title: Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.
abstract: Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS) is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs) has been developed with IC50 values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general 'ATP-engaging' binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Cho Yeow
authors.initials: CY
authors.lastname: Koh
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Allan B
authors.initials: AB
authors.lastname: Wetzel
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Will J
authors.initials: WJ
authors.lastname: de van der Schueren
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America; Department of Chemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Shibata
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Ranae M
authors.initials: RM
authors.lastname: Ranade
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Jiyun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Zhongsheng
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: J Robert
authors.initials: JR
authors.lastname: Gillespie
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Buckner
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Christophe L M J
authors.initials: CL
authors.lastname: Verlinde
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Erkang
authors.initials: E
authors.lastname: Fan
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Wim G J
authors.initials: WG
authors.lastname: Hol
mesh_terms: D000981:Antiprotozoal Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D008718:Methionine-tRNA Ligase / Q000737:chemistry*; D008958:Models, Molecular; D021261:Parasitic Sensitivity Tests; D011487:Protein Conformation; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D014508:Urea / Q000737:chemistry*
chemical_list: D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D014508:Urea; D008718:Methionine-tRNA Ligase
references.citation: Expert Opin Investig Drugs. 2007 May;16(5):573-93
references.pmid: 17461733
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: Lancet. 2010 Jan 9;375(9709):148-59
references.pmid: 19833383
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Antimicrob Agents Chemother. 2003 Jun;47(6):1784-9
references.pmid: 12760849
references.citation: PLoS Med. 2008 Feb;5(2):e55
references.pmid: 18303943
references.citation: Nature. 2013 Feb 7;494(7435):121-4
references.pmid: 23263184
references.citation: Science. 2005 Jul 15;309(5733):416-22
references.pmid: 16020726
references.citation: Annu Rev Biochem. 2000;69:617-50
references.pmid: 10966471
references.citation: J Med Chem. 2012 Jul 26;55(14):6342-51
references.pmid: 22720744
references.citation: Nat Struct Mol Biol. 2005 Oct;12(10):931-2
references.pmid: 16155581
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: Antimicrob Agents Chemother. 2011 May;55(5):1982-9
references.pmid: 21282428
references.citation: Antimicrob Agents Chemother. 2013 Jul;57(7):3021-8
references.pmid: 23587950
references.citation: Antimicrob Agents Chemother. 2009 Jan;53(1):86-94
references.pmid: 19015366
references.citation: Nat Struct Mol Biol. 2012 Jun 10;19(7):677-84
references.pmid: 22683997
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):12-21
references.pmid: 20057044
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Structure. 2012 Oct 10;20(10):1681-91
references.pmid: 22902861
references.citation: N Engl J Med. 2007 Sep 6;357(10):1018-27
references.pmid: 17804846
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Eur J Med Chem. 2011 Nov;46(11):5227-36
references.pmid: 21968372
references.citation: J Mol Biol. 1999 Dec 17;294(5):1287-97
references.pmid: 10600385
references.citation: Nat Chem Biol. 2012 Feb 12;8(3):311-7
references.pmid: 22327401
references.citation: Trends Biochem Sci. 2010 Oct;35(10):539-46
references.pmid: 20541943
references.citation: Curr Drug Targets Infect Disord. 2004 Dec;4(4):261-72
references.pmid: 15578968