Crystallization of a Mos1 transposase-inverted-repeat DNA complex: biochemical and preliminary crystallographic analyses

A complex formed between Mos1 transposase and its inverted-repeat DNA has been crystallized. The crystals diffract to 3.25 Å resolution and exhibit monoclinic (P2(1)) symmetry, with unit-cell parameters a = 120.8, b = 85.1, c = 131.6 Å, β = 99.3°. The X-ray diffraction data display noncrystallographic twofold symmetry and characteristic dsDNA diffraction at ∼3.3 Å. Biochemical analyses confirmed the presence of DNA and full-length protein in the crystals. The relationship between the axis of noncrystallographic symmetry, the unit-cell axes and the DNA diffraction pattern are discussed. The data are consistent with the previously proposed model of the paired-ends complex containing a dimer of the transposase

Inhibitors of Leishmania mexicana phosphoglycerate mutase identified by virtual screening and verified by inhibition studies

Cofactor-independent phosphoglycerate mutase has been proposed as a therapeutic target for the treatment of trypanosomatid diseases. In this paper, we report the identification of compounds that could potentially be developed as selective inhibitors of cofactor-independent phosphoglycerate mutase from Leishmania mexicana (LmiPGAM). Virtual screening was used in this search, as well as compounds identified by high-throughput screening. A ligand-based virtual screen programme, ultra fast shape recognition with atom types (UFSRAT), was used to screen for compounds resembling the substrate/product, before a structure-based approach was applied using AutoDock 4 and AutoDock Vina in a consensus docking scheme. In this way eight selected compounds were identified. In addition, three compounds from the Library of Pharmacologically Active Compounds (LOPAC) were selected from the published results of high-throughput screening of this library. The inhibitory effects of these compounds were tested at a fixed concentration of 1 mM. The results showed that seven compounds inhibited LmiPGAM activity and of these, two compounds (one each from high-throughput and virtual screening) showed substantial inhibition (i.e. 14% and 49% remaining activity, respectively). Taken together, the findings from this study indicate that these compounds have potential as novel inhibitors that specifically target LmiPGAM

Structure- and ligand-based virtual screening identifies new scaffolds for inhibitors of the oncoprotein MDM2

A major challenge in the field of ligand discovery is to identify chemically useful fragments that can be developed into inhibitors of specific protein-protein interactions. Low molecular weight fragments (with molecular weight less than 250 Da) are likely to bind weakly to a protein's surface. Here we use a new virtual screening procedure which uses a combination of similarity searching and docking to identify chemically tractable scaffolds that bind to the p53-interaction site of MDM2. The binding has been verified using capillary electrophoresis which has proven to be an excellent screening method for such small, weakly binding ligands

Demonstration of the anthelmintic potency of marimastat in the Heligmosomoides polygyrus rodent model

In the course of a structure based drug discovery program the known anticancer candidate marimastat was uncovered as a potent inhibitor of an enzyme in nematode cuticle biogenesis. It was shown to kill Caenorhabditis elegans, and the sheep parasites Haemonchus contortus and Teladorsagia circumcinta via an entirely novel nematode-specific pathway, specifically by inhibiting cuticle-remodelling enzymes that the parasites require for the developmentally essential moulting process. This discovery prompted an investigation of the compound's effect on Heligmosomoides polygyrus parasites in a mouse model of helminth infection. Mice were administered the drug via oral gavage daily from day of infection for a period of 2 wk. A second group received the drug via intra-peritoneal implantation of an osmotic minipump for 4 wk. Control groups were administered identical volumes of water by oral gavage in both cases. Counts of H. polygyrus faecal egg and larval load showed that marimastat effected a consistent and significant reduction in egg laying, and a consistent but minor reduction in adult worm load when administered every day, starting on the first day of infection. However, the drug failed to have any significant effect on egg counts or worm burdens when administered to mice with established infections. Therefore, marimastat does not appear to show promise as an anthelmintic in gastrointestinal nematode infections, although other metalloproteases such as batimastat may prove more effective

Structures of pyruvate kinases display evolutionarily divergent allosteric strategies

The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (k(cat)/S(0.5)). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (k(cat)/S(0.5)) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors

Structural and kinetic characterisation of Trypanosoma congolense pyruvate kinase

Trypanosoma are blood-borne parasites and are the causative agents of neglected tropical diseases (NTDs) affecting both humans and animals. These parasites mainly rely on glycolysis for their energy production within the mammalian host, which is why trypanosomal glycolytic enzymes have been pursued as interesting targets for the development of trypanocidal drugs. The structure-function relationships of pyruvate kinases (PYKs) from trypanosomatids (Trypanosoma and Leishmania) have been well-studied within this context. In this paper, we describe the structural and enzymatic characterization of PYK from T. congolense (TcoPYK), the main causative agent of Animal African Trypanosomosis (AAT), by employing a combination of enzymatic assays, thermal unfolding studies and X-ray crystallography