title: Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase.
abstract: Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals-including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics-that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition.
  
  
  OBJECTIVE
  The stark differences between the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus in pathogenic protozoa, fungi, and viruses and those of their metazoan hosts highlight RTPase as a target for anti-infective drug discovery. Protozoan, fungal, and DNA virus RTPases belong to the triphosphate tunnel metalloenzyme family. This study shows that a protozoan RTPase, TbCet1 from Trypanosoma brucei, is essential for growth of the parasite in culture and identifies, via in vitro screening of chemical libraries, several classes of potent small-molecule inhibitors of TbCet1 phosphohydrolase activity.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Smith
authors.affiliation: Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York, USA.
authors.forename: C Kiong
authors.initials: CK
authors.lastname: Ho
authors.affiliation: Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York, USA.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Takagi
authors.affiliation: High Throughput Screening Core Facility, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology; D001081:Apyrase / Q000378:metabolism; D001665:Binding Sites; D002109:Caffeic Acids / Q000737:chemistry / Q000494:pharmacology; D020134:Catalytic Domain; D002934:Cinnamates / Q000737:chemistry / Q000494:pharmacology; D053630:Depsides / Q000737:chemistry / Q000494:pharmacology; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D005960:Glucosides / Q000737:chemistry / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D011794:Quercetin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D011801:Quinic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D034622:RNA Interference; D054852:Small Molecule Libraries / Q000737:chemistry; D014344:Trypanocidal Agents / Q000737:chemistry / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D000097123:Rosmarinic Acid
chemical_list: D017766:Acid Anhydride Hydrolases; D000975:Antioxidants; D001081:Apyrase; D002109:Caffeic Acids; D002934:Cinnamates; D053630:Depsides; D004791:Enzyme Inhibitors; D005707:Gallic Acid; D005960:Glucosides; D015800:Protozoan Proteins; D011794:Quercetin; D011801:Quinic Acid; D054852:Small Molecule Libraries; D014344:Trypanocidal Agents; D000097123:Rosmarinic Acid; C000611671:1,3,4,5-O-tetracaffeoylquinic acid; C000611672:5-galloylgalloylquinic acid; C023090:RNA triphosphatase; C110309:quercetin 3-O-glucopyranoside