Aspects of development of an HT assay for identifying inhibitors of the Tryp target protein REL 1: Potential microbial therapeutics by 'hit to lead'

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RNA editing as a drug target in trypanosomes:
Development of a high throughput screening
assay for RNA editing ligase 1
Conference Paper · June 2012
DOI: 10.13140/2.1.3455.9362
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Laurence Stuart Dawkins-Hall
University of Leicester
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Achim Schnaufer
The University of Edinburgh
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FRET (Fluorescence Resonance Energy Transfer) is achieved
when two fluorophores with overlapping spectra are fixed in
close proximity, such that virtual photons are transferred
between a ‘donor’ (‘D’) and ‘acceptor’ fluorophore (‘A’),
resulting in acceptor fluorescence when the donor is excited,
which can be quantified by spectrophotometry
Annealing ofAnnealing of fluorophorefluorophore--labelledlabelled 55’’ and 3and 3’’ RNA substrates toRNA substrates to
an RNA bridge (thean RNA bridge (the ““guide RNAguide RNA””) anchors the donor and) anchors the donor and
acceptor fluorophores in close proximityacceptor fluorophores in close proximity
Consequently, transfer of virtual photons results in FRETConsequently, transfer of virtual photons results in FRET
After ligation and denaturation (to disruptAfter ligation and denaturation (to disrupt unligatedunligated dsRNAdsRNA),),
FRET emission is measured byFRET emission is measured by spectrophotometryspectrophotometry::
In the presence of active REL1, ligation occurs, resulting in
denaturation-resistant FRET
In the absence of active REL1, the dsRNA dissociates with
denaturation, abrogating FRET
Thus, inhibitors of REL1 will abrogate FRET as compared with a
control
'' (( ! #! #
RNA oligos annealed resolved on aRNA oligos annealed resolved on a 20%20% acrylamideacrylamide/ 5%/ 5%
glycerol /1 x TBE nonglycerol /1 x TBE non--denaturing geldenaturing gel
55’’ and 3and 3’’ RNA substrates annealed to bridge show efficientRNA substrates annealed to bridge show efficient
FRETFRET
Ligation by REL1 results in denaturationLigation by REL1 results in denaturation--resistant FRETresistant FRET
Absence ofAbsence of ligationligation abrogates FRETabrogates FRET
Furthermore, absence of theFurthermore, absence of the gRNAgRNA bridge precludes annealingbridge precludes annealing
and thus FRETand thus FRET
)) ** ++
A)A) rREL1rREL1 InductionInduction
B)B) Assay conditions (buffer pH)Assay conditions (buffer pH)
Induction of soluble protein improved by optimising [ITPG]Induction of soluble protein improved by optimising [ITPG]
Induction of soluble protein further improved by inclusion ofInduction of soluble protein further improved by inclusion of
heat shock prior to IPTG facilitating correct protein foldingheat shock prior to IPTG facilitating correct protein folding
Ligation and annealing efficiency optimised with respect toLigation and annealing efficiency optimised with respect to
assay buffer pHassay buffer pH
pH 8.0pH 8.0 most conducive to annealing and ligationmost conducive to annealing and ligation
,, --
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RNA editing by uridine insertion/deletion is essential for mitochondrial gene
expression in all trypanosomatids pathogens.
Editing is catalyzed by multiprotein complexes, the editosomes.
A key catalytic component of editosomes is RNA editing ligase 1 (REL1).
REL1 has been validated as a drug target in T. brucei in vitro and in vivo
(Schnaufer et al., Science 2001).
The 1.2 crystal structure of REL1 revealed a deep ATP binding pocket with
numerous opportunities for specific binding of small molecule inhibitors
(Deng, Schnaufer et al., J Mol Biol 2004).
There are no close REL1 homologs in the mammalian host and thus this
enzyme represents a target of potentially high specificity.
Computational screening efforts based on molecular dynamics simulations
resulted in the identification of several REL1 inhibitors with IC50 values in the
single-digit µM range (Amaro, Schnaufer et al., PNAS 2008; Durrant, Hall et al.,
PLoS NTD 2010).
Here we describe the development of a high throughput screening (HTS)
compatible, fluorescence-based REL1 assay.
