preliminary quantification of radio mimetic anti REL 1 hits by titration: Scoping likely IC50

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 intially inferred from virtual screening programs

2. CompoundCompound
ClassClass
SetSet # substances# substances Drug discovery algorithmDrug discovery algorithm
1. NCI #1 #1 16 Compounds screened with ‘Auto
dock 4’
Ranked
Verified with so-called
‘relaxed complex scheme’
2. FDA #2 1 in house (sigma)
11 more predicted but
financially prohibitive
1 pending (Dec 2009)
3. NCI #2 #3 8 Identified using a fragment
based approach:
 Initially docked small fragments
into REL 1
 Identified favourable fragments
 Linked together and several
databases screened
4. Hit 2 lead #3 15
5. Sigma #3 2
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
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 !
2 Nominal_100uM2 Nominal_100uM

3. = 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

4. Future WorkFuture Work
1) Screen 1 available FDA candidates @ 10uM & 100uM respectively
2) Concomitantly 2 remaining Sigma substances @ 10uM & 100uM respectively
2) Perform Titrations on NCI batch #1 compounds with proven efficacy @100uM
3) Complete titrations on NCI batch #1 & #2 compounds to yield 3 requisite data
sets

5. =10=10µMµM
=100=100µMµM
Low S:N precluded quantitation !
This was even true of depicted assay #0026, wherein cassette left for 6 days !
BAC 1 prep with low intrinsic activity (also common to H2L compounds)

6. #1#1 #2#2 #3#3 #4#4
#5#5
#6#6
1ul 1ul 1ul 1ul 1ul
1ul
0.5ul 0.5ul 0.5ul 0.5ul 0.5ul
0.5ul
0.25 0.25 0.25 0.25 0.25
0.25~17 hours:~17 hours:
~3 hours:~3 hours:
#1#1 #2#2 #3#3 #4#4
Counts yielded byCounts yielded by
0.250.25µl of preps #1-4µl of preps #1-4
(~4 x10(~4 x1077
) approx to) approx to
>10 x average counts>10 x average counts
from 50% glycerolfrom 50% glycerol
diluted prepsdiluted preps

7. % (Putative) Inhibition of REL 1 Ligation (Adenylation) by Mordant Black (solute) relative to DMSO
(solvent)_# Mordant Black Molarity Titration
100uM
100uM
30uM
30uM
10uM
10uM
DMSO
DMSO
3uM
3uM
1uM
1uM
300nM
300nM
100nM
100nM
30nM
30nM
0
20
40
60
80
100
120
140
Prep #3_050203 Prep #1_080802
Mordant Black Molarity Titration: 100uM-30nM
SpecificSignal/DMSO_%
3.4%3.4%
10%10%
23.7%23.7%
52%52%
81%81%
<0.5%<0.5% 5%5%
26.5%26.5%
55.8%55.8%
75%75%
1ul Enzyme1ul Enzyme 0.25ul Enzyme0.25ul Enzyme
Although both REL 1 BAC preps differ with regard to
intrinsic activity both yield putative IC50’s in the range
of 3uM-1uM
This concurs with prior assays100uM100uM 30nM30nM
50S50S-1-1
100S100S-1-1

8. 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 !

9. % (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
Although both REL 1 BAC preps differ with regard to
intrinsic activity both yield putative IC50’s in the range
of 100uM-30uM
This concurs with prior assays50S50S-1-1
100S100S-1-1

10. # 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

11. CompoundCompound
TypeType
Compound #Compound # 1010µµMM 100100µMµM Titrated ?Titrated ? ICIC5050
RangeRange
CommentsComments
NCI Batch #2NCI Batch #2 # 45609 ! 95 % (78 %) (88%) 10uM-
1uM
Identified using
fragment based
algorithm
Fig. in red derive
from Titrations’
# 162535 !!# 162535 !! 85_90 % (85%) (90 %) 3uM-
300nM
# 1698 ! 80 % (50 %) (90%) 30uM-
3uM
NCI Batch #1NCI Batch #1 # 117079 30_80% >99% (~80 %) 100uM
-30uM
Identified using
Auto Dock 4
relaxed complex
scheme# 125908 20_70% ~95 %
# 45201 NA ~70%
Sigma rareSigma rare
LibraryLibrary
MordantMordant
Black !!Black !!
Evident but
not quantified
Evident but
not quantified
3uM-
1uM
Identified using
fragment based
algorithm
Low S:N !Low S:N !
CompleteComplete

12. #3_0.25ul #1_0.25ul #3_1ul Comments
#0027  Both BAC REL preps undiluted
 70o
C; Frozen; 70o
C
#0028 ?  BAC prep #3 now has 50% glycerol
 70o
C; run
#0029A  REL 1 Prep #1 still undiluted !
 70o
C; Frozen; 70o
C
#0029B
#0030 ?  BAC prep #3 now has 50% glycerol
 90o
C; run
38,209,15638,209,15636,105,90736,105,907 321,906,245
444,913444,913
151,883,726151,883,726 28,493,807
8,146,98433,698,59933,698,599
427,533427,533
Undiluted preps in original assay
manifest similar activity
Assuming no denaturing variable
evidently not a linear relationship
between (glycerol) dilution of REL1
BAC prep and intrinsic activity, cf.
# 0027 vs. # 0028 & # 0030
Also compare activity from 1ul #3
without (# 0027) and with glycerol
(# 0029)
Conclusion: Require ~1ul 50% dil.
Prep. #3 !
Contrast this with apparent situation
for (still) undiluted prep. #1 where
activity is seemingly retained:
Compare # 0027 with # 0029
Apparently not a linear relationship
between prep vol. and activity
Single denaturation
Coincidence ?
Single denaturation
Coincidence ?

