Procedural steps for developing a FRET based assay to screen for inhibitors of the Tryp enzyme REL 1: Potential microbial therapeutics

1. RNA editing as a drug target inRNA editing as a drug target in Trypanosoma bruceiTrypanosoma brucei::
Development of a (high throughput) fluorescence assayDevelopment of a (high throughput) fluorescence assay
to identify inhibitors of REL1to identify inhibitors of REL1
Laurence Hall,Laurence Hall, Achim SchnauferAchim Schnaufer
Institute of Immunology & Infection Research, 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 multi protein 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 congeners were identified and demonstrated to inhibit
REL1 with IC50s in the single-digit µM range based on a radio labelling
(adenylation) in vitro assay (see ‘References’)
Current efforts are focused on expediting compound screens by developing a
high throughput (FRET based) fluorescence assay (scalable 96 well plate
format) to screen compound libraries for REL1 inhibitors
This assay is being optimised with respect to FRET and dynamic range as a
precursor to implementing (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) Experiment with utility of 384 well plate format
FRET (Fluorescence Resonance Energy Transfer) is achieved
when 2 fluorophores with overlapping spectra are fixed in close
proximity, such that photons are transferred between a ‘donor’
(‘D’) & ‘acceptor’ fluorophore (‘A’), resulting in acceptor
fluorescence when the donor is excited, which can be quantified
by spectrophotometry
Annealing of fluorophore labelled 5’ and 3’ RNA substrates to anAnnealing of fluorophore labelled 5’ and 3’ RNA substrates to an
RNA bridge anchors the donor and acceptor fluorophores inRNA bridge anchors the donor and acceptor fluorophores in
close proximityclose proximity
Consequently, photon transfer occurs and FRET is evidentConsequently, photon transfer occurs and FRET is evident
After putative ligation & denaturation, FRET emission isAfter putative ligation & denaturation, FRET emission is
measured (in a plate based format) by spectrophotometry:measured (in a plate based format) by spectrophotometry:
In the presence of active REL1, ligation occurs and a
contiguous dual labelled species is generated, demonstrating
FRET
In the absence of active REL1, the scaffold dissociates with
denaturation, abrogating FRET
Thus, competitive inhibitors of REL1 will abrogate FRET as
compared with a ‘ligation only’ control
Finally, a molar excess of competitor oligo (complementary toFinally, a molar excess of competitor oligo (complementary to
the RNA bridge) ensures that dissociation is efficient and stablethe RNA bridge) ensures that dissociation is efficient and stable
(2)(2) Visualisation of FRETVisualisation of FRET
100 ng
REL1
1000 ng
REL1
DenDen DenDenNDND NDND
RNA oligos annealed & resolved on a 10% acrylamide/glycerol /TBERNA oligos annealed & resolved on a 10% acrylamide/glycerol /TBE
non denaturing gelnon denaturing gel
Individual fluorophore labelled oligos show very weak backgroundIndividual fluorophore labelled oligos show very weak background
emission at acceptor wavelengthemission at acceptor wavelength
5’ and 3’ RNA substrates annealed to bridge show efficient FRET,5’ and 3’ RNA substrates annealed to bridge show efficient FRET,
whereas subsets of the above do notwhereas subsets of the above do not
Nevertheless, appropriate signals are discernable from individualNevertheless, appropriate signals are discernable from individual
dyes when examined by multi channel (plex) filtersdyes when examined by multi channel (plex) filters
A)A) Fluorescence measurements of individual and annealed RNAs after PAGEFluorescence measurements of individual and annealed RNAs after PAGE B)B) Annealing + LigationAnnealing + Ligation
FRET signal onlyFRET signal only Multiplex (false color) imageMultiplex (false color) image
#1#1
#2#2
Centricon column fractionation
(> 30KD) of fractions from
LC peaks #1 & #2
BL21(DE3) cells transformed with REL1 expression construct (NBL21(DE3) cells transformed with REL1 expression construct (N
terminal His tag on interacting partner protein A2)terminal His tag on interacting partner protein A2)
Induction of expression (from pET construct) initiated with IPTGInduction of expression (from pET construct) initiated with IPTG
Soluble protein purified by LC using aSoluble protein purified by LC using a Ni-NTANi-NTA affinity columnaffinity column
Peak #2 represents active rREL1Peak #2 represents active rREL1
(4)(4) Assay Optimisation (examples)Assay Optimisation (examples)
A)A) InductionInduction
B)B) Assay buffer pHAssay 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) Chemical denaturationChemical denaturation
(5)(5) Assay validationAssay validation
Assay statistically validated as suitable forAssay statistically validated as suitable for
high throughput screeninghigh throughput screening
Ligation & annealing efficiency optimised with respect to assayLigation & annealing efficiency optimised with respect to assay
buffer pHbuffer pH
pH 8.0pH 8.0 most conducive to annealing and ligationmost conducive to annealing and ligation
Efficient low temperature denaturation of scaffold afforded byEfficient low temperature denaturation of scaffold afforded by
use of denaturing chemical in conjunction with empiricallyuse of denaturing chemical in conjunction with empirically
verified/optimised amount of competitor oligoverified/optimised amount of competitor oligo
The latter contributes to stable and efficient denaturationThe latter contributes to stable and efficient denaturation
Different purified fractions of REL1-A2 demonstrated significantDifferent purified fractions of REL1-A2 demonstrated significant
activity in the context of this radio labelling assayactivity in the context of this radio labelling assay
A)A) REL1 expression & LC purificationREL1 expression & LC purification B)B) PAGE examination of REL1 fractionsPAGE examination of REL1 fractions
C)C) Enzymatic activity validationEnzymatic activity validation
KeyKey
1)1) ‘‘Den’ = Denatured before PAGEDen’ = Denatured before PAGE
2)2) ‘‘NDND’ = Not denatured before PAGE’ = Not denatured before PAGE
ResultsResults
1)1) Denaturation liberates unligated oligosDenaturation liberates unligated oligos
2)2) The magnitude of specific ligated productThe magnitude of specific ligated product
(FRET signal) increases with the amount of(FRET signal) increases with the amount of
REL1 (L1)REL1 (L1)
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): 17278-83.. (2008), PNAS 105 (45): 17278-83.