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 ?

Why REL 1 ?Why REL 1 ?

1) No human homolog2) Required in both insect and blood stream stage of life cycle3) Mg 2+:ATP binding pocket of REL1 has a unique structure relative to other ligases4) 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

CompoundCompoundClassClass

SetSet # substances# substances Drug discovery Drug discovery algorithmalgorithm

1. NCI #1 #1 16 Compounds screened with ‘Auto dock 4’

RankedVerified with so-called

‘relaxed complex scheme’ 2. FDA #2 1 in house (sigma)

11 more predicted but financially prohibitive1 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

= 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

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

=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)

#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 by0.250.25µl 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

100uM

100uM30uM 30uM

10uM10uM

DMSO

DMSO

3uM 3u

M

1uM 1u

M

300n

M

300n

M

100n

M

100n

M

30nM

30nM

0

20

40

60

80

100

120

140

Prep #3_050203 Prep #1_080802

Mordant Black Molarity Titration: 100uM- 30nM

Spec

ific

Sign

al/D

MSO

_%

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 assays100uM 100uM 30nM 30nM

50S50S-1-1 100S100S-1-1

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 !

% (Putative) Inhibition of REL 1 Ligation (Adenylation) by #117079 (solute) relative to DMSO (solvent)_# 117079 Molarity Titration

DM

SO

DM

SO

100u

M

100u

M

30uM

30uM

10uM

10uM 3u

M

3uM

1uM

1uM

300n

M

300n

M

100n

M

100n

M

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

Spec

ific

Sign

al/D

MSO

_%

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

# 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

Compound Compound TypeType

Compound #Compound # 1010µµMM 100100µMµM Titrated ?Titrated ? ICIC50 50

RangeRangeCommentsComments

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 rareLibraryLibrary

Mordant Mordant Black !!Black !!

Evident butnot quantified

Evident butnot quantified

3uM-1uM

Identified using fragment based algorithm

Low S:N !Low S:N !CompleteComplete

#3_0.25ul #1_0.25ul #3_1ul Comments

#0027 Both BAC REL preps undiluted 70oC; Frozen; 70oC

#0028 ? BAC prep #3 now has 50% glycerol 70oC; run

#0029A REL 1 Prep #1 still undiluted ! 70oC; Frozen; 70oC

#0029B

#0030 ? BAC prep #3 now has 50% glycerol 90oC; 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 denaturationCoincidence ?

Single denaturationCoincidence ?

LH_#2LH_#2

LH_#4LH_#4

ΔΔΔΔCt AnalysisCt Analysis1) ΔCt Sub unit gene – ΔCt 18S rRNA: BS2) ΔCt Subunit gene – ΔCt 18S rRNA: PCF 3) ΔΔCt = 1) – 2)

4) Expression = 2(- ΔΔCt)

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_

Neat

cDN

A

PCF_

Neat

cDN

A

PCF_

Neat

cDN

A

PCF_

1:10

0 cD

NA

PCF_

1:10

0 cD

NA

PCF_

1:10

0 cD

NA

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/P

CF #

427

Rela

tive

Expr

essi

on

PCF_Neat cDNA

PCF_1:100 cDNA

Neat

1:100

BS 427

BS 427BS 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 efficiencyIssues expression of sub unitIssues expression of sub unit

genes in BS #427 ~ 40% ofgenes in BS #427 ~ 40% ofPCF #427PCF #427

ββ Tubulin Tubulin ββ Tubulin Tubulin

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%

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%

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 Tubulin Tubulin ββ Tubulin Tubulin

RT(+)RT(+) RT(+)RT(+)

RT(-)RT(-)NTCNTC

RT(-)RT(-)NTCNTC

1:11:11:11:1

1:1001:100 1:1001:100

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

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