The M-loop contributes to the catalytic pocket in human PDEs and is also involved in shaping the P-pocket of kinetoplastid PDEs and the related selectivity pocket of hPDE10, which both are exploited for inhibitor design [55C57]. adult population in the developed world. This study describes the single cyclic nucleotide-specific phosphodiesterase (PDE) of and assesses PDE inhibitors as a new generation of anti-giardial drugs. Methods An extensive search of the genome database identified a single gene coding for a class I PDE, GlPDE. The predicted protein sequence was analyzed to characterize its domain structure and catalytic domain. Enzymatic activity of GlPDE was established by complementation of a PDE-deficient strain, and enzyme kinetics were characterized in soluble yeast lysates. The potency of known PDE inhibitors was tested against the activity of recombinant GlPDE expressed in yeast and against proliferating trophozoites. Finally, the localization of epitope-tagged and Rabbit Polyclonal to KITH_HHV1C ectopically expressed GlPDE in cells was investigated. Results encodes a class I PDE. Catalytically important residues are fully conserved between GlPDE and human PDEs, but sequence differences between their catalytic domains suggest that designing PDE activity leads to a profound inhibition of parasite proliferation and that GlPDE is a promising target for developing novel anti-giardial drugs. Author summary Cellular signaling by the cyclic nucleotides cAMP and cGMP is ubiquitously found in organisms from human to unicellular parasites. Cyclic nucleotide-specific phosphodiesterases (PDEs) are pivotal regulators of these signaling processes and these enzymes represent important drug targets for a variety of diseases. Eleven PDE families are distinguished in humans and selective inhibition of a single human PDE family without targeting others is feasible. In parasites, interference in the signaling mechanism by PDE inhibition may be fatal. The diarrhea-causing parasite contains only one single PDE, named GlPDE. GlPDE activity is highly impaired by a Istaroxime range of PDE inhibitors, which also suppress parasite proliferation is a protozoan parasite that causes giardiasis, an intestinal disease with symptoms such as diarrhea, nausea, and malabsorption . Trophozoites are the disease-causing stage and colonize the upper small intestine of humans and other vertebrates. They form cysts, which are shed into the environment via the fecal route and which are then orally transmitted, mostly via contaminated water. Giardiasis occurs worldwide, predominantly in resource-poor countries with low standards of sanitation, and represents a major cause of non-bacterial diarrhea with 280 million symptomatic human cases every year . In developing countries, infection rates of 10% to 30% are common, though rates of 40% and higher have been reported in some instances [3,4]. Chronic or recurrent giardiasis in early childhood is associated with poor cognitive function and failure to thrive . Metronidazole (commercially known as Flagyl) and other nitroimidazoles are being used as a therapy of choice since the 1960s. However, resistance against metronidazole has been described [6,7]. Consequently, substitution therapies including the benzimidazole albendazole, the acridine derivative quinacrineor the aminoglycoside paromomycin, alone or in combination with metronidazole [8,9], are of increasing importance. New therapies are urgently needed because current treatments (i) depend on repeated dosing schedules (suboptimal for developing countries), (ii) have adverse effects, (iii) are ineffective in up to 20% of cases and (iv) clinical or laboratory-induced resistance has been reported for most of the current anti-giardial drugs [10,11]. Phosphodiesterases (PDEs) are key enzymes of cyclic nucleotide signaling. They constitute the only enzymes for hydrolyzing the signaling molecules cAMP and cGMP and thus are crucially important regulators of the temporal and spatial shape of the cyclic nucleotide signals. Three structurally distinct classes of PDEs have been described . Thereof only class I enzymes have been identified in protozoan parasites and their mammalian hosts so far. Human PDEs (hPDEs) comprise eleven class I families (hPDE1C11), Istaroxime which differ with respect to substrate-specificity, regulation and distribution in tissues as well as in intracellular Istaroxime compartments. The catalytic domains of class I PDEs are highly conserved at the level of their three-dimensional structures, though the different families talk about just 20C50% amino acidity sequence identity of their.