1. Titration of REL 1 antagonist hits identified by radio mimetic assay to quantify efficacy (IC50) 2. Exposition of HT FRET assay principles for large scale compound library screens to empirically identify REL 1 antagonist hits

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 ?
Why REL 1 ?Why REL 1 ?
1) No human homolog
2) Required in both insect and blood stream stage of life cycle
3) Mg 2+
:ATP binding pocket of REL1 has a unique structure relative to other ligases
4) Only 1 new drug has been realised in the last 50 years
General Overview/ObjectivesGeneral Overview/Objectives
…..i.e. substances with the potential capacity to bind within and exclude ATP from the
Binding pocket as initially inferred from virtual screening programs

2. 20S Editosome: Core Catalytic Complex20S Editosome: Core Catalytic Complex
These 4 enzymes constitute 20S Core catalytic Editing comples
Insertion and deletion editing structurally and functionally compartmentalised:
In particular, Yeast 2 hybrid and coimmunoppt have identified an ExoUase-REL 1
subcomplex
The same is true for TUTase-REL 2 in context of insertion editing
REL 2 !REL 2 ! REL 1 !REL 1 !

3. REL 1-AMP* intermediateREL 1-AMP* intermediate
Auto Adenylation
In initial step enzymes auto adenylates by covalent condensation between lysine
residue in ATP binding pocket and ATP
Covalent adenylation co ordinated by Mg 2+ ions in ATP pocket
Subsequently, AMP moiety transferred to 5’ phosphate of 3’ RNA species
Finally, 3’ OH displaces AMP and phosphodiester bond is restored

4. 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:

5. CompoundCompound
ClassClass
SetSet # substances# substances Drug discovery algorithmDrug discovery algorithm
1. NCI batch
#1
#1 16 total
PNAS Compounds screened with
‘Auto dock 4’
Ranked
Verified with so-called
‘relaxed complex scheme’
Identified leads ‘discovered’ by
screening multiple data bases for
compounds with similar scaffolds to
PNAS compounds
2. FDA #2 1 in house (sigma)
11 more predicted but
financially prohibitive
1 pending (Mar 2010?)
3. NCI batch
#2
#1 8 total
4. Hit 2 lead #3 15 total
5. Sigma #3 2 total
Identify compounds that bind to ATP binding pocket of REL 1, viz. :Identify compounds that bind to ATP binding pocket of REL 1, viz. :
1 Strong lead !1 Strong lead !
3 Strong leads !3 Strong leads !
Also,Tetracycline needs to be evaluatedAlso,Tetracycline needs to be evaluated
2 Nominal_100uM2 Nominal_100uM

6. CompoundCompound
TypeType
CompoundCompound 1010µµMM 100100µMµM TitratedTitrated N=N= ICIC5050 RangeRange Actual Computed ICActual Computed IC5050
GraphPad PrismGraphPad Prism
NCI BatchNCI Batch
#2#2
# 45609 ! 95 %
(78%)
(88%) 55  3-1uM
# 162535 !!# 162535 !! 85_90 %
(85%)
(~98%)*
(90 %)
(>99.9%)*
99 ~1uM (N=1)
 3-1uM (N=6)
 <1uM (N=2)
# 1698 ! 80 %
(50%)
(90%) 66  10uM- 3uM (N=4)
~10uM (N=2)
NCI BatchNCI Batch
#1#1
# 117079 30_80% >99%
(~80 %)
(~50-70%)
44  100uM- 30uM
(N=3)
 30uM (N=1)
# 125908 20_70% ~95 %
(~50-70%)
44  100uM- 30uM
(N=4)
# 45201 NA ~70% Ignore !Ignore !
Sigma rareSigma rare
LibraryLibrary
MordantMordant
Black !!Black !!
(~98%)* (~99.8%)* 55 ~1uM (N=1)
 >1uM (N=4)
 Values derive from titrations’
 Identified using Auto Dock 4 relaxed complex
scheme
*0.25ul undiluted prep #1_080802 1ul 50% diluted prep #3_050203
2.16µM +/- 1.20µM
(N=4)
1.53µM +/- 1.17µM
(N=6)
8.36µM +/- 1.71µM
(N=4)
78.54µM +/- 1.58µM
(N=3)
46.61µM +/- 3.02µM
(N=4)
1.59µM +/- 1.10µM
(N=3)

7. NCI Batch #2_10uMNCI Batch #2_10uM

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. 34A10S10S-1-1
50-100S50-100S-1-1
20S20S-1-1
100S100S-1-1 34B
Titration of leads from initial screensTitration of leads from initial screens

