides are ionised to allow detection

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15
Q

what are the advantages of mass spectrometry

A

-high sensitivity, accuracy and resolution

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16
Q

what is the role of a mass spectrometer

A

generates gas phase ions from a sample, separates them according to their mass to charge ratio and generates a record of their abundance, allowing:
- MW determination
- amino acid sequencing
- PTM detection
- relative quantification
- structural analysis

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17
Q

what are some of the considerations of omic studies

A
  1. generate very large amounts of data
  2. can take a lot of time and money
  3. picking conditions in transcriptomics and proteomics may be critical
  4. risk of errors
  5. amount of data often exceeds amount of understanding
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18
Q

what is DNA sequencing used for

A
  • nucleic acid sequencing can be used in diagnostics
  • sequencing of genes amplified by PCR allows identification
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19
Q

give examples of next generation sequencing

A
  • Illumina, SOLiD
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20
Q

Give an example of nanopore sequencing

A

minION

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21
Q

outline the drug discovery process

A
  1. target identification
  2. determine DNA and protein sequence
  3. elucidate structure and function of the target
  4. proof of concept and assay development
  5. assay for high throughput molecular screen
  6. high throughput screening/ directed synthesis programme
  7. select lead structures
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22
Q

how can drug targets be identified

A
  1. genetic association studies
  2. gene expression studies- transcriptomics
  3. protein expression studies- proteomics
23
Q

what do genetic association studies involve

A

looking for presence of DNA sequence variation in diseased vs healthy individuals/cells
- identify mutations and polymorphisms associated with disease

24
Q

what do gene expression studies involve

A

looking for differences in gene expression in diseased vs healthy individuals

25
what do protein expression studies involve
looking for differences in protein expression and modification in diseased vs healthy individuals
26
what properties does a good drug target need to have
1. unique to pathogen/cell type - otherwise higher risk of side effects 2. essential - inhibition/antagonism needs to affect cell or tissue 3. non redundant (no other molecules with similar function) - otherwise the other gene/protein may compensate 4. susceptible to modification - drug like molecules need to modify activity
27
what are the 2 key steps involved in validation
1. reproducibility- once identified via specific technique 2. introduction of variation to ligand target environment
28
what should variation to ligand target environment lead to
- modulation of drug molecule activity results in changes in affinity for target - variation in cell or tissue type should/should not vary effect - mutation of binding domain of protein target should result in loss or modulation of drug effect
29
what is overexpression
making more of the protein than normal
30
what is gene knockout
deleting the gene and therefore stop protein production
31
how are animal models used in human disease
animal models can be genetically modified so that they develop human disease - eg. knockouts, transgenes
32
what techniques are used in making the target
express recombinant protein in a suitable host - E.coli - Saccharomyces cerevisiae - cells in culture (mammalian cells)
33
what are the advantages of using recombinant DNA techniques
1. get large amounts of protein 2. enables structural studies, functional studies and screening 3. can make altered forms (change DNA to change resulting protein) - study isolated domains/regions - alter single amino acid residues and test hypotheses about drug binding
34
what is the genome project
- determined the DNA sequence for humans - many G protein coupled receptor genes identified - many of them of unknown function
35
what are downstream effectors
1. if we know that a protein is overexpressed in a diseased tissue, whole cell transcriptome can elucidate the cascade that follows 2. this can be used to identify potential drug targets
36
what are genetic polymorphisms
different in DNA sequence between individuals - chance mutations - induced mutations (viral infections, chemical agents)
37
what is a genotype
what is in the DNA - inherited from parents - deduced from gene sequence
38
give an example of a genotype
genes encoding eye colour
39
what is a phenotype
what is observed in an individual - protein expression - environmental factors - epigenetics
40
give an example of a phenotype
an individual with brown eyes
41
what are the types of genetic polymorphisms
1. single nucleotide polymorphisms 2. copy number variants 3. INDELS
42
what are single nucleotide polymorphisms
- most common type of polymorphism - differ in base pair in DNA sequence
43
what are copy number variants
- segment of DNA can have multiple copies - important in drug metabolism
44
what are INDELS
- insertion or deletion of a base pair - often results in non functional protein
45
what are genome wide association studies
- collect very large data sets on individuals genetic makeup - look for associations between genetic variations and different disease states
46
what is a Manhattan plot
- type of scatter plot - helps identify which SNPs are linked to the condition
47
what are the clinical implications of genome wide association studies
1. can establish correlation but not necessarily causation 2. can be used for diagnosis and prognosis decisions 3. starting point for further biological study
48
what are enteroviruses
responsible for several significant diseases (polio, common cold)
49
why is the common cold difficult to treat or prevent
rapid mutation impedes vaccine development
50
what is required for enterovirus pathogenesis
requires the host methyltransferase SETD3 - virus infects human cell - uses host enzymes for replication - cell bursts releasing new virus particles
51
describe the study for enterovirus pathogenesis
1. studied the transcriptome of infected cells and worked out what proteins were being expressed 2. produced a gene knockout strain of mice 3. compared disease progression with wild type mice
52
what were the implications of this study
1. not producing SETD3 enzyme appears to prevent virus replication 2. gene knockout mice lived normally
53
what are pharmacogenetics
study of gene variations in drug response

PY366 Drug discovery (15 decks)

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