Membrane-bound pyrophosphatases (mPPases) are large homodimeric integral membrane proteins found in archaea, bacteria, plants and protist parasites. These enzymes couple the hydrolysis of pyrophosphate (PPi) to the pumping of H+ or Na+ ions, generating an electrochemical potential across a membrane. They are essential for many organisms since PPi is a by-product from many biosynthetic pathways and too high concentrations may disturb physiological reactions. Although mPPases can be found in many pathogenic parasites, such as Leishmania spp. (leishmaniasis), Toxoplasma gondii (toxoplasmosis), Trypanosoma spp. (trypanosomiasis) and Plasmodium spp. (malaria), there are no homologous proteins in humans, thereby making them promising drug targets. In addition the structures of the Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata mPPase were recently solved. Our aim is to develop novel protozoan mPPase inhibitors capable of disrupting the essential electrochemical potential of the pathogenic parasites in order to decrease their viability. So far only phosphorus-containing inhibitors of mPPases have been reported, limiting their therapeutic utility. Through sequential screening efforts we found novel organic inhibitors of the Thermotoga maritima PPase and our best hit compound inhibited the enzyme activity uncompetitively with an IC50 of 1.7 μM. The binding mode was solved by X-ray crystallography at 3.7 Å resolution together with the substrate analogue imidodiphosphate. As the hit compound binds to the protein monomer near the exit channel, it forms a hydrophobic clamp that locks the enzyme conformation in the closed state thus preventing hydrolysis and sodium pumping activity.

Structure of membrane bound pyrophosphatase from Thermotoga maritima in complex with imidodiphosphate and N-[(2-aminobenzo[d]thiazol-6-yl)methyl]-1H-indole-2-carboxamide

2019 Spring National Meeting

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