Rna editing as a drug target identification of inhibitors of rel 1 bsp 210
•
0 likes•32 views
Low through put radiomimetic assay to identify putative inhibitors of the Tryp enzyme REL 1 'in vitro' predicted by 'in silico' modelling
![RNA editing as a drug target inRNA editing as a drug target in Trypanosoma bruceiTrypanosoma brucei::
Identification of inhibitors of the essential enzyme REL1Identification of inhibitors of the essential enzyme REL1
Laurence HallLaurence Hall11
, Jacob D. Durrant, Jacob D. Durrant22
, Rommie Amaro, Rommie Amaro33
, Achim Schnaufer, Achim Schnaufer11
(1) Institute of Immunology & Infection Research, University of Edinburgh, UK
(2) Biomedical Sciences Program, University of California San Diego, La Jolla, USA
(3) Department of Pharmaceutical Sciences, University of California, Irvine, USA
SynopsisSynopsis
There is an urgent need to identify new targets and drugs to comThere is an urgent need to identify new targets and drugs to combatbat
trypanosomatid pathogenstrypanosomatid 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.Editing is catalyzed by multi protein complexes, the editosomes.
A key component of editosomes is RNA editing ligase 1 (REL1).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 a no close REL1 homologs in the host and thus this enzymeThere a no close REL1 homologs in the host and thus this enzyme
represents a target of potential high efficacy and specificity.represents a target of potential high efficacy and specificity.
Molecular dynamics simulations identified potential REL1 inhibitors by
virtual screening.
Initially, threethree napthalenenapthalene--based compounds that inhibited REL1based compounds that inhibited REL1
activity with ICactivity with IC5050 values in the low micromolar range werevalues in the low micromolar range were
experimentally validated.experimentally validated.
Subsequently, substructure searches against databasessubstructure searches against databases ofof
commercially available compoundscommercially available compounds utilising napthalene scaffolds
identified another 588 candidate moleculesidentified another 588 candidate molecules.
25 top-ranked candidates were subjected to biochemical assays
(adenylylation assay).
4 compounds inhibited REL1 with IC4 compounds inhibited REL1 with IC5050 values between 1 and 10values between 1 and 10 μμMM
Efforts are under way to improve the in vitro activity and selectivity of
these compounds and to establish their effect on trypanosomes.
AMP
..
(2)(2) Experimental validationExperimental validation in vitroin vitro
(3)(3) Enzymatic ICEnzymatic IC5050ss
Dose-Response # 1698
-2 -1 0 1 2 3
0
50
100
150
Log(10) concentration [M]
Activity[%]
IC50 (GraphPad v.5)
8.36 +/- 1.71µM
R2
0.9955
Dose-Response # 45609
-2 -1 0 1 2 3
0
50
100
150
Log(10) Concentration [M]
Activity[%]
IC50 (GraphPad v.5)
2.16 +/- 1.20µM
R2
0.9983
Dose-Response # 117079
0 1 2 3 4
0
50
100
150
Log (10)concentration [M]
Activity[%]
IC50 (GraphPad v.5)
78.54 +/-1.58µM
R2
0.9918
Dose-Response # 125908
0 1 2 3 4
0
50
100
150
Log (10)concentration [M]
Activity[%]
IC50 (GraphPad v.5)
46.61 +/- 3.02µM
R2
0.9675
Dose-Response Mordant Black
-2 -1 0 1 2 3
0
50
100
150
Log(10) Concentration [M]
Activity[%]
IC50 (GraphPad v.5)
1.59 +/- 1.10µM
R2
0.9998
Dose-Response #162535
-2 -1 0 1 2 3
0
50
100
150
Log(10) concentration [M]
Activity[%]
25 top25 top--ranked compounds initially screened @ 10ranked compounds initially screened @ 10 μμMM
& 100& 100 μμM using anM using an in vitroin vitro enzymatic (enzymatic (adenylylationadenylylation))
assayassay
6 compounds demonstrated inhibition of enzyme6 compounds demonstrated inhibition of enzyme
activityactivity
IC50 (GraphPad v.5)
1.53 +/- 1.17µM
R2
0.9991
DoseDose--response curves for these 6 compounds wereresponse curves for these 6 compounds were
established usingestablished using molaritymolarity titrationstitrations
ICIC5050 values were calculated using nonvalues were calculated using non--linear regressionlinear regression
analysis withanalysis with GraphPadGraphPad ®® software and are depictedsoftware and are depicted
oppositeopposite
(4)(4) TrypanocidalTrypanocidal ActivityActivity
Future workFuture work
1)1) Mordant black 25 (Sigma_4310) has a demonstrableMordant black 25 (Sigma_4310) has a demonstrable trypanocidaltrypanocidal effect on bloodstream formeffect on bloodstream form Trypanosome bruceiTrypanosome brucei
bruceibrucei cells (strain 427)cells (strain 427) in vitroin vitro
2)2) Thus, studies are under way to quantify the potential trypanocidal efficacy of this compound based on EC50 measurements
exploiting metabolic reduction of a commercial reazurin variant(Alamar blue®)
3)3) In the near future, high throughput screening of small compound libraries will be attempted using a (96 well plate) ‘mix &
measure’ format exploiting FRET-based detection of fluorophore-labeled substrates
(1) Virtual Screening Approach1) Virtual Screening Approach
A virtual screen over the NCI Diversity Set was performedA virtual screen over the NCI Diversity Set was performed with the high resolution TbREL1with the high resolution TbREL1
crystal structure to identify potential lead compounds.
