Drug Discovery 12

Question 0

define pharmacodynamics:

Answer 0

it is the study of the effects of drugs on biological processes
for drugs to produce an effect they must interact with biomolecules =drug targets

Question 1

what are the different types of drug targets ?

Answer 1

enzymess
plasma membrane receptors
transporters
ion channels
nucleic acids
nuclear receptors
many drugs have been discovered without knowing the structure of their target

Question 2

what does structure based drug discovery require ?

Answer 2

requires knowing the structure of the drug target

Question 3

where do you get atomic resolution structures of proteins?

Answer 3

x-ray crystallography or nuclear magnetic resonance

• x ray crystallography requires crystals of pure protein and NMR requires pure protein in solution
• both also require lots of protein

Question 4

how does x-ray crystallography work ?

Answer 4

this process is difficult for oily membrane proteins
fire x-ray beam through the crystal and then collect the diffraction pattern - the greater the amount of diffraction the greater the resolution
- this process produces an electron density map which you fit a model of he known polypeptide sequence of the protein to

Question 5

what is NMR limited to ?

Answer 5

it is limited to smaller proteins, <35kDa

Question 6

how does NMR work ?

Answer 6

the pure protein solution is placed in a very strong magnetic field enabling the distance between atoms to be estimated
powerful magnets and electromagnetic radiation are used to yield structural information
it calculates the inter-atomic distances within the protein structure and this enables you to determine the structure of the protein

Question 7

with NMR what does the solving to the structure also incorporate ?

Answer 7

incorporates all the known chemical/bonding/chirality properties of the protein

Question 8

what does an ensemble of structures produced by the protein enable ?

Answer 8

it demonstrates the numerous possible solutions of the distance matrix data so it can be used to locate/understand regions of conformational flexibility - demonstrates the flexibility of the protein

Question 9

what are the advantages and disadvantages of x-ray crystallography ?

Answer 9

advantages
- better resolution
- not limited by protein size
disadvantages
- requires crystals
- more difficult for membrane proteins- hard to crystallise

Question 10

what are the advantages and disadvantages of NMR?

Answer 10

advantages
- solution structure - so no crystals
- can study dynamics 
disadvantages
- limited by protein size <35kDa

Question 11

what are the requirements for pharmacological screening ?

Answer 11

-small amounts of protein are required
-protein can be impure
but
- human/mammalian cell lines are used which are expensive to culture and the yield is poor

Question 12

what are the requirements of structure determination screening ?

Answer 12

• bacterial, yeast or insect expression systems are used
  but
• requires large amounts of protein
• requires pure homogenous protein

Question 13

what are the stages involved in protein expression and what is the crucial difference ?

Answer 13

1- obtain cDNA clone to be expressed
2- clone into an appropriate expression vector
3- introduce into a host cell/organism
4- express and isolate the expressed protein
crucial difference is in our choice of expression host and therefore an appropriate vector

Question 14

what is present in a mammalian expression vector ?

Answer 14

multiple cloning site
polyA signal- if protein is to be expressed in human/mammalian/eukaryotic cell line
promoter
colE1 origin of replication - required for replication in bacteria
antibiotic resistance gene- required for selection in bacteria
SV40 origin of replication- required for replication in human/mammalian cell lines

Question 15

what is present in a bacterial expression vector ?

Answer 15

ribosome binding site
multiple cloning site 
inducible promoter- it needs to be recognised by bacterial RNA polymerase 
colE1 origin of replication 
antibiotic resistance gene

Question 16

how does protein synthesis initiation occur in bacteria ?

Answer 16

the 30s subunit of the ribosome recognises the ribosome binding site of the mRNA
start signal codon AUG is situated about 10 bases on the 3’ side of RBS which is important because otherwise it will not be expressed
ribosome binds to the RBS and then locates AUG

Question 17

what is an inducible promoter?

Answer 17

it means that the promoter has to be induced before it starts making mRNA of the target gene
the most common method for inducing bacterial expression is by using IPTG to induce the lacZ promoter - based on the natural way that bacteria induce the enzyme beta-galactosidase

Question 18

what does e.coli usually rely on for protein expression, however if it is not present what does it then rely on ?

Answer 18

glucose
however if glucose is not present then it relies on lactose
it uses lactose by cleaving this disaccharide using the enzyme beta-galactosidase to produce galactose and glucose

Question 19

what does it means by beta-galactosidase levels are nutrient dependent ?

