FBDD Flashcards
what is fragament based discovery
identification of scaffolds that bind a protein of interest which allows:
- growth of that scaffold to increase affinity and specificity
- linking of scaffold to the same ends
what is the is the rule of lipinkis 5 for a small molecule
mw/Da= less than or 500
H bond donors =less than or 5
H bond acceptors = less than or 10
clogP= less than or 5
what is the rule of 3 for fragments
mw/Da= less than or 3
H bond donor = less than or 3
H bond acceptor = less than or 3
clogP = less than or 3
what steps are part of the FBDD
- fragemnt library
- screening methods
- confirmaiton methods
- fragement growth
why are multiple methods used in screening methods
the low affintiy can give false positives
what are the three most commonly used screening methods
- NMR
- Thermal shift assay
- surface plasmon resonanace
what is X-ray crystallography required for
verify binding and binding mode
how does differential scanning fluorimtry work?
when heated the proteins unfold at Tm dtermined by the buffer composition and sequence.
fragments bind and stabilize the structure and increase Tm
syproorange dye is added which bind to the hydrophobic portions of the protein and flourscence increases
In DSF how do the proteins stabilise from the fragments
the fragemnts form H bonds with residues and hydrophobic bonds with the activesite residues
what are the disadvantages of DSF
doenst show you where or how tightly it binds
what is HIT
the inital fragment taht binds and inhibits the activity of the enzyme of you interest
what is HIT to lead
improving its ability to inhibit
what is the advantage of DSF over SRP
higher throughput
how does surface plasmon resonance work
protein is bound covalently to the surface which is sitting on a gold leaf
a light is shined onto surface and measure the light that is refelcted
the fragments flow over the surface
when there is a binding there is a shift in light reflected
what are the advantages of SRP over DSF
- more precise
-shows the kinetics of binding and dissociation - shows the exact binding measurements
- need less protein
what are the advantages of DSF over SRP
- less expensive
- higher throughput
- less time needed
what is the main disadvantge of NMR
need small proteins as 1d NMR can become very complex
what is the easiest eay to look at the NMR
to look at fragment as they are normally at a differnet shift compared to the protein. Fragments have flourine present most of the time whihc naturally reacts with the NMR. if it is bound to water gives off one signal but if it is bound the protein this signal will change
what is the gold standrard to measure ligand affinity
isotheramal calorimetry
what are the disadvantges of isothermal calorimetry
low through put
doesnt directly measure inhibition
what are functional assays
screening methods that directly measure enzyme activity
what is the most common fucntional assay used
flourscent assays
when would a FRET assay be used
proteases
what are the two uses for structural biology
- hit indentification
- indentification of binding mode for fragment linking/ growth
what is the resoution of x-ray crytaogrpahy
<3Å
What is the most common method for protein structure determination
X-ray crystallogrpahy
how are high levels of pure protein made for X-ray crytallogrpahy
using e. coli to produce protein and histag it to make it easier to purify
what information does X-ray crystallogrpahy provide
- provides infomration on key interactions
- provides infomrtaiton on how to grow the hit
- provides binding location
- can provide infromtaion on how to bind fragmnets
- surface representation looks at the shape of the active site
when is docking used
- when the ligand soaking isn’t possible (would cause craking or disintergration)
- when there is no crystal structure of the protein available
What are the three methods used after crystallography
growing -> make modification to chemical structures to make it bigger (this is for a single hit)
linking -> link them together, if the two hits are quite close togetehr
mergining -> two hits with a related group that binds to similar location