AutoDock4 was used to dock ~2000 compounds to the crystal structAutoDock4 was used to dock ~2000 compounds to the crystal structure active siteure active site. The top 30
candidates (top 2%) were selected based on predicted binding energy and docking pose, re ranked
using the Relaxed Complex Scheme (RCS) and filtered based on drug-like properties.
Enzymatic assays identified two compounds with micromolar activiEnzymatic assays identified two compounds with micromolar activity against REL1.ty against REL1.
A subsequent hierarchical similarity search with the most active compound resulted in identification
of NSC #16209, a naphthalene-sulphonic acid compound.
A substructure search for relatedA substructure search for related napthalenesnapthalenes in databases of commercially availablein databases of commercially available
compounds resulted in identification of 588 compoundscompounds resulted in identification of 588 compounds, which were then ranked with AutoDock4 and
RCS according to their predicted average binding energy.
The 25 topThe 25 top--ranked compounds were selected for experimental testing.ranked compounds were selected for experimental testing.
Effect of 100uM solute (NCI compound) on BS # 427 cells versus DMSO (only)
solvent
162535
MordantBlack
45609
1698
125908
117079
0.4%DMSO
MediumOnly
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
7.00E+06
8.00E+06
NCI Drug #
NumberofCells_Coultercounter
162535
Mordant Black
45609
1698
125908
117079
0.4% DMSO
Medium Only
1% viable cells compared
with untreated controls
100µM drug
8 compounds
enzymatic
assays
2 compounds
similarity search
against full NCI data set,
RCS rescoring,
Lipinski
NCI 16209
Lipinski
rules
enzymatic
assays
substructure search
for related scaffolds in
HIT2LEAD, ZINC, NCI,
Sigma etc.
588 compounds
AD4 docking
45 compounds
RCS
25 compounds
8 compounds
enzymatic
assays
enzymatic
assays
2 compounds
similarity search
against full NCI data set,
RCS rescoring,
Lipinski
NCI 16209
Lipinski
rules
enzymatic
assays
enzymatic
assays
substructure search
for related scaffolds in
HIT2LEAD, ZINC, NCI,
Sigma etc.
