Radio ligand assay to validate putative antagonists of REL1 predicted by in silico modelling

1. Identify REL 1 InhibitorsIdentify REL 1 Inhibitors
Assay (by covalent labelling of recombinant ligase with α_32
P ATP: Adenylation)
& substantiate compounds which confer consistent and significant inhibition of REL 1
(identified as potential candidates by a priori screening)
Molarity titrate consequent (putative) inhibitors to determine IC50
Do substances favourable in terms of the above confer a growth phenotype ?
Potential Inhibitor InvantryPotential Inhibitor Invantry
A. Set #1 NCI compounds: ~ 20 substances already screened @ 10uM & 100uM
(batch #1) & ~10 (batch #2) @ 10uM (100uM pending)
B. Set #2 FDA Approved Drugs: 13 compounds from various sources. Thus far 2
ordered (1 already in house)
C. Set #3 compounds: 17 so called ‘hit 2 lead’ substances; Ordered and in house
IC50 Considerations…IC50 Considerations…
Some set #1 Batch #1 compounds may bind exert effect by binding to and stabilising
apo enzyme. This is one reason for efficacy screens @100uM. Virtual drug discovery
algorithms are conducive to screens @100uM in the case of set #3 compounds

2. Skeleton MethodSkeleton Method
Set up radio ligation reactions, cf. REL 1; α32
P_ATP; compound in DMSO
For each compound set up duplicate sets of reactions per assay
Include DMSO only negative control and 1 x published + ive control (e.g. #16209)
Resolve labelled products on Novex bis tris acrylamide gels (10%)
Run to a specified point based on dye front (~ 45 minutes)
Open gel stack and cut away gel front below 40kD mark
Fix gel with Methanol acetic acid and subsequently rehydrate/ equilibrate with
glycerol_fix solution
Vacuum dry gel for 1 hour
Expose dryed gel to Phospho imaging cassette o/n
Visualise radioligation using Typhoon
Obtain 3 independent data sets from each compound cohort
Quantify data using Image quant:

3. Batch # 1 NCI compoundsBatch # 1 NCI compounds
10uM10uM

4. ~ 70 % efficacy based on this assay
100uM100uM

5. # 117079 has variable but substantial efficacy (when manifest)
Similarly for # 125908
Thus @ 100uM it is likely that a definite and substantive efficacy should be seen
10uM10uM

6. Eureka !!: #117079 exhibits > 99% efficacy in context of this assay
Similarly, @ 100uM # 125908 exhibits ~ 95 % inhibition relative to DMSO only
Batch #1_100uMBatch #1_100uM

7. Reaction conditionsReaction conditions
10uM compound
1ug BSA per rxn
70o
C 10 minutes
denaturation
Cassette #3
Reaction conditionsReaction conditions
10uM compound
1ug BSA per rxn
70o
C 10 minutes
denaturation
Cassette #2
Low S:N precludedLow S:N precluded
Quantitation !Quantitation !
Batch #2 NCI compoundsBatch #2 NCI compounds

8. Putative REL 1 inhibition by # 162535 & # 45609 consistent and substantial:
~ 95 % in both cases
Similarly, inhibition associated with #1698 approximates to 80 %
Finally, # 16209 induces similar levels of inhibition to # 1698 but << 162535/45609 !
Batch #2Batch #2
10uM10uM

9. To establish IC50 the 3 candidates will be molarity titrated @ 0, 1 uM, 3 uM, 10 uM,
30 uM,100 uM, 300 uM, 1 mM respectively

10. Preventing signal ‘tailing’ within a duplicate set
In particular, eliminating 8-9 x signal disparity between duplicate sets
Increasing specific signal by reducing background
Reducing non specific signal, cf. unincorporated 32
P_dATP in wells

11. Splitting samples between gels
nullifies significant signal differences
This alludes to reaction set up
differences
ATP DMSO preincubation and/ or
enzyme denaturation between sets ?

12. Original 10 x adenylationOriginal 10 x adenylation
buffer
REL 1 master mix made
fresh just prior to use
0.5ul of REL 1 prep added
(like #0002/3 & unlike #0004/5/6
Fresh 10 x adenylationFresh 10 x adenylation
buffer
REL 1 master mix made
fresh just prior to use
0.5ul of REL 1 prep added
(like #0002/3 & unlike #0004/5/6
T0 minT0 min T11T11 T22T22 T33T33 T44T44 T55T55 T66T66 T90T90
Signal from A at any given time point 8-9 x B
Gel drying artifact, i.e. significant and/ or differential leaching ?
Between 30-40 minutes for both (10 x) buffers signal starts to diminish
Up until then no significant loss of activity
S:N for this assay comparable to #0002/3 implying REL1 has not denatured
Putative loss of activity asspciated with #0004/5/6 in fact caused by adding
0.25ul of REL 1 instead of 0.5ul !!
30-40 mins 30-40 mins

13. REL master mixes fresh with respect to each duplicate set
ATP added to drug (in DMSO) just prior to commencing ligation
Gel cut nearer 40kD marker instead of 30_40kD
Novex gels equilibrated in Glycerol fix for ~ 30minutes instead of 10min
This culminates in more even drying and possibly lower background

14. Ligations performed at progressively
delayed times with respect to 32
P_dATP/
DMSO ‘pre incubation’
No progressive drop in ligation signal
However, significant (and inexplicable) drop
in signal at T + 34 minutes ?