Answer 19

e.coli growing on an agar plate containing glucose will only have about 10 copies of beta-galactosidase whereas on a media containing no glucose the e.coli ill contain several thousand copies of the beta-galactosidase enzyme

Question 20

how is beta galactosidase controlled?

Answer 20

it is only expressed when its required which is when glucose is not present and therefore it needs to cleave lactose

Question 21

define operon:

Answer 21

a collection of genes that are controlled as a unit

Question 22

what is the lactose operon ?

Answer 22

it a a collection of genes= lacZ, lacY and lacA - these 3 genes are transcribed to give a single polygenic transcript
the gene for beta-galactosidase enzyme is called lacZ

Question 23

why is the transcription of the lactose operon different from other genes?

Answer 23

because another gene called the regulator encodes a repressor protein when glucose is present and this binds strongly to a region of DNA between the promoter region and the gene called the operator and when this binds to this region it prevents RNApolymerase transcribing the gene
- a relatively low level of repressor is able to prevent the large scale expression of beta-galactosidase

Question 24

why is it beneficial that a low level of expression of the repressor gene is needed to prevent the expression of the lacZ gene ?

Answer 24

because it saves the bacterial cell a lot of energy because if glucose is present it doesnt need to cleave lactose therefore it doesnt require beta galactosidase expression

Question 25

what happens to the lactose operon when there is low levels of glucose ?

Answer 25

if lactose is present it enters the cell and a natural variant allo-lactsse can bind to the repressor protein an inhibit it or the inducer IPTG can inhibit it and enable the expression of beta galactosidase
- the inducer binds to the repressor protein so it cant bind to the operator

Question 26

what would happen if the the lac operon was replaced with the cDNA of our target ?

Answer 26

we could induce the bacteria to express the cDNA protein

Question 27

what is the advantage of inducing expression ?

Answer 27

it means that the cell culture can be grown without the need to produce the foreign protein- then when the culture is healthy and in its exponential growth phase the inducer can be added to obtain maximum yield of the target protein

Question 28

what are the stages involved in bacterial expression ?

Answer 28

1- first sub clone your cDNA into an appropriate vector
2- transform it into an appropriate strain of cDNA for high levels of expression (BL21)
3- culture the cells and induce protein expression
4- centrifuge the cells to obtain a cell protein
5- break open the cell pellet and purify your protein

Question 29

what is the quickest way to purify proteins ?

Answer 29

affinity chromatography - need a ligand capable of binding to the protein needed to be extracted

• a common approach is to use a column that contains agarose bead that have attached to them a compound which specifically binds to your protein of interest or if you add 6 histidine residues to your protein, nickel-NTA affinity column can be used
• important feature is that the column only binds the target protein and so the rest of the bacterial proteins are washed away and the protein is eluted by adding something that competes for the binding column

Question 30

what are the advantages of expression in bacteria?

Answer 30

inexpensive - bacteria grow easily and rapidly

large amounts of protein can be made

Question 31

what are the disadvantages of expression in bacteria ?

Answer 31

no post translational modifications - therefore some proteins wont be expressed properly
aggregation into inactive proteins- this can occur because the bacteria doesnt know how to fold up the protein properly
disulphide bonds often not formed - however this can be overcome with secretion vectors

Question 32

how was HIV proteinases structure determined and how was the protein expressed?

Answer 32

determined the structure of the protein by x-ray crystallography
produced the protein by bacterial expression

Question 33

what is the protein structure of HIV proteinase like ?

Answer 33

it is related to aspartic proteinases found in human e.g. renin however it is perfect symmetrical dimer
protein binds and cleaves peptides- binding site being a pocket with 2 flaps hanging over it

Question 34

what is produced when the HIV proteinase binds a peptide?

Answer 34

firstly a tetrahedral intermediate is produced and and then a bond is cleaved to produce the product complex

Question 35

what can yeast expression enable?

Answer 35

can enable the expression of drug targets- its inexpensive and easy and yields moderate to high levels of expression
it also is able to carry out some post translational modifications that bacteria cannot do e.g. glycosylation

Question 36

what other features are present in yeast vectors compared to bacterial vectors ?

Answer 36

Ura3- this enables transformed Ura3 yeast to grow in media with no uracil - it is sort of selection
has a yeast origin of replication

Question 37

what are the advantages of expression in yeast ?

Answer 37

easy and inexpensive to grow
levels of expression moderate to high
some post translational modifications- the yeast are eurkaryotic so they can do things to proteins which bacterial cells cannot

Question 38

what are the disadvantages of expression in yeast ?

Answer 38

proteolytic degradation

not all post translational modifications occur in yeast