588 compounds
AD4 docking
45 compounds
RCS
25 compounds
DMSO
#45577
# 45609
#75908
# 162535
# 1698
#8674
#12363
#42067
#117079
# 16209
0
20
40
60
80
100
120
NCI #
MeanSpecificSignal_%DMSO
DMSO
100uM 30uM
10uM
3uM
1uM
300nM 100nM 30nM
0
20
40
60
80
100
120
MeanSpecificSignal/DMSO_%
DMSO
100uM
30uM
10uM
3uM
1uM
300nM
100nM
30nM
0.8% +/-
0.1%
1.7% +/-
0.2%
7.3% +/-
1.4%
34.4% +/-
2.0%
67.4% +/-
3.1%
IC50 (GraphPad v.5)
2.16 +/- 1.20µM
R2
0.9983
Viability of BS #427 (Try brucei brucei ) cells after 3 days
incubation with Mordant Black Titration series
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
7.00E+06
8.00E+06
100uM
MB
10uM
MB
1uM MB 100nM
MB
0.4%
DMSO
Medium
Only
Mordant Black Titration
Cellcount_Coulter
counter](https://image.slidesharecdn.com/rnaeditingasadrugtargetidentificationofinhibitorsofrel1bsp210-170405085102/85/rna-editing-as-a-drug-target-identification-of-inhibitors-of-rel-1-bsp-210-1-320.jpg)
Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Rna editing as a drug target identification of inhibitors of rel 1 bsp 210
Similar to Rna editing as a drug target identification of inhibitors of rel 1 bsp 210 (20)
More from Laurence Dawkins-Hall
More from Laurence Dawkins-Hall (20)
Recently uploaded
Recently uploaded (20)
Rna editing as a drug target identification of inhibitors of rel 1 bsp 210
- 1. RNA editing as a drug target inRNA editing as a drug target in Trypanosoma bruceiTrypanosoma brucei:: Identification of inhibitors of the essential enzyme REL1Identification of inhibitors of the essential enzyme REL1 Laurence HallLaurence Hall11 , Jacob D. Durrant, Jacob D. Durrant22 , Rommie Amaro, Rommie Amaro33 , Achim Schnaufer, Achim Schnaufer11 (1) Institute of Immunology & Infection Research, University of Edinburgh, UK (2) Biomedical Sciences Program, University of California San Diego, La Jolla, USA (3) Department of Pharmaceutical Sciences, University of California, Irvine, USA SynopsisSynopsis There is an urgent need to identify new targets and drugs to comThere is an urgent need to identify new targets and drugs to combatbat trypanosomatid pathogenstrypanosomatid 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.Editing is catalyzed by multi protein complexes, the editosomes. A key component of editosomes is RNA editing ligase 1 (REL1).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 a no close REL1 homologs in the host and thus this enzymeThere a no close REL1 homologs in the host and thus this enzyme represents a target of potential high efficacy and specificity.represents a target of potential high efficacy and specificity. Molecular dynamics simulations identified potential REL1 inhibitors by virtual screening. Initially, threethree napthalenenapthalene--based compounds that inhibited REL1based compounds that inhibited REL1 activity with ICactivity with IC5050 values in the low micromolar range werevalues in the low micromolar range were experimentally validated.experimentally validated. Subsequently, substructure searches against databasessubstructure searches against databases ofof commercially available compoundscommercially available compounds utilising napthalene scaffolds identified another 588 candidate moleculesidentified another 588 candidate molecules. 25 top-ranked candidates were subjected to biochemical assays (adenylylation assay). 4 compounds inhibited REL1 with IC4 compounds inhibited REL1 with IC5050 values between 1 and 10values between 1 and 10 μμMM Efforts are under way to improve the in vitro activity and selectivity of these compounds and to establish their effect on trypanosomes. AMP .. (2)(2) Experimental validationExperimental validation in vitroin vitro (3)(3) Enzymatic ICEnzymatic IC5050ss Dose-Response # 1698 -2 -1 0 1 2 3 0 50 100 150 Log(10) concentration [M] Activity[%] IC50 (GraphPad v.5) 8.36 +/- 1.71µM R2 0.9955 Dose-Response # 45609 -2 -1 0 1 2 3 0 50 100 150 Log(10) Concentration [M] Activity[%] IC50 (GraphPad v.5) 2.16 +/- 1.20µM R2 0.9983 Dose-Response # 117079 0 1 2 3 4 0 50 100 150 Log (10)concentration [M] Activity[%] IC50 (GraphPad v.5) 78.54 +/-1.58µM R2 0.9918 Dose-Response # 125908 0 1 2 3 4 0 50 100 150 Log (10)concentration [M] Activity[%] IC50 (GraphPad v.5) 46.61 +/- 3.02µM R2 0.9675 Dose-Response Mordant Black -2 -1 0 1 2 3 0 50 100 150 Log(10) Concentration [M] Activity[%] IC50 (GraphPad v.5) 1.59 +/- 1.10µM R2 0.9998 Dose-Response #162535 -2 -1 0 1 2 3 0 50 100 150 Log(10) concentration [M] Activity[%] 25 top25 top--ranked compounds initially screened @ 10ranked compounds initially screened @ 10 μμMM & 100& 