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Indole-pyridinyl-ethanones as novel inhibitors of indoleamine-2,3dioxygenase (IDO), a promising target for anti-cancer immunotherapy Eduard Dolušić,a Pierre Larrieu,b Sébastien Blanc,c Laurence Moineaux,a Frédéric Sapunaric,d Jenny Pouyez,a Delphine Colette,c Graeme Fraser,c Vincent Stroobant,b Luc Pilotte,b Didier Colau,b Jean-Marie Frère,d Bernard Masereel,a Benoît Van den Eyndeb, Johan Wouters,a and Raphaël Frédéricka* aDrug design and Discovery Center, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium ; bLudwig Institute for Cancer Research, Université Catholique de Louvain, 74 Avenue Hippocrate, 1200 Brussels, Belgium ; cEuroscreen SA, 47 Rue Adrienne Boland, 6041 Gosselies, Belgium ; dCentre d'Ingénierie des Protéines, Université de Liège, Allée du 6 août, 4000 Liège, Belgium E-mail : [email protected] Introduction Detailed binding analysis of 1a IDO is an extrahepatic, tryptophane (Trp) metabolizing enzyme that catalyzes the initial and ratelimiting step along the kynurenine pathway. 0.7 0.6 1/Vmax 0.5 Trp metabolism results in a local Trp depletion that severely affects the proliferation of T lymphocytes and is thereby profoundly immunosuppressive. Recently, the team of Van den Eynde and colleagues showed that many human tumours express IDO in a constitutive manner.1 IDO thus noticeably protects foreign cells against immune rejection. Based on its implication in immunosuppression, and particularly in tumors,2 IDO clearly represents an attractive target for the development of inhibitors.3 The most frequently used IDO inhibitor, 1-methyl-tryptophane (1MT) has a reported Ki of 34 µM. A phase 1 clinical trial is currently investigating the safety, toxicity, pharmacokinetics and efficacy of 1MT in subjects with advanced malignancies. Although more potent IDO inhibitors were recently reported,4,5,6 only a limited number of different scaffolds are available. Representative inhibitors are derived from the indole moiety (1-MT, brassinin and methylthiohydantoine-tryprtophane (MTHTrp)), the naphtoquinone nucleus (annulin B or C and exiguamine A), or the phenylimidazole (PIM), a weak IDO inhibitor. O 0.4 0.3 0.2 0.1 0 -250 -200 -150 -100 -50 0 50 -0.1 [Inhibiteur] µM Possible pharmacomodulations for preliminary SARs OH HN S S substitution of the indole in the 1-, 4and 5-position through introduction of small goups such as halogens, methyl, methoxy or nitro moieties S NH NMe NH2 appraise the importance of the ketone function through replacement of the ethanone linker with an ethylene, a hydroxyethylene or an amide O N Me N Me MTH-Trp 1MT O O N H Brassinin O OH O O O O 100 1a shows the best graphical match to an uncompetitive inhibition mode with a Ki value around 190 µM O OH O O O O Annulin B Annulin C O N N O Chemical Synthesis and Biological Activities NH2 O O HO N H O N N (d) NH Exiguamine A PIM Scheme 18. (i), LDA, THF / hexanes, -78 to 0°C, 1 h; (ii) THF / hexanes, 0°C to rt, 16-24 h; (iii), H2NNH2, KOH, (CH2OH)2, mW, 1h then aq. NH4Cl; (iv) HCO2NH4, Pd black, MeOH, r. t, 3 days; (v) SOCl2, D, 15 min; 3-aminopyridine, DIPEA, THF, 0°C -> r. t, 30 min. The two three-dimensional structures of IDO, in complex with PIM and the cyanide ion (CN¯), provide important results for the structure-based drug design of novel IDO inhibitors.7 As the enzyme active site is now well known, it represents a very good tool to undertake a direct approach of receptor-based drug discovery and design. Pocket A Pocket B Biological activities of the newly designed IDO inhibitors This preliminary SAR suggests, in agreement with the docking