10. % (Putative) Inhibition of Ligation (Adenylation) by #45609 (solute) relative to DMSO
(solvent)_# 45609 Molarity Titration
DMSO
DMSO
100uM
100uM 30uM30uM
10uM
10uM
3uM
3uM
1uM
1uM
300nM
300nM
100nM
100nM 30nM
30nM
0
20
40
60
80
100
120
# 0035B # 0037A
Molar Titration
MeanSpecificSignal_%DMSO
IC50 resides here
199,000,000 counts ! 65,000,000 counts !
0.9%
1.6%
7.9%
36%
68%
IC50 resides here
0.7%
1.8%
6.2%
33%
67%
Both assay sets generated with undiluted enzyme prep #1 & concord
Thus, these assays were pooled for purposes of generating IC50 data via GraphPad Prism
0.25ul undiluted Prep #1_080802

11. % (Putative) Inhibition of Ligation (Adenylation) by # 45609 (solute) relative to DMSO (solvent)_#
45609 Molarity Titration: Mean data assays # 0035B & # 0037A
DMSO
100uM 30uM
10uM
3uM
1uM
300nM 100nM 30nM
0
20
40
60
80
100
120
#0035B_# 0037A
Molarity Titration: 100uM-30nM
MeanSpecificSignal/DMSO_%
DMSO
100uM
30uM
10uM
3uM
1uM
300nM
100nM
30nM
0.8% +/-
0.1%
1.7% +/-
0.2%
7.3% +/-
1.4%
34.4% +/-
2.0%
67.4% +/-
3.1%
IC50 ~3-1uM
IC50 (GraphPad v.5)
2.16 +/- 1.20µM
R2
0.9983

12. Dose-Response # 45609
-2 -1 0 1 2 3
0
50
100
150
Log(10) Concentration [µM]
Activity[%]
Data from titrations subject to non linear regression analysis and log transformation
Actual data fit to predicted (regression) fit indicated by Pearson correlation coefficient
IC50 (GraphPad v.5)
2.16 +/- 1.20µM
R2
0.9983

13. % (Putative) Inhibition of Ligation (Adenylation) by #45609 (solute) relative to DMSO
(solvent)_# 45609 Molarity Titration
DMSO
DMSO
100uM
100uM 30uM30uM
10uM
10uM
3uM
3uM
1uM
1uM
300nM
300nM
100nM
100nM
30nM
30nM
0
20
40
60
80
100
120
140
# 0035B # 0016B
Molar Titration
MeanSpecificSignal_%DMSO
IC50 resides here
199,000,000 counts ! 10,000,000 counts !
0.9%
1.6%
7.9%
36%
68%
IC50 resides here
12%
16%
22%
38%
74%
0.25ul undiluted Prep #1_080802 0.5ul 50% diluted Prep
Optimal sets generated with undiluted enzyme prep #1
In contrast earlier data derived from glycerol diluted prep yielded lower S:N

14. % (Putative) Inhibition of Ligation (Adenylation) by #1698 (solute) relative to DMSO (solvent)_# 1698
Molarity Titration
DMSO
DMSO
100uM
100uM
30uM
30uM
10uM
10uM
3uM
3uM
1uM
1uM
300nM
300nM
100nM
100nM
30nM
30nM
0
20
40
60
80
100
120
# 0018B # 0035/36A
100uM-30nM
MeanSpecificSignal/DMSO_%
DMSO
100uM
30uM
10uM
3uM
1uM
300nM
100nM
30nM
# 1698
27.1+/-
8.8%
52.7+/-
0.3%
39.1+/-
1.8%
60.5+/-
3.5%
IC50 ~ 10uM IC50 ~ 10-3uM
0.25ul Undiluted Prep #1_080802
IC50 (GraphPad v.5)
8.36 +/- 1.71µM
R2
0.9955
Submitted to GraphPad

15. Dose-Response # 1698
-2 -1 0 1 2 3
0
50
100
150
Log(10) concentration [µM]
Activity[%]
IC50 (GraphPad v.5)
8.36 +/- 1.71µM
R2
0.9955