100 μμM using anM using an in vitroin vitro enzymatic (enzymatic (adenylylationadenylylation)) assayassay 6 compounds demonstrated inhibition of enzyme6 compounds demonstrated inhibition of enzyme activityactivity IC50 (GraphPad v.5) 1.53 +/- 1.17µM R2 0.9991 DoseDose--response curves for these 6 compounds wereresponse curves for these 6 compounds were established usingestablished using molaritymolarity titrationstitrations ICIC5050 values were calculated using nonvalues were calculated using non--linear regressionlinear regression analysis withanalysis with GraphPadGraphPad ®® software and are depictedsoftware and are depicted oppositeopposite (4)(4) TrypanocidalTrypanocidal ActivityActivity Future workFuture work 1)1) Mordant black 25 (Sigma_4310) has a demonstrableMordant black 25 (Sigma_4310) has a demonstrable trypanocidaltrypanocidal effect on bloodstream formeffect on bloodstream form Trypanosome bruceiTrypanosome brucei bruceibrucei cells (strain 427)cells (strain 427) in vitroin vitro 2)2) Thus, studies are under way to quantify the potential trypanocidal efficacy of this compound based on EC50 measurements exploiting metabolic reduction of a commercial reazurin variant(Alamar blue®) 3)3) In the near future, high throughput screening of small compound libraries will be attempted using a (96 well plate) ‘mix & measure’ format exploiting FRET-based detection of fluorophore-labeled substrates (1) Virtual Screening Approach1) Virtual Screening Approach A virtual screen over the NCI Diversity Set was performedA virtual screen over the NCI Diversity Set was performed with the high resolution TbREL1with the high resolution TbREL1 crystal structure to identify potential lead compounds. AutoDock4 was used to dock ~2000 compounds to the crystal structAutoDock4 was used to dock ~2000 compounds to the crystal structure active siteure active site. The top 30 candidates (top 2%) were selected based on predicted binding energy and docking pose, re ranked using the Relaxed Complex Scheme (RCS) and filtered based on drug-like properties. Enzymatic assays identified two compounds with micromolar activiEnzymatic assays identified two compounds with micromolar activity against REL1.ty against REL1. A subsequent hierarchical similarity search with the most active compound resulted in identification of NSC #16209, a naphthalene-sulphonic acid compound. A substructure search for relatedA substructure search for related napthalenesnapthalenes in databases of commercially availablein databases of commercially available compounds resulted in identification of 588 compoundscompounds resulted in identification of 588 compounds, which were then ranked with AutoDock4 and RCS according to their predicted average binding energy. The 25 topThe 25 top--ranked compounds were selected for experimental testing.ranked compounds were selected for experimental testing. Effect of 100uM solute (NCI compound) on BS # 427 cells versus DMSO (only) solvent 162535 MordantBlack 45609 1698 125908 117079 0.4%DMSO MediumOnly 0.00E+00 1.00E+06 2.00E+06 3.00E+06 4.00E+06 5.00E+06 6.00E+06 7.00E+06 8.00E+06 NCI Drug # NumberofCells_Coultercounter 162535 Mordant Black 45609 1698 125908 117079 0.4% DMSO Medium Only 1% viable cells compared with untreated controls 100µM drug 8 compounds enzymatic assays 2 compounds similarity search against full NCI data set, RCS rescoring, Lipinski NCI 16209 Lipinski rules enzymatic assays substructure search for related scaffolds in HIT2LEAD, ZINC, NCI, Sigma etc. 588 compounds AD4 docking 45 compounds RCS 25 compounds 8 compounds enzymatic assays enzymatic assays 2 compounds similarity search against full NCI data set, RCS rescoring, Lipinski NCI 16209 Lipinski rules enzymatic assays enzymatic assays substructure search for related scaffolds in HIT2LEAD, ZINC, NCI, Sigma etc. 588 compounds AD4 docking 45 compounds RCS 25 compounds DMSO #45577 # 45609 #75908 # 162535 # 1698 #8674 #12363 #42067 #117079 # 16209 0 20 40 60 80 100 120 NCI # MeanSpecificSignal_%DMSO DMSO 100uM 30uM 10uM 3uM 1uM 300nM 100nM 30nM 0 20 40 60 80 100 120 MeanSpecificSignal/DMSO_% DMSO 100uM 30uM 10uM 3uM 1uM 300nM 100nM 30nM 0.8% +/- 0.1% 1.7% +/- 0.2% 7.3% +/- 1.4% 34.4% +/- 2.0% 67.4% +/- 3.1% IC50 (GraphPad v.5) 2.16 +/- 1.20µM R2 0.9983 Viability of BS #427 (Try brucei brucei ) cells after 3 days incubation with Mordant Black Titration series 0.00E+00 1.00E+06 2.00E+06 3.00E+06 4.00E+06 5.00E+06 6.00E+06 7.00E+06 8.00E+06 100uM MB 10uM MB 1uM MB 100nM MB 0.4% DMSO Medium Only Mordant Black Titration Cellcount_Coulter counter