7) High Input Screening

Question 1

What is high input screening, HTS

Answer 1

Lab techniques used to quickly test thousands (50,000 approx) of compounds for biological activity to generate a “hit” compound for any biological target

Question 2

What considerations are required before carrying out HTS

Answer 2

• firstly, is it even required because it is a very expensive process so is it necessary
• how many HTS campaigns are required
• can we quantify the results

Question 3

What’s the gartner cycle

Answer 3

Model that shows how new technologies evolve over time in terms of hype, expectations and adoption.

Question 4

What is key for HTS to succeed

Answer 4

1) chemical compound collection - size, diversity in chemical structure, quality and integrity (making sure they haven’t degraded overtime)
2) assay reagents - proteins, cells against making sure the quality, amount, integrity and engineered readouts are sufficient
3) assay, screening & IT - technologies, making sure the data is handled correctly, the physics and lab robotics are reliable

Question 5

How do we assess quality of our compounds

Answer 5

Using the physiochemical properties- using Lipinksi’s rule of 5 :
The molecular weight should be less than 500Da bc smaller molecules cross the membrane more easily
cLogP value should be equal to or less than 5 bc this indicates moderate lipophilicity meaning a balance of solubility (how well the drug will dissolve) and lipophilicity (how well the drug will be able to cross the membrane)
Then u can go on to talk about bioavailability and all that ladada

Question 6

Name some hit-finding strategies

Answer 6

Diversity screening, structural biology and fragments, phenotypic drug discovery, DNA encoded library, etc…

Question 7

What drug targets are suitable for HTS

Answer 7

Kinases, GPCR- peptides and proteases - others haven’t had good success rates

Question 8

What do we use to validate “hit” findings

Answer 8

Counter screens and orthogonal screens

Question 9

What are new HTS technologies

Answer 9

Using stem cells, 3D tumor spheroids, NSCLC, precise genome editing (CRISPR-cas9) and others stufff

Question 10

What’s neutral control and scale reference control

Answer 10

Neutral control = controls in an assay at steady state e.g. In an inhibitor assay represent maximal activity in absence of an inhibitor (no activity)
Scale reference control = The activity in presence of a supra-maximal concentration of a compound or no added enzyme (max activity)

Question 11

What is the primary screen

Answer 11

= each compound that has been tested once at a single concentration, it is vital that the assay is reliable enough that a single test is a true representation of the activity of the compound and that each plate in the screen is valid . You have 2 different controls (neutral = 0 and scale reference= 100) and you ideally want the compounds to be in the middle

Question 12

Why do we have to make sure our assays are reliable

Answer 12

Otherwise we could get false positives which would lead to huge cost implications if you mistakenly take this compound to further developing stages . Or you could get a false negative = potentially huge missed opportunity for a drug that works

Question 13

What makes a good assay APP THIS COMES UP IN SECTION B AS WELL AS THE EQUATION!

Answer 13

1) a clear separation between the tails of the distribution of the controls
2) a large signal window or separation band (>3 fold)

Question 14

What’s the equation for the Z factor and why is it used

Answer 14

Z factor = identifies the separation band (if it’s good or not)

Z = 1 - (3-SD(N)+3-SD(SR))/|mean(N)-mean(SR)| pls just look at summary notes it’s hard to type it out
Or z = 1 - (3-SD(Cpd)+3-SD(SR)/|mean(Cpd)-mean(SR|

Question 15

What does a Z factor value of 1, 1-0.5 , 0.5-0 and less than 0 tell us

Answer 15

1 = ideal perfect assay
1-0.5 = excellant assay, separation band is large
0.5-0 = separation band is small, data is not reliable
0 = no separation band, variation bands touch not reliable
<0 = single point screening is impossible

Question 16

What are the drawbacks of using the Z factor + the solution (HIGH CHANCE OF BEING IN SECTION B js saying)

Answer 16

The mean and standard deviation are influenced are outliers = affecting the Z factor = leading to poor estimate of Z’ (Z prime) . HTS data contains outliers and the purpose of the screen is to find these outliers
Solution= we use medium instead of of the mean this is called Robust statistics = these robust measures are resistant to outliers and provide good reliable estimates from HTS data! The median is robust towards the outlier.

Question 17

What’s the equation for Robust z’/Z factor

Answer 17

RZ’=1-・ 3•RSD (N) + 3•RSD(SR)
|<N> - <SR>||
RZ = 1 - 3•RSD (Cpd) + 3-RSD (SR) Pls look at summary notes
|<Cpd> - <SR>|</SR></Cpd></SR></N>

Question 18

What do we have to do after calculating Z factor/Z’

Answer 18

ASSAY VALIDATION = experiments to ensure that the assay is reliably detecting compounds having an effect on the biological target using equipment and reagents to be used in the primary screen. (We usually have known inhibitors that can be used to determine if assay is reliable but in HTS we have no known inhibitors).
Minimum of 2 assays across 2 days - to test reliability of assay

Question 19

What are the 2 approaches that can be used in assay validation and what do we look for

Answer 19

1) Construct a validation set that is representative of the diversity in your screening collection
• For large scale HTS this is typically around 10, 000 compounds
2) Select at random a number of plates from your collection, screen these plates at least twice across 2 days
We look for:
the level of reproducibility across the repeated data sets
• False discovery rate (active once out of 2 replicates)
• Median of the differences between the 2 replicate values
We need some active compounds to assess validation across the activity range. Add in some known inhibitors or construct a set with known frequent hitters.

Question 20

Name the 8 analytical validation characteristics

Answer 20

Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Range

Question 21

What is precision - in terms of an assay

Answer 21

How close the measured mean is to the true real mean, large deviation = imprecise

The precision (VARIABILITY) of an analytical procedure is usually expressed as the standard deviation
(S), variance (S), or coefficient of variation (= relative standard deviation, RSD%.) of a series of measurements.
The

Question 22

What’s repeatability- in terms of an assay

Answer 22

Repeatability expresses the precision (spread of the data, variability) under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

Question 23

What’s a sensitive vs robust assay

Answer 23

Sensitive = small changes e.g. different company producing the same buffer, producing largely different results
Robust = changes have little effect on the output - not good as well
Ideally an assay would have a balance of both

Question 24

What’s the Bland Altman Plots - IMPORTANT FOR EXAM APP

Answer 24

this plots the difference between pairs of measurements vs the means of the pairs to assess agreement between data sets. 95% confidence interval is useful to judge reproducibility of the assay assuming average difference is close to or on 0