Aspects of HT assay development for identifying putative inhibitors of the Tryp RNA editing enzyme REL 1 by compound library screens

1. RNA editing as a drug target in trypanosomes:RNA editing as a drug target in trypanosomes:
Development of a high throughput screening assayDevelopment of a high throughput screening assay
for RNA editing ligase 1for RNA editing ligase 1
Laurence Hall & Achim SchnauferLaurence Hall & Achim Schnaufer
Institute of Immunology & Infection Research and Centre of Immunity, Infection & Evolution, University of Edinburgh, UK
Project backgroundProject background
There is an urgent need to identify new targets and drugs to combat
trypanosomatid pathogens (human African trypanosomiasis, Chagas disease,
leishmaniases)
RNA editing by uridine insertion/deletion is essential for mitochondrial gene
expression in trypanosomatids
Editing is catalyzed by multiprotein complexes, the editosomes
A key component of editosomes is RNA editing ligase 1 (REL1). The crystal
structure of this enzyme revealed a deep pocket that binds to and orients the
essential ATP cofactor
There are no close REL1 homologs in the host and thus this enzyme
represents a target of (potential) high efficacy and specificity
Molecular dynamics simulations identified potential REL1 inhibitors by virtual
screening of approximately 2000 compounds (NCI diversity set):
Resulting naphtalene-based congeners were identified and demonstrated to
inhibit REL1 with IC50s in the single-digit µM range based on a radiolabelling
(adenylylation) in vitro assay [1,2]
Current efforts are focused on expediting compound screens by developing
a high throughput fluorescence-based assay (scalable 96-well plate format)
to screen compound libraries for REL1 inhibitors
This assay is being optimised with respect to signal-to-noise and other
parameters to enable (small compound) library screens
(1)(1) FRET Assay: Principles & PracticeFRET Assay: Principles & Practice
(3)(3) Production of rREL1Production of rREL1
Future WorkFuture Work
1) Determine optimal [substrate] = Km for actual compound
library screens
2)Optimise assay buffer composition to maximise S:N
3)Transfer to 384-well plate format
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
(2) Gel(2) Gel--basedbased VisualisationVisualisation of FRETof FRET
RNA oligos annealed & resolved on a 20%RNA oligos annealed & resolved on a 20% acrylamideacrylamide/ 5% glycerol/ 5% glycerol
/1 x TBE non/1 x TBE non--denaturing geldenaturing gel
IndividualIndividual fluorophorefluorophore--labelled oligos show very weak backgroundlabelled oligos show very weak background
at acceptor emission wavelength upon donor excitationat acceptor emission wavelength upon donor excitation
55’’ and 3and 3’’ RNA substrates annealed to bridge show efficient FRETRNA substrates annealed to bridge show efficient FRET
Ligation by REL1 results in denaturationLigation by REL1 results in denaturation--resistant FRETresistant FRET
FRET signal onlyFRET signal only
#1#1
#2#2
Centricon column fractionation
(> 30KD) of fractions from
LC peaks #1 & #2
BL21(DE3) cells transformed with REL1 expression constructBL21(DE3) cells transformed with REL1 expression construct
Induction of expression (fromInduction of expression (from pETpET construct) initiated with IPTGconstruct) initiated with IPTG
Soluble protein purified by LC using aSoluble protein purified by LC using a NiNi--NTANTA affinity columnaffinity column
Peak #2 represents active rREL1Peak #2 represents active rREL1
(4)(4) AssayAssay OptimisationOptimisation (examples)(examples)
A)A) 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
C)C) Assay conditions (detergent)Assay conditions (detergent)
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
A)A) REL1 expression & LC purificationREL1 expression & LC purification
B)B) PAGE examination of rREL1 fractionsPAGE examination of rREL1 fractions
ReferencesReferences
1)1) Durrant, HallDurrant, Hall et al.et al. (2010), PLoS, Neglected Tropical Diseases(2010), PLoS, Neglected Tropical Diseases
4(8): e803.4(8): e803.
2)2) Amaro et alAmaro et al. (2008), PNAS 105 (45): 17278. (2008), PNAS 105 (45): 17278--83.83.
(5)(5) Assay ValidationAssay Validation
Assay statistically validated as suitable forAssay statistically validated as suitable for
high throughput screeninghigh throughput screening
Addition of Triton XAddition of Triton X--100 increases REL1 activity100 increases REL1 activity
fluorophorefluorophore--labelledlabelled
REL1 substrateREL1 substrate