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Title: Optimization of a binding fragment targeting the “enlarged methionine pocket” leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1416585
Grant/Contract Number:
AC02-76SF00515
Resource Type:
Journal Article: Publisher's Accepted Manuscript
Journal Name:
Bioorganic and Medicinal Chemistry Letters
Additional Journal Information:
Journal Volume: 27; Journal Issue: 12; Related Information: CHORUS Timestamp: 2018-01-11 00:23:50; Journal ID: ISSN 0960-894X
Publisher:
Elsevier
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Huang, Wenlin, Zhang, Zhongsheng, Ranade, Ranae M., Gillespie, J. Robert, Barros-Álvarez, Ximena, Creason, Sharon A., Shibata, Sayaka, Verlinde, Christophe L. M. J., Hol, Wim G. J., Buckner, Frederick S., and Fan, Erkang. Optimization of a binding fragment targeting the “enlarged methionine pocket” leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors. United Kingdom: N. p., 2017. Web. doi:10.1016/j.bmcl.2017.04.048.
Huang, Wenlin, Zhang, Zhongsheng, Ranade, Ranae M., Gillespie, J. Robert, Barros-Álvarez, Ximena, Creason, Sharon A., Shibata, Sayaka, Verlinde, Christophe L. M. J., Hol, Wim G. J., Buckner, Frederick S., & Fan, Erkang. Optimization of a binding fragment targeting the “enlarged methionine pocket” leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors. United Kingdom. doi:10.1016/j.bmcl.2017.04.048.
Huang, Wenlin, Zhang, Zhongsheng, Ranade, Ranae M., Gillespie, J. Robert, Barros-Álvarez, Ximena, Creason, Sharon A., Shibata, Sayaka, Verlinde, Christophe L. M. J., Hol, Wim G. J., Buckner, Frederick S., and Fan, Erkang. Thu . "Optimization of a binding fragment targeting the “enlarged methionine pocket” leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors". United Kingdom. doi:10.1016/j.bmcl.2017.04.048.
@article{osti_1416585,
title = {Optimization of a binding fragment targeting the “enlarged methionine pocket” leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors},
author = {Huang, Wenlin and Zhang, Zhongsheng and Ranade, Ranae M. and Gillespie, J. Robert and Barros-Álvarez, Ximena and Creason, Sharon A. and Shibata, Sayaka and Verlinde, Christophe L. M. J. and Hol, Wim G. J. and Buckner, Frederick S. and Fan, Erkang},
abstractNote = {},
doi = {10.1016/j.bmcl.2017.04.048},
journal = {Bioorganic and Medicinal Chemistry Letters},
number = 12,
volume = 27,
place = {United Kingdom},
year = {Thu Jun 01 00:00:00 EDT 2017},
month = {Thu Jun 01 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.bmcl.2017.04.048

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  • Cited by 8
  • A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNA/sup fMet/). Incubation of the affinity-labeling tRNA/sup fMet/ derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNA/sup fMet/, but not by noncognate tRNA/sup Phe/. The covalentmore » complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.« less
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  • Initiator tRNA molecules modified at the 3{prime}-end and lacking either A{sub 76} (tRNA-C{sub 75}), the C{sub 75}-A{sub 76} (tRNA-C{sub 74}), the C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 73}), or the A{sub 73}-C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 72}) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTS{sub T}), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C{sub 75}ox, tRNA-A{sub 73}ox, and tRNA-A{sub 72}ox) abolished both the isotopic ({sup 32}P)PP{sub i}ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the variousmore » tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C{sub 75}ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C{sub 75}. The peptides of MTS{sub T} labeled with either tRNA-C{sub 75}ox or tRNA-C{sub 74}ox were identified. In a previous work all these peptides but one (peptide D) had been already found labeled upon MTS{sub T} incubation with ({sup 14}C)tRNA-A{sub 76}ox. According to the crystallographic structure of MTS{sub T}, the labeled residues K335, K61, K142, K147, and K149 are within a sphere of about 5.5-{angstrom} radius. The present results therefore argue for a marked flexibility of the 3{prime}-end of the enzyme-bound tRNA, enabling it to contact any of the identified reacting residues. Such a cluster of basic amino acids may reflect ionic requirements in the guiding of the negatively charged CCA arm of tRNA toward enzyme-bound methionyl-adenylate.« less