Browsing by Subject "ACIDOCALCISOMES"

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  • Vidilaseris, Keni; Kellosalo, Juho; Goldman, Adrian (2018)
    Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes. They occur in bacteria, archaea, plants, and protist parasites. As they are essential in protist parasites and there are no homologous proteins in animals and humans, these enzymes represent an excellent drug target for treating protistal diseases. Experimental screening to find drug candidates is an important step to discover new hit compounds. For that, a cheap, simple, and robust assay is needed. Here we report the application of the molybdenum blue reaction method for a medium throughput microplate activity assay of the hyperthermophilic bacterium Thermotoga maritima mPPase and the possible application of the assay to screen inhibitors of membrane-bound pyrophosphatases.
  • Vidilaseris, Keni; Kiriazis, Alexandros; Turku, Ainoleena; Khattab, Ayman Abdelnaby Shaaban; Johansson, Niklas G; Leino, Teppo Olavi; Kiuru, Paula Sinikka; Boije af Gennäs, Per Gustav; Meri, Seppo Kalevi; Yli-Kauhaluoma, Jari Tapani; Xhaard, Henri Guillaume Michel; Goldman, Adrian (2019)
    Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of beta-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.
  • Johansson, Niklas G; Turku, Ainoleena; Vidilaseris, Keni; Dreano, Loic; Khattab, Ayman; Ayuso Perez, Daniel; Wilkinson, Aaron; Zhang, Yuezhou; Tamminen, Matti; Grazhdankin, Evgeni; Kiriazis, Alexandros; Fishwick, Colin W. G.; Meri, Seppo; Yli-Kauhaluoma, Jari; Goldman, Adrian; Boije af Gennäs, Gustav; Xhaard, Henri (2020)
    Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in pathogenic protozoan parasites and lack human homologues, which makes them promising targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here, we explore an isoxazole fragment hit, leading to the discovery of small mPPase inhibitors with 6-10 mu M IC50 values in the Thermotoga maritima test system. Promisingly, the compounds retained activity against Plasmodium falciparum mPPase in membranes and inhibited parasite growth.
  • Johansson, Niklas G; Dreano, Loic; Vidilaseris, Keni; Khattab, Ayman; Liu, Jianing; Lasbleiz, Arthur; de Castro Ribeiro, Orquidea Marilia; Kiriazis, Alexandros; Boije af Gennäs, Gustav; Meri, Seppo; Goldman, Adrian; Yli-Kauhaluoma, Jari; Xhaard, Henri (2021)
    Inhibition of membrane-bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure-activity relationships around a new scaffold having a pyrazolo[1,5-a]pyrimidine core. The most promising pyrazolo[1,5-a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay.
  • Shah, Nita R.; Wilkinson, Craig; Harborne, Steven P. D.; Turku, Ainoleena; Li, Kun-Mou; Sun, Yuh-Ju; Harris, Sarah; Goldman, Adrian (2017)
    Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PPi) to the pumping of Na+, H+, or both these ions across a membrane. Recently solved structures of the Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata mPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5-6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures. Furthermore, creating an apo model of TmPPase by removing ligands from the TmPPase: IDP: Na structure in MD simulations resulted in increased dynamics in loop 5-6, which results in this loop moving to uncover the active site, suggesting that interactions between loop 5-6 and the imidodiphosphate and its associated Mg2+ are important for holding a loop-closed conformation. We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase. (C) 2017 Author(s).