Journal of Medicinal Chemistry ARTICLE Figure 6. Summary of the development of hit compound 1a by an initial chemistry-driven approach and subsequent structure-based inhibitor design strategy. The units for ligand eﬃciencies (Lig. eﬀ.) are kcal mol 1 per non-hydrogen atom. despite both sets of compounds having similar TryR inhibitory activities (Tables 3 and 5). The current level of antitrypanosomal activity and selectivity against mammalian cells is insuﬃcient to test these compounds in an animal model of infection. Therefore, continued develop- ment is required to identify a lead compound from this hit series. ’ CONCLUSIONS Large and open active site pockets such as that found in TryR are diﬃcult targets to eﬀectively inhibit with small drug-like molecules. To date, no high-resolution, noncovalent enzyme inhibitor complexes of TryR have been structurally characterized, further limiting attempts to develop potent, low molecular weight inhibitors. In this study, we have reported the crystal structures of T. brucei TryR in complex with a noncovalently bound inhibitor series based around a 3,4-dihydroquinazoline scaﬀold. In addition, the structure of T. brucei TryR in complex with the cofactors FAD, NADPH, and reduced trypanothione is presented. The results describe a novel and small hydrophobic pocket generated upon inhibitor binding that lies within a “privileged” region of the large active site pocket. It is anticipated that these results will facilitate the area of TryR inhibitor design and assist in the generation of further protein inhibitor complex structures which until now have relied mainly on in silico modeling and docking studies. Given the urgent need for new HAT therapeutics, we hope that the structural information reported herein will accelerate the development of TryR inhibi- tors and possibly identify new drug leads. In addition to the crystallographic studies, we have reported the identiﬁcation and characterization of a novel TryR inhibitor series based upon the 3,4-dihydroquinazoline ring system. From hit compounds 1a and 1b, a systematic SAR study has expanded the series to include >75 analogues (Tables 1 5, Figures 3 and 6, and Figure S3 of the Supporting Information). A chemistry- driven approach was used to identify compounds which were more potent than 1a in both the TryR biochemical and T. brucei cell assays (cf. 1a and 13h, Figure 6). However, the analogues developed by this strategy contained large N3-substitutions, resulting in high molecular weight inhibitors which were un- suitable for further optimization. The structural elucidation of ligand TryR complexes allowed us to subsequently adopt a structure-based inhibitor design approach, which was used to develop compounds with both improved TryR and T. brucei inhibitory activities (cf. 1a and 29a, Figure 6) while maintaining ligand eﬃciencies >0.3 kcal mol 1 per non-H atom (6k, 29a, Figure 6). As the dihydroquinazoline series underwent develop- ment, the mode of inhibition changed from competitive to mixed (cf. 1a and 13h/29a, Figure 6 and see Figure S2 of the Supporting Information). The development of a mixed inhibitor is potentially advantageous, as these are more resistant to substrate accumulation, a feature that could be important due to the high intracellular concentration of trypanothione. To our knowledge, compound 29a is the most potent, drug- like inhibitor of TryR reported to date. However, this series requires further optimization to improve its cellular potency and selectivity. Because of their high ligand eﬃciencies and low molecular weights, analogues 29a and 6k represent good starting 6524 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530