Selective Inhibition of Trypanosoma brucei 6-Phosphogluconate Dehydrogenase by High-Energy Intermediate and Transition-State Analogues Christophe Dardonville, † Eliana Rinaldi, ‡ Michael P. Barrett, § Reto Brun, # Ian H. Gilbert,* ,† and Stefania Hanau ‡ Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, United Kingdom; Dipartimento di Biochimica e Biologia Molecolare, Universita ´ di Ferrara, Via Luigi Borsari 46, 44100 Ferrara, Italy; IBLS, Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, United Kingdom; and Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland Received October 20, 2003 Two series of compounds were designed to mimic the transition state and high-energy intermediates (HEI) of the enzymatic reaction of 6-phosphogluconate dehydrogenase (6PGDH). Sulfoxide analogues (7-11) were designed to mimic the transition state during the oxidation of the substrate to 3-keto-6-phosphogluconate, an enzyme-bound intermediate of the enzyme. Hydroxamate and amide derivatives of D-erythronic acid were designed to mimic the 1,2-cis- enediol HEI of the 6PGDH reaction. These two series of compounds were assayed as competitive inhibitors of the Trypanosoma brucei and sheep liver enzymes, and their selectivity value (ratio sheep/parasite) was calculated. The sulfoxide transition-state analogues showed weak and selective inhibition of the T. brucei enzyme. The hydroxamic derivatives showed potent and selective inhibition of the T. brucei 6PGDH with a K i in the nanomolar range. Introduction Human African trypanosomiasis is an important disease that has recently become resurgent in Africa. The disease is caused by parasitic protozoa of the brucei group of the genus Trypanosoma. In our search for new antitrypanosomal agents, we decided to target 6-phos- phogluconate dehydrogenase (6PGDH), whose essential nature to growth of the bloodstream form of T. brucei has been recently demonstrated by RNA interference. 1 6PGDH is the third enzyme of the pentose phosphate pathway 2 (PPP), which generates NADPH and ribulose- 5-phosphate by oxidative decarboxylation of 6-phospho- gluconate (6PG) (Figure 1). Hence, 6PGDH helps to maintain a pool of NADPH, which serves to protect the parasite against oxidative stress, and it generates carbohydrate intermediates used in nucleotide and other biosynthetic pathways. We have previously reported the design and synthesis of noncarbohydrate substrate analogues of 6PGDH as potential inhibitors of this enzyme. 3,4 Such deoxy and/ or protected analogues of 6PG were designed as poten- tial inhibitors with improved pharmacokinetic proper- ties compared to phosphorylated carbohydrates, which are unlikely to possess good druglike characteristics due to metabolic instability and impermeability at biological membranes. However, such strategy afforded only a few weak inhibitors with K i values larger than the K m value of 6PG. 3,4 These results prompted us to use another approach: that of transition-state analogues and ana- logues of high-energy intermediates formed during the reaction. It has been postulated and proved in many cases that such analogues may be good enzyme inhibi- tors. This is because enzymes increase reaction rates by stabilizing the transition state and/or high-energy intermediates. 5,6 According to the mechanism of 6PGDH described in the literature, 7,8 there are two intermediate species in the enzyme reaction (Figure 1): a 3-keto-6PG entity and a putative 1,2-enediol high-energy intermediate (HEI). The recent report of some phosphate, phosphonate, and hydroxamate sugar derivatives showing selective inhi- bition of 6PGDH from T. brucei over the sheep liver enzyme, 9 and especially the powerful and selective inhi- bition displayed by 4-phospho-D-erythronate (K i(T.brucei) ) 0.13 μM and K i(sheep) ) 10.7 μM), reinforced the idea that analogues of the enediol intermediate could give potent inhibitors of 6PGDH. We present in this paper the design and synthesis of two series of inhibitors (i.e., sulfoxide and hydroxamate derivatives) mimicking the transition-state species of 6PGDH (Figure 1). Inhibitor Design. The “sulfoxide” analogues were designed to mimic the polarization of the CdO bond in the 3-keto-6PG species (Figure 1). Site-directed mu- tagenesis and crystallographic studies suggest that the carbonyl group of the 3-keto-6PG intermediate accepts a proton from the general acid of the enzymatic reaction (i.e., Lys183 and Glu190 of the sheep liver 6PGDH and Lys185 of the T. brucei enzyme), which facilitates the decarboxylation step. 10 Because 2-deoxy-3-keto-6PG was previously found to bind 6PGDH from T. brucei with high affinity and competitively inhibit the oxidation of 6PG, 11 we considered the preparation of 2,4-dideoxy sulfoxide TSA (11) in which the sulfoxide group would mimic the polarization of the CdO bond. Moreover, the tetrahedral geometry around the sulfur atom renders the C3-position more alike to an sp 3 hybridized carbon, such as in the proposed transition state (Figure 2). Some protected analogues and the thioether analogues (com- * Author to whom correspondence should be addressed [e-mail gilbertih@cf.ac.uk; fax +44 (0) 29 2087 4149]. † Cardiff University. ‡ Universita ´ di Ferrara. § University of Glasgow. # Swiss Tropical Institute. 3427 J. Med. Chem. 2004, 47, 3427-3437 10.1021/jm031066i CCC: $27.50 © 2004 American Chemical Society Published on Web 05/22/2004