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 ?
tential Inhibitor Invantrytential Inhibitor Invantry
Set #1 NCI compounds: ~ 30 substances already screened @ 10uM & 100uM
( ~20 batch #1 & ~10 batch #2). Optimal IC50S ~low uM range (see caveat below)
Set #2 FDA Approved Drugs: 13 compounds from various sources. Thus far 3
ordered (1 already in house)
Set #3 compounds: 15 so called ‘hit 2 lead’ substances; Ordered and in house
2 preliminary screens @10uM
C50 Considerations…50 Considerations…
ome set #1 Batch #1 compounds may bind exert effect by binding to and stabilising
o enzyme. This is one reason for efficacy screens @100uM. Virtual drug discovery
gorithms 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 radio ligation using Typhoon
Obtain 3 independent data sets from each compound cohort
Quantify data using Image quant:

3. Hit 2 LeadHit 2 Lead
No evidence of significant REL 1 inhibition from these 1st
7 H2L compounds
However, 1st
screen

4. To establish IC50 the 3 candidates will be molarity titrated @ 0, 30nM, 100nM,
300nM, 1uM, 3uM, 10uM, 30uM & 100uM (respectively)
NCI Batch #2_10uMNCI Batch #2_10uM

5. 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

6. Data Summary-Last timeData Summary-Last time
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

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

8. = Remainder of original REL 1 prepRemainder of original REL 1 prep
= Fresh REL 1 prep (May 2Fresh REL 1 prep (May 2ndnd
2003)2003)
# 125908
# 117079
Strong inhibition in common with # 0011
# 45201 Evidence of efficacy @100uM in common with # 0011
# 1# 1
NCI Batch #1NCI Batch #1
100uM100uM

9. # 1# 1
# 117079 & #125908 exhibit putative efficacy to the order of 95_99%
& 88_97 % respectively based on these assays
NCI Batch # 1NCI Batch # 1

10. # 1# 1
# 45201 exhibits putative efficacy to the order of 70%
based on these assays
NCI Batch #1NCI Batch #1

11. 100uM100uM
100uM100uM
Like # 0013, # 42067 & # 45577 appear to exert significant inhibition
In addition, # 75908 appears to be exerting some magnitude of inhibition
In general background is higher for # 0013 & # 0014 than previous assays in
last 2 months. Why ?
NCI Batch #2NCI Batch #2
Reaction #1 Reaction #2
Reaction #1 Reaction #2
# 2# 2

12. # 2# 2
NCI Batch # 2NCI Batch # 2
# 45577 exhibits significant inhibition in the order
~90-95% @100uM
Variation due to variable & variagated background

13. # 45577 manifests (putative) inhibition approximating to 92_95%
# 42067 manifests (putative) inhibition in the range of 65_89%
# 0014B_1 excluded from collated data: Hence, no DMSO control !
SD computed from relative not actual specific counts: Hence no error bars for
DMSO
# 2# 2
NCI Batch #1NCI Batch #1

14. # 16209# 16209
# 45609# 45609
10S10S-1-1
50S50S-1-1
50S50S-1-1 10S10S-1-1
Formely @ 10uM (putative) inhibition associated with # 45609 was ~ 95 %
Now, putative inhibition associated with # 45609 @ 10uM ~ 78 %# 3.# 3.

15. # 16209# 16209
10S10S-1-1
50S50S-1-1
IC50 somewhere in the
range 3uM – 10uM
In next assay focus on
10uM_2uM range in 1uM
increments
Variation in signal due to
elevated and variable back-
ground, particularly @ higher
titrants
Also may result from evident
variable signal in duplicates
# 3.# 3.

16. # 45609# 45609
50S50S-1-1
10S10S-1-1
IC50 resides somewhere
between 10uM & 1uM approx.
to 3uM
In next assay focus on 10uM
to 2uM range
# 3.# 3.

17. 20S20S-1-1
20 - 50S20 - 50S-1-1
10 - 20S10 - 20S-1-1
20 - 50S20 - 50S-1-1
#162535#162535
#1698#1698
Gel # 0018B loaded from behind incurring less torque (?)
Thus, does pushing tips into gel enable localised diffusion and/or electro endosmosis of label culminating
in higher and variegated background ?
# 3.# 3.

18. 20S20S-1-1
20 - 50S20 - 50S-1-1
#162535#162535
IC50 somewhere between
3uM & 300nM
In both this and prior assays
inhibition @10uM ~ 85 – 90%
Signal variation exacerbated by
background
Potential for signal variation
between duplicates most evident
where efficacy most significant
i.e. in the IC50 range !

