Drug Discovery 12 Flashcards
define pharmacodynamics:
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
what are the different types of drug targets ?
enzymess plasma membrane receptors transporters ion channels nucleic acids nuclear receptors many drugs have been discovered without knowing the structure of their target
what does structure based drug discovery require ?
requires knowing the structure of the drug target
where do you get atomic resolution structures of proteins?
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
how does x-ray crystallography work ?
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
what is NMR limited to ?
it is limited to smaller proteins, <35kDa
how does NMR work ?
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
with NMR what does the solving to the structure also incorporate ?
incorporates all the known chemical/bonding/chirality properties of the protein
what does an ensemble of structures produced by the protein enable ?
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
what are the advantages and disadvantages of x-ray crystallography ?
advantages - better resolution - not limited by protein size disadvantages - requires crystals - more difficult for membrane proteins- hard to crystallise
what are the advantages and disadvantages of NMR?
advantages - solution structure - so no crystals - can study dynamics disadvantages - limited by protein size <35kDa
what are the requirements for pharmacological screening ?
-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
what are the requirements of structure determination screening ?
- bacterial, yeast or insect expression systems are used
but - requires large amounts of protein
- requires pure homogenous protein
what are the stages involved in protein expression and what is the crucial difference ?
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
what is present in a mammalian expression vector ?
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
what is present in a bacterial expression vector ?
ribosome binding site multiple cloning site inducible promoter- it needs to be recognised by bacterial RNA polymerase colE1 origin of replication antibiotic resistance gene
how does protein synthesis initiation occur in bacteria ?
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
what is an inducible promoter?
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
what does e.coli usually rely on for protein expression, however if it is not present what does it then rely on ?
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
what does it means by beta-galactosidase levels are nutrient dependent ?
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
how is beta galactosidase controlled?
it is only expressed when its required which is when glucose is not present and therefore it needs to cleave lactose
define operon:
a collection of genes that are controlled as a unit
what is the lactose operon ?
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
why is the transcription of the lactose operon different from other genes?
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
why is it beneficial that a low level of expression of the repressor gene is needed to prevent the expression of the lacZ gene ?
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
what happens to the lactose operon when there is low levels of glucose ?
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
what would happen if the the lac operon was replaced with the cDNA of our target ?
we could induce the bacteria to express the cDNA protein
what is the advantage of inducing expression ?
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
what are the stages involved in bacterial expression ?
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
what is the quickest way to purify proteins ?
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
what are the advantages of expression in bacteria?
inexpensive - bacteria grow easily and rapidly
large amounts of protein can be made
what are the disadvantages of expression in bacteria ?
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
how was HIV proteinases structure determined and how was the protein expressed?
determined the structure of the protein by x-ray crystallography
produced the protein by bacterial expression
what is the protein structure of HIV proteinase like ?
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
what is produced when the HIV proteinase binds a peptide?
firstly a tetrahedral intermediate is produced and and then a bond is cleaved to produce the product complex
what can yeast expression enable?
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
what other features are present in yeast vectors compared to bacterial vectors ?
Ura3- this enables transformed Ura3 yeast to grow in media with no uracil - it is sort of selection
has a yeast origin of replication
what are the advantages of expression in yeast ?
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
what are the disadvantages of expression in yeast ?
proteolytic degradation
not all post translational modifications occur in yeast
