(i) Lipinski-style rules - (ii) Docking in the hTDO model (GOLD) - (iii) Scoring : - Goldscore
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-(i) Lipinski-style rules - (ii) Docking in the hTDO model (GOLD) - (iii) Scoring : - Goldscore - Chemscore - ASP - (iii) Ranking by ligand efficiency - (iv) Visual analysis and selection of 49 molecules Tryptophan 2,3-dioxygenase (TDO) inhibitors : Identification of new scaffolds using virtual screening Laurence Moineaux , a Caroline Charlier, a Eduard Dolušić, a Pierre Larrieu, b Luc Pilotte, b Didier Colau, b Vincent Stroobant, b Moreno Galleni, c Bernard Masereel, a Benoît Van den Eynde b Johan Wouters, a Raphaël Frédérick a a Drug Design and Discovery Center, University of Namur (FUNDP), 61 Rue de Bruxelles, B-5000 Namur, Belgium; b Ludwig Institute for Cancer Research, de Duve Institute, Université Catholique de Louvain, 74 Avenue Hippocrate B-1200 Brussels, Belgium; c Center for Protrein Engineering (CIP), University of Liège, Institut de Chimie B6, Sart-Tilman, Liège, Belgium [email protected] ; [email protected] Acknowledgements L.M. thanks the Fonds de la Recherche Scientifique-FNRS for the award of a Télévie grant (7.4.543.07F). C.C. and R.F. are greatly indebted to the FRS-FNRS for the award of a postdoctoral fellowship. 8. Discussion and Conclusions 9. References • (1) Batabyal and Yeh (2007), “Human Tryptophan Dioxygenase: A comparaison to Indoleamine 2,3- Dioxygenase”, Jacs, 129 (50), 15690-15701 • (2) Zhang and al. (2007), “Crystal structure and mechanisme of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis”, Biochemistry, 46, 145-155 • (3) Forouhar and al. (2006), “Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase”, PNAS, 104, 473-478 • (4) Van den Eynde, B., and al., (2010), WO2010008427. • (5) Salter and al. (1994), “The effect of an inhibitor of tryptophan 2,3-dioxygenase and a combined inhibitor of tryptophan 2,3-dioxygenase and 5-HT reuptake in the rat”, Neuropharmacology, 34, 217-227 • (6) Madge and al., (1996), “Novel tryptophan dioxygenase inhibitors and combined tryptophan dioxygenase/5-HT reuptake inhibitors”, Bioorganic and Medicinal Chemistry Letters, 6(7), 857-860 • (7) Zhang and al. (2007), “Crystal structure and mechanisme of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis”, Biochemistry, 46, 145-155 • (8) Forouhar and al. (2006), “Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase”, PNAS, 104, 473-478 In conclusion, a virtual screen combining various filter including high-throughput docking in a 3-D model of hTDO was used to search for novel TDO inhibitors. From the 49 find compounds identified and arranged, three compounds exhibited > 50% TDO inhibition at 100µM. Analogues were purchased in two series. Two novel hits were confirmed in the analogues. Theses modifications will be improve the inhibition of theses series. 1. Introduction Figure 1: model of hTDO 2. Objectives Fragment-like library 65640 structures TDO 32,5 kD Purification by IMAC MW 9 10 11 12 13 14 15 100 25 20 150 250 kD 75 50 37 15 A 450ml culture yielded 3ml of pure rmTDO (25mg/ml). TDO 375µM in tris (R =408/280=1.68) 0 0.5 1 1.5 2 2.5 3 3.5 4 215 265 315 365 415 465 515 565 w avelenght(nm ) Absorbance Soret band (a) (b) 24 compounds display a percentage of inhibition greater than 50% at 100µM Figure 2: (a) 5111103 and (b) 5212151 docked into the model of hTDO. 3. Virtual screening flowstart 4. TDO inhibition Like IDO, TDO catalyses the oxidative cleavage of the L-trp indole ring to form N-formyl-L-kynurenine (1) . TDO is an homotetrameric enzyme (2-3) . Located exclusively in the liver (1) . Expressed in many tumor cells (4) . It prevents tumor surveillance by the immune system (4) . It prevents tumor rejection by locally degrading tryptophan (4) . Only one series of compounds reported so far and characterized by a (fluoro)indole scaffold substituted in the 3-position by a pyridinyl-vinyl side chain (5-6) . N H NH 2 H CO 2 H NHCHO O NH 2 H CO 2 H IDO or TDO L-Tryptophan N-formyl-L-Kynurenine O 2 Overexpression and purification of rmTDO O N H O O OH NH 2 O NH 2 NH 2 O OH O NH 2 O OH N N CH 3 CH 3 N H O O H NH 2 TCA 30% P-DMAB 4% Measurement of the absorbance at 490 nm 37°C – 10min 37°C – 30min Premix = Ascorbic Acid, methylene blue and phosphate buffer L-tryptophan N-formyl-L-kynurenine L-Kynurenine Schiff basis TDO •DMSO •Phosphate Buffer pH=7.5 •Catalase •hemin •Premix Fluorimetric assay (9) 