study reported above, that only rather small and lipophilic groups such as halogens are tolerated in the 5-position. Nmethylation of the indole is also tolerated In the present work, we performed a virtual screen to identify novel IDO inhibitors. From the few scaffolds that display IDO inhibition, preliminary SARs were investigated around the keto-indole scaffolds. Virtual screening (i) (ii) (iii) (iv) Replacement of the ethanone linker with an ethylene (2) or even a hydroxylethylene (3) totally suppresses the IDO inhibitory potency thus confirming the importance of the ketone pharmacophore for IDO inhibition in this series. Only its replacement with an amide (4) afforded a compound that is still active against IDO. Lipinski-style rules Goldscore > 50 Cscore ≥ 4, (iv) Visual analysis and commercial availability Concerning the ability of these derivatives to inhibit tryptophan degradation in cells expressing murine IDO, 1c, 1d and 1e are the most potent derivatives with ~25% inhibition of tryptophane degradation at 20 µM. With the best hits being as potent as 1MT, these data corroborate the potency of this series for further structure-based design effort. The biological activity of the 39 compounds was evaluated using a colorimetric in vitro IDO inhibition assay.1 CN N 90 N 80 70 N N O (1a) 60 50 40 30 20 10 0 zi nc zi 002 nc 7 6 zi 010 33 nc 8 1 1 zi 002 36 n 3 3 zi c00 77 nc 6 0 2 0 10 zi 030 34 nc 8 3 1 zi 001 57 nc 5 1 0 55 zi 432 63 n 3 zi c00 38 nc 3 3 6 zi 000 59 nc 8 7 3 zi 000 55 nc 7 9 9 zi 052 82 nc 3 0 4 zi 015 72 nc 9 5 9 zi 001 00 nc 1 1 7 zi 024 59 nc 9 5 2 zi 010 42 nc 7 9 6 zi 010 09 nc 5 0 2 zi 006 35 nc 1 5 7 zi 005 31 nc 7 8 1 zi 004 95 nc 4 6 8 zi 004 87 nc 4 9 0 zi 002 37 nc 7 6 6 zi 002 84 nc 7 7 6 zi 002 10 nc 7 6 5 zi 002 98 nc 3 6 7 zi 002 70 nc 7 2 2 zi 002 61 nc 4 3 0 47 zi 009 02 nc 9 zi 04 02 nc 0 1 9 zi 020 96 nc 2 6 3 zi 016 28 nc 8 5 9 zi 004 95 nc 4 2 8 zi 015 87 nc 9 9 9 zi 046 00 nc 6 1 7 zi 041 84 nc 0 0 0 zi 045 31 nc 6 2 6 zi 004 58 nc 4 4 8 zi 003 87 nc 8 9 7 zi 043 47 nc 2 8 08 33 76 83 31 02 1M T IDO inhibition (%) @ [I] = 100 µM Discussion and conclusion H N 100 In conclusion, a virtual screen combining various filters including high-throughput docking was used to search for new IDO inhibitors.9-10 From the 39 final compounds identified and assayed, six derivatives displayed an inhibitory potency > 30% at a concentration of 100µM, 2 of them possessing more than 50% IDO inhibition at this concentration. Detailed kinetics revealed an uncompetitive inhibition profile for the best hit 1a. Its binding mode inside the IDO binding cleft was evaluated by means of docking and revealed essential features responsible for the IDO inhibition potency in this series. Preliminary SARs around 1a corroborated this putative binding orientation and support the interest of this series for further drug design effort. Bibliography 1) Uyttenhove, C. et al. Nat Med 2003, 9, 1269-1274; (2) Löb, S. et al. Nat Rev Cancer 2009, 9, 445-452; (3) Macchiarulo, A. et al. Amino Acids 2009, 37, 219-229; (4) Röhrig, U. et al. J Med Chem 2010, 53, 1172-1189; (5) Kumar, S. et al. J Med Chem 2008, 51, 4968-4977; (6) Yue, E. et al. J Med Chem 2009, 52, 7364-7367; (7) Sugimoto, H. PNAS 2006, 103, 2611-2616; (8) Sundberg, R. J Org Chem 1978, 43, 4859-4865; (9) Dolusic, et al. Bioorg Med Chem 2011, 19(4), 1550-61; (10) Dolusic, et al. Eur J Med Chem 2011, 46, 3058-65 This work is supported in part by the FNRS and Biowin (Cantol : convention n°5678). RF is greatly indebted to the Belgian “Fonds de la Recherche Scientifique – FNRS” for the award of a postdoctoral research grant.