scholarly journals Urea-Based Inhibitors of Trypanosoma brucei Methionyl-tRNA Synthetase: Selectivity and in Vivo Characterization

2012 ◽  
Vol 55 (14) ◽  
pp. 6342-6351 ◽  
Author(s):  
Sayaka Shibata ◽  
J. Robert Gillespie ◽  
Ranae M. Ranade ◽  
Cho Yeow Koh ◽  
Jessica E. Kim ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Trna Synthetase ◽  
In Vivo Characterization ◽  
Methionyl Trna Synthetase

scholarly journals Structure-based drug design against Trypanosoma brucei methionyl-tRNA synthetase

2014 ◽  
Vol 70 (a1) ◽  
pp. C708-C708
Author(s):  
Cho Yeow Koh ◽  
Jasmine Nguyen ◽  
Sayaka Shibata ◽  
Zhongsheng Zhang ◽  
Ranae Ranade ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Trypanosoma Brucei ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protozoan Parasite ◽  
Trna Synthetase ◽  
Structure Based Drug Design ◽  
Chemical Structures ◽  
Ic50 Values ◽  
Methionyl Trna Synthetase

Infection by the protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, commonly known as sleeping sickness. The disease is fatal without treatment; yet, current therapeutic options for the disease are inadequate due to toxicity, difficulty in administration and emerging resistance. Therefore, methionyl-tRNA synthetase of T. brucei (TbMetRS) is targeted for the development of new antitrypanosomal drugs. We have recently completed a high-throughput screening campaign against TbMetRS using a 364,131 compounds library in The Scripps Research Institute Molecular Screening Center. Here we outline our strategy to integrate the power of crystal structures with high-throughput screening in a drug discovery project. We applied the rapid crystal soaking procedure to obtain structures of TbMetRS in complex with inhibitors reported earlier[1] to approximately 70 high-throughput screening hits. This resulted in more than 20 crystal structures of TbMetRS·hit complexes. These hits cover a large diversity of chemical structures with IC50 values between 200 nM and 10 µM. Based on the solved structures and existing knowledge drawn from other in-house inhibitors, the IC50 value of the most promising hit has been improved. Further development of the compounds into potent TbMetRS inhibitors with desirable pharmacokinetic properties is on-going and will continue to benefit from information derived from crystal structures.

scholarly journals Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model

2020 ◽  
Vol 75 (5) ◽  
pp. 1218-1227
Author(s):  
Samantha A Michaels ◽  
Han-Wei Shih ◽  
Bailin Zhang ◽  
Edelmar D Navaluna ◽  
Zhongsheng Zhang ◽  
...  
Keyword(s):  
Giardia Lamblia ◽  
Trna Synthetase ◽  
Luciferase Reporter ◽  
Infection Model ◽  
Cell Assay ◽  
Mouse Infection Model ◽  
Mouse Infection ◽  
Methionyl Trna Synthetase

Abstract Background Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. Objectives To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Methods Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. Results Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. Conclusions The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

scholarly journals Corrigendum

2016 ◽  
Vol 21 (6) ◽  
pp. 653-653
Keyword(s):  
Trypanosoma Brucei ◽  
High Throughput ◽  
Trna Synthetase ◽  
Methionyl Trna Synthetase

Pedró-Rosa, L.; Buckner, F.; Ranade, R. M.; et al. Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening. J Biomol Screen. 2015, 20, 122–130. (Original DOI: 10.1177/1087057114548832 )

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

2017 ◽  
Vol 27 (12) ◽  
pp. 2702-2707 ◽  
Author(s):  
Wenlin Huang ◽  
Zhongsheng Zhang ◽  
Ranae M. Ranade ◽  
J. Robert Gillespie ◽  
Ximena Barros-Álvarez ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Trna Synthetase ◽  
Methionyl Trna Synthetase

scholarly journals Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening

2014 ◽  
Vol 20 (1) ◽  
pp. 122-130 ◽  
Author(s):  
Laura Pedró-Rosa ◽  
Frederick S. Buckner ◽  
Ranae M. Ranade ◽  
Christina Eberhart ◽  
Franck Madoux ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Trypanosoma Brucei ◽  
High Throughput ◽  
High Throughput Screening ◽  
Adenosine Monophosphate ◽  
Trna Synthetase ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Cytotoxic Compounds ◽  
Methionyl Trna Synthetase

Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.

scholarly journals Structure-guided design of novel Trypanosoma brucei Methionyl-tRNA synthetase inhibitors

2016 ◽  
Vol 124 ◽  
pp. 1081-1092 ◽  
Author(s):  
Wenlin Huang ◽  
Zhongsheng Zhang ◽  
Ximena Barros-Álvarez ◽  
Cho Yeow Koh ◽  
Ranae M. Ranade ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Trna Synthetase ◽  
Methionyl Trna Synthetase

scholarly journals Visualization and Identification of Bioorthogonally Labeled Exosome Proteins Following Systemic Administration in Mice

2021 ◽  
Vol 9 ◽  
Author(s):  
Eric Zhang ◽  
Yanwen Liu ◽  
Chaoshan Han ◽  
Chengming Fan ◽  
Lu Wang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Trna Synthetase ◽  
Biologically Active ◽  
Metabolic Labeling ◽  
Paracrine Factors ◽  
Beneficial Effects ◽  
Canonical Amino Acid ◽  
Covalently Linked ◽  
Methionyl Trna Synthetase

Exosomes transport biologically active cargo (e.g., proteins and microRNA) between cells, including many of the paracrine factors that mediate the beneficial effects associated with stem-cell therapy. Stem cell derived exosomes, in particular mesenchymal stem cells (MSCs), have been shown previously to largely replicate the therapeutic activity associated with the cells themselves, which suggests that exosomes may be a useful cell-free alternative for the treatment of cardiovascular disorders. However, the mechanisms that govern how exosomes home to damaged cells and tissues or the uptake and distribution of exosomal cargo are poorly characterized, because techniques for distinguishing between exosomal proteins and proteins in the targeted tissues are lacking. Here, we report the development of an in vivo model that enabled the visualization, tracking, and quantification of proteins from systemically administered MSC exosomes. The model uses bioorthogonal chemistry and cell-selective metabolic labeling to incorporate the non-canonical amino acid azidonorleucine (ANL) into the MSC proteome. ANL incorporation is facilitated via expression of a mutant (L274G) methionyl-tRNA-synthetase (MetRS∗) and subsequent incubation with ANL-supplemented media; after which ANL can be covalently linked to alkyne-conjugated reagents (e.g., dyes and resins) via click chemistry. Our results demonstrate that when the exosomes produced by ANL-treated, MetRS∗-expressing MSCs were systemically administered to mice, the ANL-labeled exosomal proteins could be accurately and reliably identified, isolated, and quantified from a variety of mouse organs, and that myocardial infarction (MI) both increased the abundance of exosomal proteins and redistributed a number of them from the membrane fraction of intact hearts to the cytosol of cells in infarcted hearts. Additionally, we found that Desmoglein-1c is enriched in MSC exosomes and taken up by ischemic myocardium. Collectively, our results indicate that this newly developed bioorthogonal system can provide crucial insights into exosome homing, as well as the uptake and biodistribution of exosomal proteins.

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