16. % (Putative) Inhibition of Ligation (Adenylation) by #162535 (solute) relative to DMSO (solvent)_#
162535 Molarity Titration
DMSO
DMSO
DMSO
100uM
100uM
100uM
30uM
30uM
30uM
10uM
10uM
10uM
3uM
3uM
3uM
1uM
1uM
1uM
300nM
300nM
300nM
100nM
100nM
100nM
30nM
30nM
30nM
0
20
40
60
80
100
120
140
160
180
# 0036B # 0037B # 0034B
Molarity Titration: 100uM-30nM
MeanSpecificsignal/DMSO_%
DMSO
100uM
30uM
10uM
3uM
1uM
300nM
100nM
30nM
# 162535
0.25ul Undiluted Prep #1_080802 0.25ul Undiluted Prep #1_080802
21%
42%
42%
90%
36%
74%141,000,000 90,000,0000.4%+/-0.03%
0.8%+/-0.05%
4.3%+/-0.3%
0.5%+/-0.2%
1.4%+/-0.4%
7.0%+/-2.4%
0.7%+/-0.1%
1.9%+/-
9.5%+/-0.5%
64%
IC50 ~ 1uM-300nM !
0.25ul Undiluted Prep #1_080802
IC50 ~ 3uM-1uM !
200,000,000
All data sets generated with undiluted enzyme prep #1
Two sets concord but one is more disparate with respect to predicted IC50
However, because all 3 represent replicate cohorts all data pooled for purposes of IC50
elucidation

17. % (Putative) Inhibition of Ligation (Adenylation) by #162535 (solute) relative to DMSO (solvent)_#
162535 Molarity Titration
DMSO
100uM 30uM
10uM 3uM
1uM
300nM
100nM
30nM
0
20
40
60
80
100
120
140
160
Assays #0034B_0036B_0037B
Molarity Titration:100uM-30nM
MeanSpecificSignal/DMSO_%
DMSO
100uM
30uM
10uM
3uM
1uM
300nM
100nM
30nM
0.5% +/-
0.2% 1.4% +/-
0.6%
7.0% +/-
2.6%
33.0% +/-
10.9%
68.5% +/-
23.2%
IC50 ~ 3-1uM
● Inclusion of assay # 0036B reduces
putative IC50 but increases variance
(relative to assays # 0034B_36B
alone)
● Nevertheless, putative IC50 still
resides between 3µM-1µM
● In context of assay # 0036B (N=2)
putative IC50 < 1µM and variance
lowest of all
0.25ul Undiluted Prep #1_080802
IC50 (GraphPad v.5)
1.53 +/- 1.17µM
R2
0.9991
Error bars reflect disparity between assay #0036B and other two assays

18. Dose-Response #162535
-2 -1 0 1 2 3
0
50
100
150
Log(10) concentration [µM]
activity[%] IC50 (GraphPad v.5)
1.53 +/- 1.17µM
R2
0.9991
Error bars reflect disparity between assay # 0036B and other two assays
Nevertheless regression fit to actual data, i.e. R2
excellent

19. % (Putative) Inhibition of Ligation (Adenylation) by Mordant Black (solute) relative to DMSO (solvent)_# Mordant Black
Molarity Titration
0
20
40
60
80
100
120
140
1ul Prep #1_080802 0.25ul Prep #1_080802
SpecificSignal/DMSO_%
26.6+/-0.2%
23.7%
51.9%
● For 0.25ul Prep #1_080802, N=3 =
1 #0029A_1
2 #0034A_1
3 #0034A_2
● For 1ul Prep #1_080802, N=1 =
1 #0033A
● For Prep #3_050203 N=1
1 #0029A_3
● For assay #0029A putative IC50
~1uM
● How ever, for assays w here N=3, putative
IC50 >1uM
● For assays w here N=3, data convergence
better relative to #0033A betw een 100uM-3uM
0.25ul undiluted REL 1
4.4+/-0.2%
1.1+/-0.1%
0.7+/-0.1%
60.8+/-0.7%
53.7%
15.4
1.8%
0.4%
0.1%
1ul Prep #3_050203# 0033A # 0034A_1
# 0034A_2
# 0029A_1
# 0029A_3
100uM 30nM
IC50 (GraphPad v.5)
1.59 +/- 1.10µM
R2
0.9998
Submitted to GraphPad
Formely Mordant black assays completed but undecided as to which data
sets to pool for IC50 determination
According with other assays just those data sets derived from Prep #1 were
submitted

20. Dose-Response Mordant Black
-2 -1 0 1 2 3
0
50
100
150
Log(10) Concentration [µM]
activity[%] IC50 (GraphPad v.5)
1.59 +/- 1.10µM
R2
0.9998
I’m Happy !