19. 10 - 20S10 - 20S-1-1
20 - 50S20 - 50S-1-1
#1698#1698
IC50 somewhere between
30 & 3uM
Inhibition @ 10uM ~ 50%
In prior assays ~ 80 %
Gel loaded from the back
unlike # 0018A

20. CompoundCompound
TypeType
CompoundCompound
##
1010µµMM 100100µMµM Titrated ?Titrated ? ICIC5050 RangeRange CommentsComments
NCI BatchNCI Batch
#2#2
# 45609 ! 95 % (78 %) (88%) 10uM-1uM Values in red
pertain to
titration
assays
Compounds
in blue
original leads
# 162535 ! 85_90 %
(85%)
(90 %) 3uM-
300nM
# 1698 ! 80 % (50 %) (90%) 30uM-3uM
# 45201 NA 70%
NCI BatchNCI Batch
#1#1
# 117079 30_80% >99 % Compounds
in blue only
batch #1 with
some evident
efficacy @
10uM
# 125908 20_70% ~95 %
# 45577 NA 90_95 %
# 42067 NA 65_89 %
Hit 2 LeadHit 2 Lead # 1 - #15 Done x 2 Quantitation
pending

21. MethodMethod
1. Set up triplicate reactions with published antagonist # 45208 and DMSO control
2. Set up these triplicate reactions using 0.18ul of 32
P d ATP/rxn (std); 0.36ul & 0.54ul
3. Does the natural substrate ATP out compete #45208 @ higher conc. ?

22. ResultsResults
As 32
P_dATP increases from 0.18ul per reaction to 0.54ul specific signal from# 45208
drops from ~ 35% to 45% respectively
Thus, some inhibition but (probably) non competitive rather than competitive

23. # 45208 @100uM# 45208 @100uM
# 45208 @100uM# 45208 @100uM
# 45208 @100uM# 45208 @100uM
DMSODMSO
DMSODMSO
DMSODMSO
A = 0.18ulA = 0.18ul 3232
P_dATPP_dATP
B = 0.36ulB = 0.36ul 3232
P_dATPP_dATP
C = 0.54C = 0.54ulul 3232
P_dATPP_dATP
> 20S> 20S-1-1 20_50S20_50S--11(Putative) inhibition of #45208 @100uM relative to DMSO vs. Amount dATP
DMSO
DMSO
DMSO
#45208
#45208
#45208
0.0
20.0
40.0
60.0
80.0
100.0
120.0
0.18ul 32P_dATP 0.36ul 32P_dATP 0.54ul 32P_dATP
Amount 32
P_dATP per reaction
SpecificSignal/DMSO_%
31.8% specific signal
relative to DMSO
6 %SD about mean
34.7% specific signal
relative to DMSO
4% SD about mean
44.4% specific signal
relative to DMSO
7% SD about mean
As 32
P_dATP increases from
0.18ul per reaction to 0.54ul
specific signal from # 45208
drops from ~ 35% to 45%
respectively
SD about mean ~ 5%

24. Hit 2 Lead @ 10Hit 2 Lead @ 10µMµM
No evidence of significant REL 1 inhibition from these 1st
7 H2L compounds
However, 1st
screen
# 4.# 4.

25. Hit 2 LeadHit 2 Lead
Some DMSO solubility issues with listed H2L compounds:
 8/15 fully soluble @ 10mM
 1/15 fully soluble @ 5mM
 1/15 fully soluble @ 2.5mM
 2/15 fully soluble @2mM
 2/15 partly soluble @ 2mM
 1/15 (#2) neither soluble in water or DMSO: Seeking information
on most solubilising solvent

26. 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 ~ 30 minutes instead of 10 min
This culminates in more even drying and possibly lower background

27. Raw (Geiger) counts from
Dried gel ~ 5S-1
Raw (Geiger) counts from
Dried gel ~ 10-20S-1
Low S:N precludes actual quantification
In particular, #0013B exhibited high background, tallying with Geiger count
Although 32
P 1 month old, this exacerbated another (principal) problem
Nevertheless, # 42067 & # 45577 exhibit significant efficacy by eye
This was true based on 4x independent rxn, cf. 13A/B reactions #1/2
@ 10uM nothing significant was evident with these compounds
100uM_NCI batch #2100uM_NCI batch #2
Reaction #1Reaction #1 Reaction #2Reaction #2
Reaction #1Reaction #1 Reaction #2Reaction #2

28. # 0018# 0018
# 162535
# 1698
Gel loaded from
behind
Gel loaded from
the frontNo correlation between well
Signal & specific signal

29. Hit 2 Lead @ 10Hit 2 Lead @ 10µMµM
# 4.# 4.Signal diminutionSignal diminution
No
DMSO
signal