Indole-pyridinyl-ethanones as novel inhibitors of indoleamine-2,3- dioxygenase (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 Eynde b, Johan Wouters, a and Raphaël Frédérick a * a Drug design and Discovery Center, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium ; b Ludwig Institute for Cancer Research, Université Catholique de Louvain, 74 Avenue Hippocrate, 1200 Brussels, Belgium ; c Euroscreen SA, 47 Rue Adrienne Boland, 6041 Gosselies, Belgium ; d Centre d'Ingénierie des Protéines, Université de Liège, Allée du 6 août, 4000 Liège, Belgium Virtual screening Bibliography 1) Uyttenhove, C. et al. Nat Med 2003, 9, ; (2) Löb, S. et al. Nat Rev Cancer 2009, 9, ; (3) Macchiarulo, A. et al. Amino Acids 2009, 37, ; (4) Röhrig, U. et al. J Med Chem 2010, 53, ; (5) Kumar, S. et al. J Med Chem 2008, 51, ; (6) Yue, E. et al. J Med Chem 2009, 52, ; (7) Sugimoto, H. PNAS 2006, 103, ; (8) Sundberg, R. J Org Chem 1978, 43, ; (9) Dolusic, et al. Bioorg Med Chem 2011, 19(4), ; (10) Dolusic, et al. Eur J Med Chem 2011, 46, 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. 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. (d) Introduction  IDO is an extrahepatic, tryptophane (Trp) metabolizing enzyme that catalyzes the initial and rate- limiting step along the kynurenine pathway.  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 Detailed binding analysis of 1a Scheme 1 8. (i), LDA, THF / hexanes, -78 to 0°C, 1 h; (ii) THF / hexanes, 0°C to rt, h; (iii), H 2 NNH 2, KOH, (CH 2 OH) 2, mW, 1h then aq. NH 4 Cl; (iv) HCO 2 NH 4, Pd black, MeOH, r. t, 3 days; (v) SOCl 2, D, 15 min; 3-aminopyridine, DIPEA, THF, 0°C -> r. t, 30 min. Discussion and conclusion Chemical Synthesis and Biological Activities  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 (MTH- Trp)), the naphtoquinone nucleus (annulin B or C and exiguamine A), or the phenylimidazole (PIM), a weak IDO inhibitor.  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 (i)Lipinski-style rules (ii)Goldscore > 50 (iii)Cscore ≥ 4, (iv) (iv)Visual analysis and commercial availability The biological activity of the 39 compounds was evaluated using a colorimetric in vitro IDO inhibition assay. 1 (1a) 1a shows the best graphical match to an uncompetitive inhibition mode with a Ki value around 190 µM Possible pharmacomodulations for preliminary SARs  substitution of the indole in the 1-, 4- and 5-position through introduction of small goups such as halogens, methyl, methoxy or nitro moieties  appraise the importance of the ketone function through replacement of the ethanone linker with an ethylene, a hydroxyethylene or an amide 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. N- methylation of the indole is also tolerated Replacement of the ethanone linker with an ethylene (2) or even a hydroxyl- ethylene (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. 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. Biological activities of the newly designed IDO inhibitors In conclusion, a virtual screen combining various filters including high-throughput docking was used to search for new IDO inhibitors 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.