(i) Lipinski-style rules - (ii) Docking in the hTDO model (GOLD) - (iii) Scoring : - Goldscore

1. Tryptophan 2,3-dioxygenase (TDO) inhibitors : Identification of new scaffolds using virtual screening Laurence Moineaux,a Caroline Charlier,aEduard Dolušić,aPierre Larrieu,b Luc Pilotte,b Didier Colau,b Vincent Stroobant,b Moreno Galleni,c Bernard Masereel,aBenoît 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; bLudwig 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 Laurence.moineaux@fundp.ac.be; Raphael.frederick@fundp.ac.be 15 14 13 12 11 10 9 MW 250 kD 150 100 75 50 37 25 20 15 Purification by IMAC TDO 32,5 kD Soret band A 450ml culture yielded 3ml of purermTDO(25mg/ml). 75% 63% 62% 1. Introduction Results • 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). Compounds Compounds IDO or TDO IC50= 14,5µM [9,9à 21,4]µM IC50= 80,7µM [46,7 à 139,5]µM O2 N-formyl-L-Kynurenine L-Tryptophan speed speed Cellular assay (UCL) Cellular assay(UCL) NI@200µM 40%@200µM 5. Structural analysis (a) (b) 2. Objectives 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. 37°C – 10min 37°C – 30min TDO 3. Virtual screening flowstart TCA 30% Fragment-like library 65640 structures • (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 N-formyl-L-kynurenine L-Kynurenine L-tryptophan Figure 2: (a) 5111103 and (b) 5212151 docked into the model of hTDO. • DMSO • Phosphate Buffer pH=7.5 • Catalase • hemin • Premix 6. Purchase of analogues P-DMAB 4% Figure 1: model of hTDO Measurement of the absorbance at 490 nm 63% 75% 33 analogues purchased 68 analogues purchased 24 compounds display a percentage of inhibition greater than 50% at 100µM 49 molecules identified Schiff basis Premix = Ascorbic Acid, methylene blue and phosphate buffer 4. TDO inhibition Overexpression and purification of rmTDO (a) (b) 7.Interesting analogues IC50= 126,7µM [97,8 à 164,3]µM speed Fluorimetric assay(9) IC50= 52,9µM [45,6 à 61,3]µM 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. 9. References 8. Discussion and Conclusions • (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. 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.