This might be due to off\target effects, physical chemical properties of the compounds, or general cytotoxic effects, as indicated by cytotoxicity observed against L\6 rat myoblast cells (Table?2) and human being macrophages (data not shown), leading to low selectivity indices. Human being African trypanosomiasis threatens millions of people in about 20 sub\Saharan countries in Africa, with an estimated annual number of cases Midodrine hydrochloride Midodrine hydrochloride between 50?000 and 70?000 and an annual mortality close to 25?000.4 The emergence of multidrug\resistant parasite strains, in addition to limited available chemotherapies, demand the urgent development of new and effective medicines with novel mechanisms of actions. and offer several potential target enzymes that are implicated in pathogenesis and sponsor cell invasion, including a number of essential and closely Midodrine hydrochloride related cysteine proteases.5 The largest subfamily among them are the papain\like cysteine proteases (clan CA, family C1). In parasites communicate the cysteine protease rhodesain, a cathepsin?L\like hydrolase. Rhodesain is definitely involved in the degradation of parasitic and intracellularly transferred sponsor proteins, and is responsible for general proteolytic activity in all existence phases of the organism.13,?14 Cysteine protease inhibitors have been shown to kill African trypanosomes in vitro and in animal models.15 Various types of facipain\2 and rhodesain inhibitors have been developed in the last years, mainly based on screening methods.16,?17 However, we felt our experience in structure\based design would enable us to obtain new potent and selective inhibitors without the need for testing.18 We began our investigations based on the first X\ray crystal constructions of falcipain\2, available since 2006 (Protein Data Bank (PDB) codes: 1YVB, 2GHU, 3BPF),19C21 and of rhodesain published only recently in 2009 2009 and 2010 (PDB codes: 2P7U, 2P86).22,?23 Both falcipain\2 and rhodesain share the common features of clan CA cysteine proteases with the classical papain fold consisting of two distinct domains. Superimposition of the constructions of both enzymes shows a high degree of analogy in their overall fold, with highest conservation observed for the catalytic website (Number?1?a). Sequence alignment of the catalytic domains resulted in the task of both enzymes to the cathepsin?L\like subfamily.17 In both constructions, the catalytic dyad (falcipain\2: Cys?42, His?174; rhodesain: Cys?25, His?162) is embedded inside a channel\like junction between the two domains with a highly conserved peptide sequence (Number?1?b). The active site stretches further into the apolar S2 pocket with a strong preference for hydrophobic substituents.10,?12 Previous work suggested the S2 pocket is the key determinant of substrate specificity in papain\like cysteine proteases.24 Open in a separate window Number 1 a)?Superimposition of X\ray crystal constructions of falcipain\2 (cyan, PDB code: 2GHU) and rhodesain (magenta, PDB code: 2P86); b)?Superimposition of selected amino acids in the active site of falcipain\2 (C?skeleton: cyan) and rhodesain (C?skeleton: magenta). Color code: O?atoms: red, N?atoms: blue, S?atoms: yellow. The general structure of cysteine protease inhibitors consists of prevalently an electrophilic moiety to form a reversible, covalent thioimidate intermediate MGC79399 with the catalytic cysteine. We opted, specifically, for inhibitors featuring a nitrile residue as the electrophilic head group. More than 30 nitrile\comprising pharmaceuticals are prescribed for a variety of medicinal indications, and several are in medical development.25,?26 Unsurprisingly, nitriles are a well established class of cysteine protease inhibitors.27,?28 Oballa et?al. hypothesized the increased electrophilicity of the nitrile moiety could effect the reversibility of enzymeCinhibitor complex formation.29 According to their determined reactivities, aryl nitriles, particularly pyrimidine and triazine nitriles, should possess the most reactive nitrile moieties. Herein, we describe the structure\based design, efficient synthesis, and biological evaluation of a new series of triazine nitrile inhibitors to explore the binding properties of falcipain\2 and rhodesain. Guided by molecular modeling, we propose a binding model showing the accommodation of the different vectors in the apolar pouches of the active site. The inhibitors were tested against closely related human being and viral cysteine proteases, as well as a serine protease, to investigate their general selectivity. Additionally, in vitro activity against and parasites and cytotoxicity was analyzed. Computer\aided modeling using the MAB push field within MOLOC30 was applied to design small drug\like molecules to occupy the active Midodrine hydrochloride site. We recognized a diamino\substituted triazine as appropriate central scaffold to position vectors for the S1, S2, and S3 binding pouches and direct the thioimidate adduct into the stabilizing oxyanion opening (Number?2?a). Occupancy of the various pockets (Number?2?b) was subsequently optimized to gain high binding potency. Open in a separate window Number 2 a)?Schematic representation of the triazine nitrile core, stabilization of the thioimidate in the oxyanion hole, and positioning of the vectors; b)?Simplified diagram of the active site of falcipain\2 showing the catalytic dyad, the oxyanion hole, and the S1, S2, and S3 pockets. Active site analysis and 3D modeling exposed that a morpholine residue could act as suitable substituent to address the flat, mainly solvent\revealed S1 pocket in falcipain\2. For occupancy of the large and primarily hydrophobic S2 pocket, we recognized a 4\(and rhodesain from (Table?1), respectively, in standard fluorescence\based assays (see the Supporting Info).32,?33 For falcipain\2, investigation of substituents for the mostly solvent\exposed S1 pocket revealed a preference for the initially designed morpholine group, whereas cyclopropylamine derivative 8.