5. Structural analysis 6. Purchase of analogues (a) (b) Result s N H N F 49 molecules identified To date, the 3-D coordinates of TDO from Ralstonia metallidurans (rmTDO) (7) and Xanthomonas Campestris (xcTDO) (8) only have been reported. As these two bacterial strains share a high sequence identity with the human form, this constitutes an excellent template to build a 3-D model of the human TDO. The aim of this work is to use this model in structure-bond virtual screnning to discover novel inhibitors of this enzyme. N N H N N 75% 62% 63% Compounds 5111103 -7 -6 -5 -4 -3 0.0000 0.0005 0.0010 0.0015 0.0020 log[inh] Vitesse IC50= 14,5µM [9,9 à 21,4]µM 40%@200µM Cellular assay(UCL) Cellular assay (UCL) NI@200µM 5212151 -7 -6 -5 -4 -3 -0.0002 0.0000 0.0002 0.0004 0.0006 0.0008 log[inh] Vitesse IC50= 80,7µM [46,7 à 139,5]µM Compounds 33 analogues purchased 68 analogues purchased N N H N N 63% 75% O N H 2 N NH 2 N S 5619119 -6 -5 -4 -3 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 log[inh] Vitesse N N H N OH 7. Interesting analogues IC50= 126,7µM [97,8 à 164,3]µM IC50= 52,9µM [45,6 à 61,3]µM speed speed Two novel hits were confirmed among the analogues. Several pharmocomodulations will be realized to improve these analogues. For the first series, the unsubstituted pyridin group in R1. For the R2 position, some substitution were better like –OMe or –COOH in 4 and 5 position on the pyridin group. For the second series, a –NH group in R1, a sulphur in R2, a cyano group in R3 and R4, a amino group in R5 and finally a furan in R6. Theses modifications will be improve the inhibition of theses series. R6 R3 R4 R2 R5 R1 " N H N R1 R2 speed speed
description
Tryptophan 2,3-dioxygenase (TDO) inhibitors : Identification of new scaffolds using virtual screening Laurence Moineaux , a Caroline Charlier, a Eduard Dolušić , a Pierre Larrieu, b Luc Pilotte, b Didier Colau, b Vincent Stroobant, b - PowerPoint PPT Presentation
Transcript of (i) Lipinski-style rules - (ii) Docking in the hTDO model (GOLD) - (iii) Scoring : - Goldscore
-(i) Lipinski-style rules
- (ii) Docking in the hTDO model (GOLD)
- (iii) Scoring :- Goldscore- Chemscore- ASP
- (iii) Ranking by ligand efficiency
- (iv) Visual analysis and selection of 49 molecules
Tryptophan 2,3-dioxygenase (TDO) inhibitors : Identification of new scaffolds using virtual screening
Laurence Moineaux,a Caroline Charlier,a Eduard Dolušić,a Pierre Larrieu,b Luc Pilotte,b Didier Colau,b Vincent Stroobant,b Moreno Galleni,c Bernard Masereel,a Benoît Van den Eyndeb Johan Wouters,a Raphaël Frédéricka
a Drug Design and Discovery Center, University of Namur (FUNDP), 61 Rue de Bruxelles, B-5000 Namur, Belgium; b Ludwig Institute for Cancer Research, de Duve Institute, Université Catholique de Louvain, 74 Avenue Hippocrate B-1200 Brussels, Belgium; c Center for Protrein Engineering (CIP), University of Liège, Institut de Chimie B6, Sart-Tilman, Liège, Belgium
[email protected]; [email protected]
AcknowledgementsL.M. thanks the Fonds de la Recherche Scientifique-FNRS for the award of a Télévie grant (7.4.543.07F). C.C. and R.F. are greatly indebted to the FRS-FNRS for the award of a postdoctoral fellowship.
8. Discussion and Conclusions 9. References• (1) Batabyal and Yeh (2007), “Human Tryptophan Dioxygenase: A comparaison to Indoleamine 2,3-Dioxygenase”, Jacs, 129 (50), 15690-15701• (2) Zhang and al. (2007), “Crystal structure and mechanisme of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis”, Biochemistry, 46, 145-155• (3) Forouhar and al. (2006), “Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase”, PNAS, 104, 473-478• (4) Van den Eynde, B., and al., (2010), WO2010008427.• (5) Salter and al. (1994), “The effect of an inhibitor of tryptophan 2,3-dioxygenase and a combined inhibitor of tryptophan 2,3-dioxygenase and 5-HT reuptake in the rat”, Neuropharmacology, 34, 217-227• (6) Madge and al., (1996), “Novel tryptophan dioxygenase inhibitors and combined tryptophan dioxygenase/5-HT reuptake inhibitors”, Bioorganic and Medicinal Chemistry Letters, 6(7), 857-860• (7) Zhang and al. (2007), “Crystal structure and mechanisme of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis”, Biochemistry, 46, 145-155• (8) Forouhar and al. (2006), “Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase”, PNAS, 104, 473-478• (9) Matin and al., (2006), “A fluorescence-based assay for indoleamine 2,3-dioxygenase”, Anal.Biochem., 349, 96-102
.