21. % (Putative) Inhibition of REL 1 Ligation (Adenylation) by #117079 (solute) relative to DMSO
(solvent)_# 117079 Molarity Titration
DMSO
DMSO
100uM
100uM
30uM
30uM
10uM
10uM
3uM
3uM
1uM
1uM
300nM
300nM
100nM
100nM
30nM
30nM
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
BAC Prep #3_050203 BAC Prep #1_080802
#117079 Molarity Titration: 100uM-30nM
SpecificSignal/DMSO_%
18.6%18.6%
54%54%
23.4%23.4%
75.6%75.6%
1ul Enzyme1ul Enzyme 0.25ul Enzyme0.25ul Enzyme
Predicted IC50 Prep #3 ~30uM
Predicted IC50 for prep #100uM-30uM
The latter concurs with previous assays50S50S-1-1
100S100S-1-1

22. % (Putative) Inhibition of REL 1 Ligation (Adenylation) by #117079 (solute) relative to DMSO (solvent):
#117079 Molarity Titration. Assay #0032A/B
DMSO
1mM 300uM
100uM
30uM 10uM
3uM
1uM
0
20
40
60
80
100
120
N = 3
Molarity Titration #117079: 1mM - 1uM
MeanSpecificSignal/DMSO_%
DMSO
1mM
300uM
100uM
30uM
10uM
3uM
1uM
IC50 (GraphPad v.5)
78.54 +/- 1.58µM
R2
0.9918
1ul Prep #3_050203

23. Dose-Response # 117079
0 1 2 3 4
0
50
100
150
Log (10)concentration [µM]
Activity[%]
IC50 (GraphPad v.5)
78.54 +/- 1.58µM
R2
0.9918

24. % (Putative) Inhibition of REL 1 Ligation (Adenylation) by #125908 (solute) relative to DMSO (solvent):
#125908 Molarity Titration. Assay #0031A/B
DMSO
1mM
300uM
100uM
30uM
10uM
3uM
1uM
0
20
40
60
80
100
120
140
N = 4
Molarity Titration #125908: 1mM-1uM
MeanSpecificSignal/DMSO_%
DMSO
1mM
300uM
100uM
30uM
10uM
3uM
1uM
1ul Prep #3_050203
IC50 (GraphPad v.5)
46.61 +/- 3.02µM
R2
0.9675

25. Dose-Response # 125908
0 1 2 3 4
0
50
100
150
Log (10)concentration [µM]
Activity[%]
IC50 (GraphPad v.5)
46.61 +/- 3.02µM
R2
0.9675

26. #16209
#45609
IC50 = 1.01 uM
IC50 = 2.16 +/- 1.20uM
#1698
IC50 = 8.36 +/-1.71 uM
#162535
IC50 = 1.53 +/- 1.17 uM
LogP = -1.043
LogP = 0.492
Log P values for # 45609
More encouraging than for #16209
This bodes well for successful
EC50 determination by Alamar
Blue viability assay

27. A fluorescence-based high-throughput ligase assay
Donor (D) = FAM, Acceptor (A) = Cy5, 250 nM
Assay will utilise a 104bp RNA fragment
(CYb) in concert with labelled 20mers
Coupling efficiency varies with fluorophore distance
70-100A is where FRET is achieved
Forster distance = Separation at which 50% photon transfer achieved

28. Assay EstablishmentAssay Establishment
1) Purify catalytic domain (L151-324
) of REL 1 (in association with TEV cleavable His
Tag A2) by FPLC utilising NTA columns in concert with imidazole gradient
2) Express 104bp RNA (CYb) species from linear pBlueScript clone by In Vitro
transcription
3) Anneal labelled 20mers
4) Test E Coli purified REL1 batch versus adenylation verified leads
5) Independently verify FRET by radioactive ligation assays
Assay OptimisationAssay Optimisation
Various parameters associated with ‘Mix & measure’ assay will be optimised, e.g.
 Gap distance: 16 donor/acceptor fluorophore combinations assayed over 0-12bp
hiatus
 RNA Substrate: Is CYb best ?
 Fluorophore species: TAMRA, Cy3, Cy5 etc
 Dynamic detection/sensitivity range: Serial substrate dilutions versus detection
 DMSO Concentration: 0.1% - 10%
Assay ImplementationAssay Implementation
1) ~3000 SMDC compounds to screen
2) Initial primary screen @ 10uM + 1% DMSO
3) Compounds >/- 3 x SD from (collective) median inhibition will be deemed ‘active’
and subject to counter screens

29. #1#1 #2#2 #3#3 #4#4
50KD
40KD
30KD
20KD
PP PP PP P?P?SS PP PP PP P?P?SS SS SS SS SS S?S? S?S?
+ + + +- - - -
Catalytic domainCatalytic domain
REL1REL1
Specific and high level expression of L1 (catalytic REL 1 domain) in pellet
No obvious sign of soluble protein !
Is this due to a lack of A2 (N-terminal TEV His tag) rendering L1 soluble ?
+/+/-- 0.5mM IPTG0.5mM IPTGWestern blotting
with:
 α REL 1Ab
 α His tag Ab