RNA editing as a drug target in trypanosomes:

Development of a high throughput screening assay

for RNA editing ligase 1
Laurence Hall & Achim Schnaufer
Institute of Immunology & Infection Research and Centre of Immunity, Infection & Evolution, University of Edinburgh, UK

(1) FRET Assay: Principles & Practice

Project 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 M 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

fluorophorefluorophore-labelled
REL1 substrate

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 of fluorophorefluorophore-labelled 5 and 3
3 RNA substrates to
an RNA bridge (the guide RNA
RNA) anchors the donor and
acceptor fluorophores in close proximity
Consequently, transfer of virtual photons results in FRET
After ligation and denaturation (to disrupt unligated dsRNA),
dsRNA),
FRET emission is measured by spectrophotometry:
spectrophotometry:
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) GelGel-based Visualisation of FRET

(4) Assay Optimisation (examples)

RNA oligos annealed & resolved on a 20% acrylamide/

acrylamide/ 5% glycerol
/1 x TBE nonnon-denaturing gel
Individual fluorophorefluorophore-labelled oligos show very weak background
at acceptor emission wavelength upon donor excitation

A) Induction

5 and 3
3 RNA substrates annealed to bridge show efficient FRET
Ligation by REL1 results in denaturationdenaturation-resistant FRET

FRET signal only

(3) Production of rREL1

A) REL1 expression & LC purification
Induction of soluble protein improved by optimising [ITPG]
Induction of soluble protein further improved by inclusion of
heat shock prior to IPTG facilitating correct protein folding
B) Assay conditions (buffer pH)

#1
#2

(5) Assay Validation

BL21(DE3) cells transformed with REL1 expression construct

Induction of expression (from pET construct) initiated with IPTG
Soluble protein purified by LC using a NiNi-NTA affinity column
Peak #2 represents active rREL1

Centricon column fractionation

(> 30KD) of fractions from
LC peaks #1 & #2

Ligation and annealing efficiency optimised with respect to

assay buffer pH
pH 8.0 most conducive to annealing and ligation

B) PAGE examination of rREL1 fractions

C) Assay conditions (detergent)

Assay statistically validated as suitable for

high throughput screening

Future 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
Addition of Triton XX-100 increases REL1 activity

References
1) Durrant, Hall et al. (2010), PLoS, Neglected Tropical Diseases
4(8): e803.
2) Amaro et al.
al. (2008), PNAS 105 (45): 1727817278-83.