A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity

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.Peer reviewe

Integral membrane pyrophosphatases : A novel drug target for human pathogens?

Membrane-integral pyrophosphatases (mPPases) are found in several human pathogens, including Plasmodium species, the protozoan parasites that cause malaria. These enzymes hydrolyze pyrophosphate and couple this to the pumping of ions (H+ and/or Na+) across a membrane to generate an electrochemical gradient. mPPases play an important role in stress tolerance in plants, protozoan parasites, and bacteria. The solved structures of mPPases from Vigna radiata and Thermotoga maritima open the possibility of using structure-based drug design to generate novel molecules or repurpose known molecules against this enzyme. Here, we review the current state of knowledge regarding mPPases, focusing on their structure, the proposed mechanism of action, and their role in human pathogens. We also summarize different methodologies in structure-based drug design and propose an example region on the mPPase structure that can be exploited by these structure-based methods for drug targeting. Since mPPases are not found in animals and humans, this enzyme is a promising potential drug target against livestock and human pathogens. © 2016, Adrian Goldman, et al.Peer reviewe

Crystal structure of the N-terminal domain of the trypanosome flagellar protein BILBO1 reveals a ubiquitin fold with a long structured loop for protein binding

Trypanosoma brucei is a protist parasite causing sleeping sickness and nagana in sub-Saharan Africa. T. brucei has a single flagellum whose base contains a bulblike invagination of the plasma membrane called the flagellar pocket (FP). Around the neck of the FP on its cytoplasmic face is a structure called the flagellar pocket collar (FPC), which is essential for FP biogenesis. BILBO1 was the first characterized component of the FPC in trypanosomes. BILBO1's N-terminal domain (NTD) plays an essential role in T. brucei FPC biogenesis and is thus vital for the parasite's survival. Here, we report a 1.6-Å resolution crystal structure of TbBILBO1-NTD, which revealed a conserved horseshoe-like hydrophobic pocket formed by an unusually long loop. Results from mutagenesis experiments suggested that another FPC protein, FPC4, interacts with TbBILBO1 by mainly contacting its three conserved aromatic residues Trp-71, Tyr-87, and Phe-89 at the center of this pocket. Our findings disclose the binding site of TbFPC4 on TbBILBO1-NTD, which may provide a basis for rational drug design targeting BILBO1 to combat T. brucei infections.Alliance française contre les maladies parasitaire

Discovery of Membrane-Bound Pyrophosphatase Inhibitors Derived from an Isoxazole Fragment

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.Peer reviewe

Correction: BILBO1 is a scaffold protein of the flagellar pocket collar in the pathogen Trypanosoma brucei.

[This corrects the article DOI: 10.1371/journal.ppat.1004654.]

Exploration of Pyrazolo[1,5-a]pyrimidines as Membrane-Bound Pyrophosphatase Inhibitors

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.Peer reviewe

Interaction between the flagellar pocket collar and the hook complex <i>via</i> a novel microtubule-binding protein in <i>Trypanosoma brucei</i>

<div><p><i>Trypanosoma brucei</i> belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body <i>via</i> the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of <i>in vitro</i> and <i>in vivo</i> approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.</p></div