15. # 16209
# 100234
# 45208
Published Efficacy

16. No obvious difference between drug and DMSO ?

17. Affect of REL inhibitor #45208 on growth of BS #427 cells
1000
10000
100000
1000000
10000000
Day 0 Day 1 Day 2 Day 3 Day 4
Day of Sampling
Count_Log10
#45208_100uM_Plate #1
#45208_100uM_Plate #2
#45208_10uM_Plate #1
#45208_10uM_plate #2
#45208_1uM_Plate #1
#45208_1uM_Plate #2
#45208_100nM_Plate #1
#45208_100nM_Plate #2
1 % DMSO ONLY_Plate #1
1% DMSO ONLY_Plate #2
HMI-9 Medium only_Plate #1
HMI-9 Medium only_Plate #2

20. Between 0.1 & 0.4% DMSO growth inhibition by latter insignificant
However, @ 1 % DMSO growth inhibition by latter is observed
Thus, repeat growth assays for published REL 1 inhibitors @ 0.4% DMSO
to distinguish putative growth affects of compounds from definite growth
effects of DMSO @ 1 %

21. Putative growth effects of compound #100234 in 0.4% DMSO on
BS_#427 Cells
1000
10000
100000
1000000
10000000
Day 0 Day 1 Day 2 Day 3 Day 4
Day of Sampling
Realtimecount_Log10
Medium only_Plate #1
Medium Only_Plate #2
0.4% DMSO_Plate #1
0.4% DMSO_Plate #2
100uM_Plate #1
100uM_Plate #2
10uM_Plate #2
1uM_Plate #1
1uM_Plate #2
100nM_Plate #1
100nM_Plate #2
Putative Growth effect of compound #45208 in 0.4%DMSO on
BS #427 Cells
1000
10000
100000
1000000
10000000
Day 0 Day 1 Day 2 Day 3 Day 4
Day of Sampling
Realtimecounts_Log10
Medium Only_Plate #1
Medium Only_Plate #2
0.4% DMS0_Plate #1
0.4% DMSO_Plate #2
100uM_Plate #1
100uM_Plate #2
10uM_Plate #1
10uM_Plate #2
1uM_Plate #1
1uM_Plate #2
100nM_Plate #1
100nM_Plate #2
Putative Growth inhibition of compound #16209 in 0.4% DMSO
on BS #427
1000
10000
100000
1000000
10000000
Day 0 Day 1 Day 2 Day 3 Day 4
Day of Sampling
Realtimecount_Log10
Medium Only_Plate #1
Medium Only_Plate #2
0.4% DMSO_Plate #1
0.4% DMSO_Plate #2
100uM_Plate #1
100uM Plate #2
10uM_Plate #1
10uM_Plate #2
1uM_plate #1
1uM_Plate #2
100nM_Plate #1
100nM_Plate #2
Very difficult to decipher growth variables
Counts more inconsistent for all conditions
1 % assay
Reported inconsistencies with Coulter
counter at that juncture

22. Data SummaryData Summary
NCI compounds assayed by radio ligation @ 10uM (both batches) and 100uM (#1)
Regarding batch #2 clear and substantive inhibitors identified, viz.
 # 45609
 # 162535
 # 1698
~ 95 % inhibition @ 10uM
~ 80 % inhibition @ 10uM
In addition, #42067 with equivocation exerts an effect (~ 50%)
Regarding Batch #1 variable inhibition manifested by the following compounds
@10uM
 #117079
 # 125908
~ 30_80% inhibition @10uM
~ 20_70% inhibition @10uM

23. However, @ 100uM these same batch #1 compounds exhibit definite and
substantive inhibition:
 # 117079
 # 125908
~ 99 % Inhibition
~ 95 % Inhibition
Future WorkFuture Work
• Complete 1 further radio ligation assay for batch # 1 NCI compounds to yield
requisite 3 data sets for final quantitation
• Screen Batch #2 compounds @ 100uM
• For 3 promising batch #2 inhibitors perform molarity titration to determine IC50s’
• Growth assay promising NCI compounds ?
• Evaluate FDA compounds and Hit 2 lead compounds in an analogous way