Titrating preliminary hits derived from anti REL 1 radio mimetic assay: Precursor to full regression analysis of lead compounds to establish IC50's

1. 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
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
Also,Tetracycline needs to be evaluatedAlso,Tetracycline needs to be evaluated

2. CompoundCompound
TypeType
CompoundCompound 1010µµMM 100100µMµM TitratedTitrated N=N= ICIC5050 RangeRange CommentsComments
NCI BatchNCI Batch
#2#2
# 45609 ! 95 %
(78%)
(88%) 33 3-1uM  Discrepancy between
assays above predicted
IC50 range
 Repeat ?
Discrepancy between
assays
Repeated ! (#0036)
# 162535 !!# 162535 !! 85_90 %
(85%)
(~98%)*
(90 %)
(>99.9%)*
>3>3
(+2)(+2)
~1uM (N=1)
~3-1uM (N=3)
# 1698 ! 80 %
(50%)
(90%) 33
(+2)(+2)
10uM- 3uM (N=2)
3-1uM (N=1)
NCI BatchNCI Batch
#1#1
# 117079 30_80% >99%
(~80 %)
(~50-70%)
>3>3 100uM- 30uM
(N=4)
 30uM (N=1)
Discrepancy between
assays
above IC50 range
 Furthermore, IC50’s differ
between assays
 Repeat ?
# 125908 20_70% ~95 %
(~50-70%)
>3>3 100uM- 30uM
(N=3)
 ~30uM (N=1)
# 45201 NA ~70% Yet to do !!Yet to do !!
Sigma rareSigma rare
LibraryLibrary
MordantMordant
Black !!Black !!
(~98%)* (~99.8%)* >3>3 ~1uM Completed
 Values derive from titrations’
 Identified using fragment based algorithm
 Identified using Auto Dock 4 relaxed complex
scheme
Assay #0036 is an attempt to clarify IC50’s for
# 1698 & #162535
Regarding # 45609 IC50’s concur but values differ
above this threshold
Regarding #117079 & #125908 some assays have
different IC50’s
Furthermore, those with similar values differ above
IC50 titrant
* Values derived from 0.25ul undiluted prep #1

3. 34B
MBMB
#162535#162535
# 45609# 45609
# 1698# 1698
1uM1uM
>/=1uM>/=1uM
3-1uM3-1uM
>3uM>3uM

4. % (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 approx to
1uM
This concurs with prior assays100uM100uM 30nM30nM
50S50S-1-1
100S100S-1-1

5. % (Putative) Inhibition of Ligation (Adenylation) by Mordant Black (solute) relative to DMSO
(solvent)_# Mordant Black Molarity Titration
30uM
30uM
10uM
10uM
10uM
100uM
100uM
100uM
30uM
0
20
40
60
80
100
120
140
Prep #3_050203 Prep #1_080802 Prep #1_080802
Mordant Black Molarity Titration: 100uM-30nM
SpecificSignal/DMSO_%
# 0029A # 0033A
28,500,000 152,500,000 203,500,000
52%
56% 54%
1ul 50% REL 1 0.25ul 50% REL 1 1ul undiluted REL 1
17 HR17 HR 1 HR !
For assays depicted (N=3) IC50 consistently approximates to ~1uM
Undiluted prep #1 displays massively greater signal than either glycerol diluted #1 or #3

6. % (Putative) Inhibition of Ligation (Adenylation) by Mordant Black (solute) relative to DMSO
(solvent)_# Mordant Black 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
140
Prep #1_080802 Prep #3_050203
Molarity Titration: 100uM-30nM
SpecificSignal/DMSO_%
24+/-5.6%
59+/-5.2%
24%
52%
IC50 approximates
to 1uM for N=5
Combining data from assays #0029_33_34 culminates in predicted IC50 of ~1uM (N=4)
In terms of IC50 alone this is consistent with data from REL 1 prep #3 also derived from #0029

7. % (Putative) Inhibition of Ligation (Adenylation) by #162535 (solute) relative to DMSO (solvent)_#
162535 Molarity Titration
DMSO
DMSO
DMSO
3uM
3uM
3uM
1uM
1uM
1uM
0
20
40
60
80
100
120
140
160
180
# 0018A # 0033B # 0034B
Molarity Titration: 100uM-30nM
MeanSpecificsignal/DMSO_%
DMSO
100uM
30uM
10uM
3uM
1uM
300nM
100nM
30nM
# 162535
1ul Undiluted Prep #1_080802 0.25ul Undiluted Prep #1_0808020.5ul 50% Glycerol diluted prep
36%
67%
42%
90%
12%
48%
25,000,000 217,000,000 45,000,000
For N=3 predicted IC50 approximates to ~ 2uM
For N=1 predicted IC50 ~1uM (1ul undiluted prep #1)
Therefore repeated using 0.25ul undiluted prep #1
Thus, now N=4 for 0.25ul undiluted prep #1
Repeated !Repeated !

8. Variable background was a
confounding factor when it came
to signal comparison for the
different titrants
Nevertheless, evident IC50 was
approximately 1uM
This concurs with assays #34
& #35

9. % (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 # 0035A
MeanSpecificSignal/DMSO_%
IC50 resides here
# 1698
28, 000,000 190, 000,000
27%
53%
38%
61%
For N=1 predicted IC50 approximates to 10uM
For N=2 predicted IC50 lies somewhere between 10uM & 3uM
#0035A = 0.25ul undiluted prep #1_080802
Therefore repeated using (similarly) 0.25ul undiluted prep #1
Thus, for 0.25ul undiluted prep #1, now N=4 !
Repeated !Repeated !