13. LH_#2LH_#2
LH_#4LH_#4
ΔΔΔΔCt AnalysisCt Analysis
1) ΔCt Sub unit gene – ΔCt 18S rRNA: BS
2) ΔCt Subunit gene – ΔCt 18S rRNA: PCF
3) ΔΔCt = 1) – 2)
4) Expression = 2(- ΔΔCt)

14. Relative Expression (qRT PCR: LH_#2) of 3 x ATP Synthase Subunit # 9 species normalised with
reference to b Tubulin: BS/PCF in Trypanosome Brucei # 427 cells
PCF_NeatcDNA
PCF_NeatcDNA
PCF_NeatcDNA
PCF_1:100cDNA
PCF_1:100cDNA
PCF_1:100cDNA
Neat
Neat
Neat
1:1001:100
1:100
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1470 2950 6340
ATP Synthase subunit # 9 Isoform
BS/PCF#427RelativeExpression
PCF_Neat cDNA
PCF_1:100 cDNA
Neat
1:100
BS 427
BS 427
BS 427
● Values = mean of triplicates +/- 1 x SEM
● Analysis = ΔΔ Ct
● Calibrator = Procyclic form (PCF)
● This assumes approximate species
doubling during exponential phase
● Actual efficiency = 90-105%except for
PCF cDNA + # 1470 = 80 % !
● Ambiguity of expression evident for
#1470 in BS # 427 cells
● Individual Ct values for given cDNA
samples < 0.5 cycles apart
● Where this is not true analysis based
on duplicate Ct's
● β tubulin RT (DNase +) - RT(-) ~ 7 cycles
LH_#2LH_#2
LH_#4LH_#4
ConclusionConclusion
Not withstanding #1470 efficiencyNot withstanding #1470 efficiency
Issues expression of sub unitIssues expression of sub unit
genes in BS #427 ~ 40% ofgenes in BS #427 ~ 40% of
PCF #427PCF #427

15. ββ TubulinTubulin ββ TubulinTubulin
Neat cDNANeat cDNA
1:101:10
1:1001:100
1:10001:1000
Slope:-3.526
R²:0.998
Slope:-3.467
R²:0.999
 A slope of -3.1- -3.6 = Amplification efficiency of 90 %-105%
 This is conducive to ΔΔ Ct analysis
LH_#2LH_#2
90%90%90%90%

16. 1470_BS1470_BS
1470_PCF1470_PCF
slope:-3.435 R²:0.997
6340_BS6340_BS
slope:-3.403 R²:0.998
6340_PCF6340_PCF
slope:-3.552
1:1001:100
1:101:10
1:11:1
R²:0.996slope:-3.906 R²:0.995
2950_BS !2950_BS !
2950_PCF2950_PCF
slope:-3.616 R²:0.994
slope:-3.194 R²:0.954
LH_#2LH_#2
88%88%
88%88%
96%96%
96%96%
96%96%
96%96%

17. Two sets of experiments demonstrate comparable Ct’s with respect to identical replicates of β tubulin
Replicates within each experiment match well
Also, PCF and BS #427 pleiomorphs match well
For both sets of experiments, signal difference between RT (+) and RT(-) samples similar and > 20 cycles
Further, RT(-) signal concordant with NTC samples, i.e. water instead of cDNA
This suggests effective removal of genomic DNA by rDNAse
ββ TubulinTubulin ββ TubulinTubulin
RT(+)RT(+) RT(+)RT(+)
RT(-)RT(-)
NTCNTC
RT(-)RT(-)
NTCNTC
1:11:11:11:1
1:1001:100
1:1001:100

18. Two sets of experiments demonstrate comparable Ct’s with respect to identical replicates of 18s rRNA
Replicates within each experiment match well
Also, PCF and BS #427 pleiomorphs match well
For both sets of experiments, signal difference between RT (+) and RT(-) samples similar and > 20 cycles
Further, RT(-) signal concordant with NTC samples, i.e. water instead of cDNA
This suggests effective removal of genomic DNA by rDNASe
18s rRNA18s rRNA 18s rRNA18s rRNA
RT(+)RT(+)
RT(-)RT(-)
NTCNTC
RT(+)RT(+)
RT(-)RT(-)
NTCNTC
1:1
1:100
1:1
1:100

19. Future WorkFuture Work
Adenylation AssayAdenylation Assay
1) Perform 2 remaining titrations viz. #125908 & #45201
 1ul of Prep #3
 0.25ul of Prep #1
2) With remaining isotope revisit less than high quality former assays ?
Lets expedite things a bit nowLets expedite things a bit now………………
We currently have 4 strong leads to validate high throughput (FRET) assay in
addition to PNAS leads……..establish assay conditions
Evaluate alternative growth assays to coulter counting for putative REL inhibitor
phenotype
Other:
Sams cDNA ?
Perform Pfaffl analysis on LH_#2 & LH_#4 qPCR data