title: Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei adenosine transporter 1 (TbAT1) and the proposal of a structural model for the protein.
abstract: The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1(-/-) T. brucei line. Out of a set of six reported SNPs from a reported 'resistance allele', none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [(3)H]-adenosine and [(3)H]-diminazene, identical to the tbat1(-/-) control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [(3)H]-diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, UK.
authors.forename: Jane C
authors.initials: JC
authors.lastname: Munday
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Department of Laboratory Technology, University of Cape Coast, Cape Coast, Ghana.
authors.forename: Daniel N A
authors.initials: DN
authors.lastname: Tagoe
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Department of Medical Biochemistry, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Nigeria.
authors.forename: Anthonius A
authors.initials: AA
authors.lastname: Eze
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Jessica A M
authors.initials: JA
authors.lastname: Krezdorn
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Karla E
authors.initials: KE
authors.lastname: Rojas López
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Department of Biology, College of Science, Aljouf University, Sakaka, Saudi Arabia.
authors.forename: Abdulsalam A M
authors.initials: AA
authors.lastname: Alkhaldi
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Fiona
authors.initials: F
authors.lastname: McDonald
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Still
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Faculty of Medical Sciences, Taif University, Taif, Saudi Arabia.
authors.forename: Khalid J
authors.initials: KJ
authors.lastname: Alzahrani
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Department of Chemistry and Chemical Biology, 417 Egan Research Center, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
authors.forename: Luca
authors.initials: L
authors.lastname: Settimo
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Harry P
authors.initials: HP
authors.lastname: De Koning
mesh_terms: D000483:Alleles; D004133:Diminazene / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics; D007700:Kinetics; D008549:Melarsoprol / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation; D033703:Nucleoside Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D021261:Parasitic Sensitivity Tests; D010419:Pentamidine / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide; D054730:Protein Interaction Domains and Motifs; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics*
chemical_list: D033703:Nucleoside Transport Proteins; D014344:Trypanocidal Agents; C444719:adenosine transporter; D010419:Pentamidine; D004133:Diminazene; D008549:Melarsoprol