10. 108, 000, 000 counts108, 000, 000 counts
153, 000, 000 counts153, 000, 000 counts

11. % (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%
Thus, for N=3 predicted of IC50 ~3-1uM
However, above this threshold, titrants for respective assays differ considerably with respect to putative
inhibition
 Assay #0016B = 0.5ul 50 % glycerol diluted prep
 Assay #0035B = 0.25ul undiluted prep #1_080802
Repeat ??Repeat ??

12. % (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

13. IC50 resides somewhere
between 100uM & 30uM
In the main, this concurs
with previous assays, i.e.:
 #0011 & #0012
 #0029B_Prep #1_080802
However, predicted IC50
for #29B_prep #3 approx.
to 30uM rather than 100uM
To illustrate discrepancies
further:
 Putative inhibition @100uM
for assays #0011 & #0012
>99%
 For assay #0029B ~ 80%
 For assay #0032A/B ~ 50
-70%
# 117079 Molarity Titration: 1mM-1uM# 117079 Molarity Titration: 1mM-1uM
10S10S-1-1
10-20S10-20S-1-132A
32B5S5S-1-1 10S10S-1-1
1ul Prep #31ul Prep #3

14. % (Putative) Inhibition of REL 1 Ligation (Adenylation) by #125908 (solute) relative to DMSO (solvent): # 125908
Molarity Titration. Assay #0031A
1mM 1mM
DMSO
DMSO
300uM
100uM
100uM
30uM
30uM
10uM
10uM
3uM
3uM
1uM
1uM
0
20
40
60
80
100
120
140
# 31A_1 # 31A_2
Molarity Titration: 1mM-1uM
SpecificSignal/DMSO_%
32.5%
77.7%
17.4%
48.4%
86.4%
IC50 resides
here
IC50 resides
here

15. % (Putative) Inhibition of REL 1 Ligation (Adenylation) by #125908 (solute) relative to DMSO (solvent): # 125908
Molarity Titration. Assay #0031B
DMSO
DMSO
1mM1mM
300uM
300uM
100uM
100uM
30uM
30uM
10uM
10uM
3uM
3uM
1uM
1uM
0
20
40
60
80
100
120
140
# 31B_1 # 31B_2
Molarity Titration: 1mM-1uM
SpecificSignal/DMSO_%
17.4%
35.5%
60.9%
13.4%
30.2%
51.4%
IC50 resides here IC50 resides here
Predicted IC50 ~30uM: N=1
Predicted IC50 100uM-30uM: N=3
The latter concurs with previous assays

16. % (Putative) Inhibition of REL 1 Ligation (Adenylation) by #117079 (solute) relative to DMSO (solvent):
#117079 Molarity Titration. Assay #0032A/B
DMSO
1m M 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
Plan of action:Plan of action:
1. Either combine this data with #0029B_prep #1 (N=4)
2. Or repeat assay 0.25ul undiluted prep #1_080809

17. =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)

18.  Green = 1ul 50% Prep #3_050203 + 0.1% Triton X-100
 Black = 1ul undiluted prep #1_080802
 Blue = 1ul undiluted prep #1_080802
 Green = 0.25ul undiluted prep #1_080802
200S200S-1-1
1000S1000S-1-133A
33B100S100S-1-1
1000S1000S-1-1

19. Loss of Specific Signal (DMSO) with Freeze thawing: REL1 'prep #3_050203' vs 'Prep #1_080802'
#0031
#0032
#0027
#0027
#0029
#0029
#0034
#0035
#0036
0.00E+00
5.00E+07
1.00E+08
1.50E+08
2.00E+08
2.50E+08
DMSO_#1_0.25ul DMSO_#3_0.25_1ul
Assays
SpecificSignal
#0027
#0029
#0031
#0032
#0034
#0035
#0036
0.25ul undiluted Prep #3_050203
0.25ul Prep #1_080802
1ul 50 % Prep #3_050203

20. SummarySummary
1. Mordant black has evident IC50 approximating to 1uM (N=5): Best lead !!
2. Regarding #45609, assays alight on IC50 approximating to 3-1uM (N=3) but differ
with respect to putative titrant inhibition above that threshold; Repeat ?
3. With reference to #162535 & #1698, discrepancies evident between assay
predicted IC50 values and for #1698 in particular N<3 for alternative values; There
fore repeated (#0036)
4. Regarding #117079 & #125908, some assays display putative different IC50’s but
an N>/=3 exists for one value
5. However, titrant values above the predicted IC50 thresholds also differ: Repeat ?
In general, same enzyme batches concur more often and undiluted prep
#1_080802 has yielded highest signal to noise
Thus, in general and N>/= 3 derived from this REL 1 batch would constitute
the ideal
Future WorkFuture Work
1. Quantify #0036
2. Repeat other assays described to nullify IC50 and preferably titrant differences in
general, with a bias towards N>/= 3 from the same enzyme batch
3. Submit putative inhibitory values to Graph Pad Prism© to compute exact IC50s’
4. Complete titrations for #45201