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Identify REL 1 InhibitorsIdentify REL 1 Inhibitors Assay (by covalent labelling of recombinant ligase with α_32P 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: ~ 30 substances already screened @ 10uM & 100uM ( ~20 batch #1 & ~10 batch #2). Optimal IC50S ~low uM range (see caveat below) B. Set #2 FDA Approved Drugs: 13 compounds from various sources. Thus far 3 ordered (1 already in house)C. Set #3 compounds: 15 so called ‘hit 2 lead’ substances; Ordered and in house 2 preliminary screens @10uM
IC50 Considerations…IC50 Considerations…Some set #1 Batch #1 compounds may bind exert effect by binding to and stabilisingapo 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
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Skeleton MethodSkeleton Method Set up radio ligation reactions, cf. REL 1; α32P_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:
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Hit 2 LeadHit 2 Lead
No evidence of significant REL 1 inhibition from these 1st 7 H2L compounds However, 1st screen
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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
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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
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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
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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) @ 100uM3) 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 ?
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= 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 #1100uM100uM
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# 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
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# 1# 1 # 45201 exhibits putative efficacy to the order of 70%based on these assays
NCI Batch #1NCI Batch #1
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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
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# 2# 2
NCI Batch # 2NCI Batch # 2
# 45577 exhibits significant inhibition in the order ~90-95% @100uM
Variation due to variable & variagated background
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# 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
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# 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.
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# 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.
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# 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.
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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.
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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 !
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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
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Compound Compound TypeType
Compound Compound ##
1010µµMM 100100µMµM Titrated ?Titrated ? ICIC50 50 RangeRange CommentsComments
NCI Batch NCI Batch #2#2
# 45609 ! 95 % (78 %) (88%) 10uM-1uM Values in red pertain to titration assaysCompounds in blue original leads
# 162535 ! 85_90 % (85%)
(90 %) 3uM-300nM
# 1698 ! 80 % (50 %) (90%) 30uM-3uM
# 45201 NA 70%
NCI Batch NCI 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
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MethodMethod1. Set up triplicate reactions with published antagonist # 45208 and DMSO control2. Set up these triplicate reactions using 0.18ul of 32P d ATP/rxn (std); 0.36ul & 0.54ul3. Does the natural substrate ATP out compete #45208 @ higher conc. ?
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ResultsResultsAs 32P_dATP increases from 0.18ul per reaction to 0.54ul specific signal from# 45208drops from ~ 35% to 45% respectivelyThus, some inhibition but (probably) non competitive rather than competitive
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# 45208 @100uM# 45208 @100uM
# 45208 @100uM# 45208 @100uM
# 45208 @100uM# 45208 @100uM
DMSODMSO
DMSODMSO
DMSODMSO
A = 0.18ul A = 0.18ul 3232P_dATPP_dATP
B = 0.36ulB = 0.36ul 3232P_dATPP_dATP
C = 0.54C = 0.54ulul 3232P_dATPP_dATP
> 20S> 20S-1-1 20_50S20_50S--11
DM
SO
DM
SO
DM
SO
# 45
208
# 45
208
# 45
208
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
31.8% specific signal relative to DMSO 6 % SD about mean
34.7% specific signal relative to DMSO4% SD about mean
44.4% specific signal relative to DMSO7% SD about mean
As 32P_dATP increases from 0.18ul per reaction to 0.54ul specific signal from # 45208 drops from ~ 35% to 45% respectively
SD about mean ~ 5%
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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.
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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
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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
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Raw (Geiger) counts fromDried gel ~ 5S-1
Raw (Geiger) counts fromDried gel ~ 10-20S-1
Low S:N precludes actual quantification In particular, #0013B exhibited high background, tallying with Geiger count Although 32P 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 #2Reaction #1Reaction #1 Reaction #2Reaction #2
Reaction #1Reaction #1 Reaction #2Reaction #2
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# 0018# 0018
# 162535
# 1698
Gel loaded frombehind
Gel loaded fromthe frontNo correlation between well
Signal & specific signal
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Hit 2 Lead @ 10Hit 2 Lead @ 10µMµM
# 4.# 4.Signal diminutionSignal diminution
No DMSOsignal
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