The assay was optimised with respect to reaction chemistry; fluorophores;
rREL 1 production enzyme kinetics culminating in higher sensitivity; better
dynamic range higher throughput
.. /0/0
Assay statistically validated as suitable forAssay statistically validated as suitable for
high throughput screeninghigh throughput screening
Utilising two independent methods of calculating the amount ofUtilising two independent methods of calculating the amount of
ligatedligated FRET product theFRET product the [ATP] substrate corresponding to Km[ATP] substrate corresponding to Km
= 2.5= 2.5µµMM
Km = [ substrate ] @Km = [ substrate ] @ ½½ x V maxx V max
B)B) Adapting reaction conditions for actual high throughput screenAdapting reaction conditions for actual high throughput screen
A)A) Determination of [ATP] substrate concentration @ Km (=Determination of [ATP] substrate concentration @ Km (= ½½ xx VmaxVmax))
A compoundA compound independently validated as possessing REL 1independently validated as possessing REL 1
inhibitory properties demonstrates similarinhibitory properties demonstrates similar ligationligation inhibition ininhibition in
the context of this assaythe context of this assay
Furthermore, this can be demonstrated byFurthermore, this can be demonstrated by non denaturing PAGEnon denaturing PAGE
To economise on use of L1 and determineTo economise on use of L1 and determine ligationligation time yieldingtime yielding
linear order conditionslinear order conditions ligationsligations were performed @ variouswere performed @ various
[ATP]. In conclusion it was found that:[ATP]. In conclusion it was found that:
5 minute5 minute ligationsligations using 50ng of L1 @ 10using 50ng of L1 @ 10µµM ATP yielded rapidM ATP yielded rapid
sensitive reactions compatible with linear order conditions, i.esensitive reactions compatible with linear order conditions, i.e..
affording dynamic range for determining putative inhibition ofaffording dynamic range for determining putative inhibition of
rRELrREL 1 by small compound1 by small compound ligandsligands
11 ((
Plus L1
Minus L1
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
75000
80000
85000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
2
!3!#
Z = 1 - ( 3 s + 3 c)
(µs - µc)
Z = 1 - ( 16397 + 1079)
(70119 - 5909)
Z = 1 - 0.3 = 0.7 !!!
45 6 -7
50ng #003-1 + 10µM ATP
- L1 + 10 min
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
8
!3!#
Gain = 1453
C)C) Verification of standard reaction kineticsVerification of standard reaction kinetics
As depicted there is a clear correlation between reactionAs depicted there is a clear correlation between reaction
kinetics underkinetics under standard operating conditionsstandard operating conditions and FRET signaland FRET signal
evinced fromevinced from non denaturing PAGEnon denaturing PAGE
Oligo markers
! # ! #
55’’ # 2# 2
33’’ # 1# 1
DualDual
200ng
2000ng
- L1
- gR#1
Den: 200x #139 ND
200ng
2000ng
- L1
- gR#1
(+ 200ng)
(+ 200ng)
99 // ! #! #
0
10
20
30
40
50
60
70
80
90
100
L5 D L30 D Min L1
D
Min
gR#1 D
L5 ND L30 ND Min L1
ND
Min
gR#1
ND
:9;
Gel #11_Cy5 Densitometry
#114:FRET
Signal magnitude by FRET photometry compared with (gel) Cy5Signal magnitude by FRET photometry compared with (gel) Cy5
densitometrydensitometry
There is aThere is a manifest and unambiguous relationship betweenmanifest and unambiguous relationship between
signal magnitude associated with gel products and those on 96signal magnitude associated with gel products and those on 96
well plateswell plates
No L1
0uM ATP
2.5uM ATP
5uM ATP
10uM ATP
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
70000.0
80000.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0
!3
Gain = 1453
(1)(1)
(1)(1)
(2)(2)
(2)(2)
0.5 1.0 1.5 2.0
0
20
40
60
80
100
0 2 1';.)= µµµµ8
:8*
IC50 = 9.3µM +/- 0.673µM
R2 = 0.9904
0
10000
20000
30000
40000
50000
60000
70000
Tris 3.5 Tris 5.0 Tris 5.5 Tris 6.0 Tris 6.5 Tris 7.0 Hepes 8.0
; + 4 ;: ?
!3
Plus 50ng #002-5
Minus L1
Gain = 1531
9-)
%-'
.-1
)-9
%-%
1-@
%-
+ L1/ - L1
S/BS/B
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