Journal of Medicinal Chemistry ARTICLE Figure 5. The crystallographically determined binding orientations of compounds 6a, 11e, 13e, and 29a overlaid in the active site of the TryR 1a complex structure. 1a is displayed in cyan, 6a in orange, 11e in magenta, 13e in green, and 29a in yellow. The 3,4-dihydroquinazoline and phenyl ring systems overlay well, while the more variable and ﬂexible N3-substituents point out into the active site opening in a variety of orientations. This represents the first report of a high resolution noncovalent small molecule protein X-ray crystal structure for TryR. In addition, the X-ray crystal structures of nonliganded TryR (2woi), TryR in complex with NADPH (2wov), and TryR in complex with trypanothione dithiol and NADPH (2wow) have been determined. All of the TryR structures reported herein are in complex with FAD. Ramachandran analyses of the structures show more than 99% of residues are in the allowed regions in all models. The few residues outside these regions correspond to Tyr45, which is known to reside in a strained loop region of all other TryR structures and Arg331, the main chain of which is also held in a strained conformation in some TryR structures. See the Supporting Information (sections 3.2 3.5) for a detailed dis- cussion of the comparison between the T. brucei TryR X-ray structures reported in this manuscript and other TryR structures previously reported in the literature. Examination of the TryR 1a complex revealed that the ligand binds in a region of the enzyme’s active site cleft that would normally be occupied by the Gly-I and spermidine moieties of T[S]2 (Figure 4D). Therefore, the observed binding mode is consistent with the mode of inhibition of 1a being competitive with respect to T[S]2 as described above. The most interesting feature of the TryR 1a structure is the observation that the enzyme undergoes an induced conforma- tional change upon inhibitor binding. This conformational change generates a new subpocket within the active site, which accommodates the C4-phenyl of 1a (Figure 4C). Previously, the active site of TryR was believed to be a rigid structure,40 formed by residues that belong to a congregation of α-helices. Accord- ingly, the substrate T[S]2 is highly ﬂexible and adopts a con- formation to accommodate the rigid active site. However, upon binding of 1a, the Met113 side chain moves approximately 5 Å outward into the active site, creating a new hydrophobic pocket. This subpocket is lined by Leu120, Phe114, Val33, Leu17, Ala12, Val9, Val31, and Leu48. The entrance to the subpocket is “guarded” by Trp21, Met113, and Tyr110. This induced ﬁt had not previously been predicted in TryR, and the signiﬁcant movement of the Met113 side chain clearly changes the proper- ties of the active site, thus aﬀecting the binding of T[S]2. The bound inhibitor not only sterically impedes the binding of the spermidine moiety of T[S]2 but also alters the shape of the hydrophobic patch that normally binds this substrate moiety. The orientation of 1a in the active site anchors it tightly in position; the large bromine atom pushes against one end of the cleft and Trp21 interacts with the N3 substitution (see below) acting as a physical barrier, preventing the inhibitor from locating further up the opposite side of the cleft. There is only one hydrogen bonding interaction with the protein from N1 to the Oε1 of Glu18 (2.7 Å). The bromine atom of 1a sits in an area of the active site cleft surrounded by Gly13, Ser14, Leu17, Gly49, Val53, and Tyr110. The catalytic Cys52 is situated at this end of this region and sits 6.7 Å from the C6-bromine. In the non- liganded structure, the area in which the bromine atom binds is instead occupied by a water molecule. The large size of the subpocket around the bromine atom is consistent with analogues 16e and 16f that contain bicyclic aromatic substituents at C6 retaining some, albeit reduced, potency (IC50 8.0 and 8.5 μM, respectively, Table 4). The C4-phenyl group of 1a protrudes into the hydrophobic region formerly occupied by Met113. The phenyl group sits at an angle of 108 to the plane of the dihydroquinazoline core. In this position, the phenyl group makes an edge face hydrophobic interaction with Tyr110, which has rotated 62 from the non- liganded structure. Although one of the weakest hydrophobic interactions,41 it still contributes binding energy to the inhibitor complex. The residues within van der Waals interacting distances (4 Å) from this phenyl group and likely to be participating in hydrophobic interactions include Met113, Trp21, Tyr110, Leu17, Phe114, and Leu120 (Figure 4A,B). The synthetic routes to substituted dihydroquinazolines that were utilized in this study (Scheme 1) result in analogues that are racemic at C4. However, the ligand binding mode described above allows only the 4S- enantiomer to eﬀectively occupy the induced-ﬁt subpocket created by the movement of Met113. The methyl acetate moiety of 1a is directed out into the active site cleft and sits 3.1 Å from Met113Sδ. It is within 3.5 Å of, and stacks against, Trp21, which has pivoted about Cδ1 to move the indole group 1.0 Å closer to the inhibitor when compared to the nonliganded structure. This helps to wedge the inhibitor in 6521 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530