FBDD

Question 1

what is fragament based discovery

Answer 1

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

Question 2

what is the is the rule of lipinkis 5 for a small molecule

Answer 2

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

Question 3

what is the rule of 3 for fragments

Answer 3

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

Question 4

what steps are part of the FBDD

Answer 4

• fragemnt library
• screening methods
• confirmaiton methods
• fragement growth

Question 5

why are multiple methods used in screening methods

Answer 5

the low affintiy can give false positives

Question 6

what are the three most commonly used screening methods

Answer 6

• NMR
• Thermal shift assay
• surface plasmon resonanace

Question 7

what is X-ray crystallography required for

Answer 7

verify binding and binding mode

Question 8

how does differential scanning fluorimtry work?

Answer 8

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

Question 9

In DSF how do the proteins stabilise from the fragments

Answer 9

the fragemnts form H bonds with residues and hydrophobic bonds with the activesite residues

Question 10

what are the disadvantages of DSF

Answer 10

doenst show you where or how tightly it binds

Question 11

what is HIT

Answer 11

the inital fragment taht binds and inhibits the activity of the enzyme of you interest

Question 12

what is HIT to lead

Answer 12

improving its ability to inhibit

Question 13

what is the advantage of DSF over SRP

Answer 13

higher throughput

Question 14

how does surface plasmon resonance work

Answer 14

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

Question 15

what are the advantages of SRP over DSF

Answer 15

• more precise
  -shows the kinetics of binding and dissociation
• shows the exact binding measurements
• need less protein

Question 16

what are the advantages of DSF over SRP

Answer 16

• less expensive
• higher throughput
• less time needed

Question 17

what is the main disadvantge of NMR

Answer 17

need small proteins as 1d NMR can become very complex

Question 18

what is the easiest eay to look at the NMR

Answer 18

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

Question 19

what is the gold standrard to measure ligand affinity

Answer 19

isotheramal calorimetry

Question 20

what are the disadvantges of isothermal calorimetry

Answer 20

low through put
doesnt directly measure inhibition

Question 21

what are functional assays

Answer 21

screening methods that directly measure enzyme activity

Question 22

what is the most common fucntional assay used

Answer 22

flourscent assays

Question 23

when would a FRET assay be used

Answer 23

proteases

Question 24

what are the two uses for structural biology

Answer 24

• hit indentification
• indentification of binding mode for fragment linking/ growth

Question 25

what is the resoution of x-ray crytaogrpahy

Answer 25

<3Å

Question 26

What is the most common method for protein structure determination

Answer 26

X-ray crystallogrpahy

Question 27

how are high levels of pure protein made for X-ray crytallogrpahy

Answer 27

using e. coli to produce protein and histag it to make it easier to purify

Question 28

what information does X-ray crystallogrpahy provide

Answer 28

• 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

Question 29

when is docking used

Answer 29

• when the ligand soaking isn’t possible (would cause craking or disintergration)
• when there is no crystal structure of the protein available

Question 30

What are the three methods used after crystallography

Answer 30

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