ARTICLE pubs.acs.org/jmc Dihydroquinazolines as a Novel Class of Trypanosoma brucei Trypanothione Reductase Inhibitors: Discovery, Synthesis, and Characterization of their Binding Mode by Protein Crystallography Stephen Patterson,†,§ Magnus S. Alphey,†,§ Deuan C. Jones,† Emma J. Shanks,† Ian P. Street, Julie A. Frearson,† Paul G. Wyatt,† Ian H. Gilbert,† and Alan H. Fairlamb*,† ‡ †Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K. ‡The Walter and Eliza Hall Institute of Medical Research, 1G Royal Pde, Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), Parkville VIC-3052, Australia bS Supporting Information ABSTRACT: Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaﬀold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the ﬁrst report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in eﬀect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay. ’ INTRODUCTION The neglected disease human African trypanosomiasis (HAT), or African sleeping sickness, is caused by two subspecies of the protozoan parasite Trypanosoma brucei. If not successfully treated, HAT is fatal, and it is estimated to be responsible for about 30000 deaths each year in sub-Saharan Africa.1 There is no eﬀective vaccine for HAT, and the current therapeutics used to treat the disease possess a number of limitations including high toxicity, eﬃcacy against only a single T. brucei subspecies, and the emergence of resistance.1,2 Undoubtedly, there is an urgent need for the identiﬁca- tion and development of new antitrypanosomal compounds. T. brucei and a number of related parasites utilize a redox metabolism based around the dithiol trypanothione [N1,N8- bis(glutathionyl)spermidine] (Figure 1), which is absent in humans.3 Therefore, the enzymes involved in this redox path- way are considered to be attractive targets for the development of new antitrypanosomal drugs.4,5 One component of the try- panothione-based redox pathway is trypanothione reductase (TryR, EC 1.8.1.12), an NADPH-dependent disulﬁde oxidor- eductase, which is responsible for reducing trypanothione disul- ﬁde (T[S]2) to dihydro-trypanothione (T[SH]2) (Figure 1).3 This reduction is analogous to that performed by glutathione reductase (GR, EC 1.8.1.7) in most organisms, including hu- mans. However, the active sites of the two enzymes have opposite net charges, facilitating the development of inhibitors selective for TryR over GR.6 Trypanothione provides reducing equiva- lents either directly or via tryparedoxin for several essential enzymes that protect T. brucei parasites from oxidative damage.7 Genetic studies have demonstrated that TryR activity is essential for T. brucei parasites to grow in culture and to be infective in an animal disease model.8 Therefore, TryR is a genetically validated drug target, one important criterion in drug target assessment.9,10 In addition, TryR inhibitors with diverse core-scaﬀolds have been reported.9,11 19 However, many of the reported inhibitors are of low potency (TryR IC50 >1 μM), or are not drug-like (e.g., mol wt >500) and therefore do not represent suitable starting points for the development of antitrypanosomal drugs. This requirement for high molecular weight compounds to eﬀectively inhibit TryR may in part be a consequence of Received: March 18, 2011 Published: August 18, 2011 r 2011 American Chemical Society 6514 dx.doi.org/10.1021/jm200312v | J. Med. Chem. 2011, 54, 6514–6530