Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (␣␤) 2 heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E B 232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue Glu B -232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg B -310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K i of 103.57 ؎ 18.96 nM for pterin-6-aldehyde.

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( K < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.

L eishmaniasis is one of the 17 neglected tropical diseases (NTDs) assigned by the World Health Organization (WHO). NTDs affect 1 billion people worldwide (1). The primary occurrences are in low-income countries in sub-Saharan Africa, Asia, and Latin America, but the Mediterranean countries of Europe are also concerned (2). Among the NTDs is the group of "most neglected diseases," affecting the poorest, mainly rural areas, including leishmaniases, sleeping sickness (African trypanosomiasis), and Chagas' disease (3). These three NTDs have the highest rates of death. However, the NTD drug discovery pipeline is almost empty, thus leading to a lack of efficient and safe drugs (2, 4). Because of climate warming and tourism, the occurrence of leishmaniasis is also reported in states around the Mediterranean Sea (1).Leishmaniasis is caused by more than 20 species of protozoan parasites belonging to the genus Leishmania. The parasite life cycle is characterized by two morphological stages: extracellular flagellated promastigotes, occurring in the insect vector, and intracellular aflagellated amastigotes, occurring in the mammalian host. The promastigotes are transmitted by an insect bite into the skin of the host, where they are internalized by macrophages, dendritic cells, neutrophils, and fibroblasts and differentiate into amastigotes residing and replicating in parasitophorous vacuoles of these phagocytes. The parasites disseminate through the lymphatic and vascular systems. During the blood meal of an (uninfected) sand fly, amastigotes are transmitted back from the infected mammalian host to the insect vector and differentiate again into promastigotes (5, 6).The clinical outcome of leishmaniasis depends on the complex interactions between the virulence characteristics of the infecting species and the type of immune response of the host. There are three clinical forms: cutaneous, mucocutaneous, and visceral leishmaniases (6).Concerning the treatment of leishmaniasis, it is obvious that new drugs must circumvent the limitations of currently established chemotherapies, i.e., toxicity, long courses of treatment, the frequent need for parenteral administration, high costs in countries where the disease is endemic, and the emergence of resistance. Therefore, it is important not only to test and apply combinations of existing drugs to avoid resistance but also to develop new potential leishmanicidal compounds with alternative mechanisms, as well as vaccination strategies (7,8).Cysteine proteases (CPs) of parasites such as Plasmodium, Trypanosoma, and worms are druggable targets for developing a new promising strategy for chemotherapy based on protease inhi-

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