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

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:

Hit 2 LeadHit 2 Lead

No evidence of significant REL 1 inhibition from these 1st 7 H2L compounds However, 1st screen

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

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

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

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 ?

= 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

# 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

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

NCI Batch #1NCI Batch #1

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

# 2# 2

NCI Batch # 2NCI Batch # 2

# 45577 exhibits significant inhibition in the order ~90-95% @100uM

Variation due to variable & variagated background

# 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

# 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.

# 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.

# 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.

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.

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 !

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

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

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. ?

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

# 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%

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.

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

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

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

# 0018# 0018

# 162535

# 1698

Gel loaded frombehind

Gel loaded fromthe frontNo correlation between well

Signal & specific signal

Hit 2 Lead @ 10Hit 2 Lead @ 10µMµM

# 4.# 4.Signal diminutionSignal diminution

No DMSOsignal