In conclusion, a virtual screen combining various filter including high-throughput docking in a 3-D model of hTDO was used to search for novel TDO inhibitors. From the 49 find compounds identified and arranged, three compounds exhibited > 50% TDO inhibition at 100µM. Analogues were purchased in two series. Two novel hits were confirmed in the analogues. Theses modifications will be improve the inhibition of theses series.
1. Introduction
Figure 1: model of hTDO
2. Objectives
Fragment-like library 65640 structures
TDO
32,5 kD
Purification by IMAC
MW 9 101112 13 1415
100
25 20
150 250 kD
75 50 37
15
A 450ml culture yielded 3ml of pure rmTDO (25mg/ml).
TDO 375µM in tris (R=408/280=1.68)
0
0.5
1
1.5
2
2.5
3
3.5
4
215 265 315 365 415 465 515 565
w ave lenght (nm )
Abs
orba
nce
Soret band
(a) (b)
24 compounds display a percentage of inhibition greater than 50% at 100µM
Figure 2: (a) 5111103 and (b) 5212151 docked into the model of hTDO.
3. Virtual screening flowstart
4. TDO inhibition
Like IDO, TDO catalyses the oxidative cleavage of the L-trp indole ring to form N-formyl-L-kynurenine (1).
TDO is an homotetrameric enzyme(2-3).
Located exclusively in the liver(1).
Expressed in many tumor cells(4).
It prevents tumor surveillance by the immune system(4).
It prevents tumor rejection by locally degrading tryptophan(4).
Only one series of compounds reported so far and characterized by a (fluoro)indole scaffold substituted in the 3-position by a pyridinyl-vinyl side chain(5-6).
NH
NH2H
CO2H
NHCHO
O
NH2H
CO2H
IDO or TDO
L-Tryptophan N-formyl-L-Kynurenine
O2
Overexpression and purification of rmTDO
O
NH
O
O
OH
NH2 O
NH2
NH2
O
OH
O NH2
O
OH
N
N
CH3
CH3
NH
O
OH
NH2
TCA 30%
P-DMAB 4%
Measurement of the absorbance at 490 nm
37°C – 10min 37°C – 30min
Premix = Ascorbic Acid, methylene blue and phosphate buffer
L-tryptophan N-formyl-L-kynurenine L-Kynurenine
Schiff basis
TDO
•DMSO •Phosphate Buffer pH=7.5•Catalase•hemin•Premix
Fluorimetric assay(9)
5. Structural analysis
6. Purchase of analogues
(a) (b)
Results
NH
N
F
49 molecules identified
To date, the 3-D coordinates of TDO from Ralstonia metallidurans (rmTDO)(7) and Xanthomonas Campestris (xcTDO)(8) only have been reported. As these two bacterial strains share a high sequence identity with the human form, this constitutes an excellent template to build a 3-D model of the human TDO. The aim of this work is to use this model in structure-bond virtual screnning to discover novel inhibitors of this enzyme.
N
NHN N
75%
62%
63%
Compounds
5111103
-7 -6 -5 -4 -30.0000
0.0005
0.0010
0.0015
0.0020
log[inh]
Vite
sse
IC50= 14,5µM[9,9 à 21,4]µM
40%@200µM
Cellular assay(UCL) Cellular assay (UCL)NI@200µM
5212151
-7 -6 -5 -4 -3-0.0002
0.0000
0.0002
0.0004
0.0006
0.0008
log[inh]
Vite
sse
IC50= 80,7µM[46,7 à 139,5]µM
Compounds
33 analogues purchased 68 analogues purchased
N
NHN N
63%75%
O
NH2
N
NH2
N
S
5619119
-6 -5 -4 -30.0000
0.0005
0.0010
0.0015
0.0020
0.0025
log[inh]
Vite
sse
N
NHN
OH
7. Interesting analogues
IC50= 126,7µM[97,8 à 164,3]µM
IC50= 52,9µM[45,6 à 61,3]µM
spee
dsp
eed
Two novel hits were confirmed among the analogues. Several pharmocomodulations will be realized to improve these analogues. For the first series, the unsubstituted pyridin group in R1. For the R2 position, some substitution were better like –OMe or –COOH in 4 and 5 position on the pyridin group. For the second series, a –NH group in R1, a sulphur in R2, a cyano group in R3 and R4, a amino group in R5 and finally a furan in R6. Theses modifications will be improve the inhibition of theses series.
R6R3R4
R2R5 R1"
NH
NR1 R2
spee
d
spee
d
