Journal of Biomolecular Screening http://jbx.sagepub.com/
Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening Laura Pedró-Rosa, Frederick S. Buckner, Ranae M. Ranade, Christina Eberhart, Franck Madoux, J. Robert Gillespie, Cho Yeow Koh, Steven Brown, Jacqueline Lohse, Christophe L. M. Verlinde, Erkang Fan, Thomas Bannister, Louis Scampavia, Wim G. J. Hol, Timothy Spicer and Peter Hodder J Biomol Screen published online 27 August 2014 DOI: 10.1177/1087057114548832 The online version of this article can be found at: http://jbx.sagepub.com/content/early/2014/08/26/1087057114548832
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Journal of Biomolecular Screening
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>> OnlineFirst Version of Record - Aug 27, 2014 What is This?
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548832
research-article2014
JBXXXX10.1177/1087057114548832Journal of Biomolecular ScreeningPedró-Rosa et al.
Original Research
Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening
Journal of Biomolecular Screening 1–9 © 2014 Society for Laboratory Automation and Screening DOI: 10.1177/1087057114548832 jbx.sagepub.com
Laura Pedró-Rosa1*, Frederick S. Buckner2*, Ranae M. Ranade2*, Christina Eberhart1, Franck Madoux1, J. Robert Gillespie2, Cho Yeow Koh3, Steven Brown4, Jacqueline Lohse4, Christophe L. M. Verlinde3, Erkang Fan3, Thomas Bannister1, Louis Scampavia1, Wim G. J. Hol3, Timothy Spicer1, and Peter Hodder1†
Abstract 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 aminoacyltRNA 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. Keywords aminoacyl-tRNA synthetases, high-throughput screening, human African trypanosomiasis, orthogonal screening, Trypanosoma brucei
Introduction
1
Aminoacyl-tRNA synthases (aaRS) are essential in protein synthesis, catalyzing the synthesis of aminoacyl-tRNAs. The catalyzed reaction of aaRS usually proceeds via two steps: first the recognition of a specific amino acid and adenosine triphosphate (ATP) to form the aminoacyl-adenylate intermediate and, second, the recognition of associated tRNA and the transfer of the aminoacyl group to the terminal adenosine of the tRNA. Inhibition of either step results in disruption of protein biosynthesis, attenuating cell growth and compromising its viability. aaRSs are thus excellent drug targets for inhibition, delivering selective cytotoxicity versus pathogens. This has been demonstrated by the development of the drug mupirocin, which selectively inhibits isoleucyl-tRNA synthetase (IleRS) from archaea and eubacteria but does not inhibit eukaryotic IleRS.1 Other aaRSs have been targeted in high-throughput
The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA 2 Department of Medicine, University of Washington, Seattle, WA, USA 3 Department of Biochemistry, University of Washington, Seattle, WA, USA 4 Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA *These authors contributed equally to this work. † Current affiliation: Amgen, Inc., Thousand Oaks, CA. Received May 21, 2014, and in revised form July 25, 2014. Accepted for publication Aug 4, 2014. Supplementary material for this article is available on the Journal of Biomolecular Screening Web site at http://jbx.sagepub.com/supplemental. Corresponding Author: Timothy Spicer, Scripps Florida, 130 Scripps Way #1A1, Jupiter, FL 33458, USA. Email: [email protected]
Downloaded from jbx.sagepub.com at PORTLAND STATE UNIV on September 15, 2014
2
Journal of Biomolecular Screening
screening (HTS) efforts, such as phenylalanyl tRNA synthetase (PheRS) and methionyl-tRNA synthetase (MetRS) from Staphylococcus aureus.2 Herein we describe an HTS program targeting MetRS from the protozoan parasite Trypanosoma brucei, including an orthogonal counterscreen. T. brucei is the causative agent of the neglected tropical disease human African trypanosomiasis (HAT), which threatens ~60 million people in sub-Saharan Africa.3 When left untreated, HAT is fatal, and current treatment outcomes are unsatisfactory due to high toxicity, difficult administration regimes, and poor efficacy of existing drugs.4 Hence, new drugs that are effective, nontoxic, and affordable are needed. Early studies using RNA interference (RNAi) demonstrated that MetRS from T. brucei is essential for parasite survival, making MetRS inhibition an attractive strategy for treating HAT.5–7 Potent aminoquinolone-based compounds, such as 2-((3-((3,5-dichlorobenzyl)amino)propyl)amino) quinolin-4(1H)-one (CID 18353708), have been identified but present poor bioavailability.8 Moderately potent ureabased compounds and oxazolone-dipeptides have been reported.9–11 The goal of the HTS campaign presented here is to identify selective, potent, and drug-like small-molecule T. brucei MetRS inhibitors, with the ultimate potential to become novel therapeutics for HAT. To minimize false leads, a key aspect of our effort was to use two orthogonal screens early in the program, using different assay technologies to identify novel inhibitors of the MetRS catalyzed reaction in vitro. This reaction is monitored by measuring the amount of residual ATP or adenosine monophosphate (AMP) from the MetRS reaction via luminescence and fluorescence polarization (FP), respectively. Excellent performance of both assays in the 1536-well plate format was achieved throughout the screen of the Molecular Libraries Small Molecule Repository (MLSMR) collection of 364,131 compounds. More than a dozen tractable compounds with sub-micromolar potency were identified using these assays.
Materials and Methods Protein Expression and Purification The full-length gene for T. brucei MetRS (Tb927.10.1500) was amplified from T. brucei brucei strain 427, expressed in Escherichia coli, and purified as previously described.8 The N-terminal 6-His tag was retained. The human mitochondrial MetRS enzyme was expressed, as previously reported, with a plasmid provided by Dr. L. Spremulli.12
Optimization and Miniaturization of MetRS Inhibition Assays The assay for HTS required a robust, nonradiometric methodology. An ATP depletion assay based on a chemiluminescence was developed for the initial screen.13,14 Optimization
with the Kinase-Glo reagent (Promega, Madison, WI) was initially done in a 96-well format using buffer conditions previously used for the radiometric aminoacylation assay8: 10 mM MgCl2, 25 mM HEPES-KOH (pH 7.9), 50 mM KCl, 2.5 mM dithiothreitol, 0.1 mg/mL bovine serum albumin, 0.2 mM spermine, and 10 U/mL pyrophosphatase. To achieve separation between the high controls (wells not containing cold L-methionine and inhibitors) and the low controls (wells not containing inhibitors), different variables (incubation time, substrate concentration, and enzyme concentration) were manipulated independently while all other conditions were held constant. Optimized substrate concentrations were as follows: L-methionine, 32 µM; bulk E. coli tRNA (Sigma-Aldrich, St. Louis, MO), 200 µg/mL; and ATP, 100 nM. The L-methionine is in the range of the reported Km of 54 ± 42 µM,15 whereas the concentration of ATP is lower than the Km reported for the related MetRS enzyme of E. coli (20 µM).12 It was necessary to use a low concentration of ATP to achieve optimal separation between the high and low controls. Once the substrate concentrations were established, enzyme reaction progress curves (Suppl. Fig. S1A) were completed to determine the optimal enzyme concentration and incubation time, 50 nM and 120 min, respectively. Under these conditions, the amount of ATP consumed was around 85% and resulted in excellent assay reproducibility and statistics with an average Z′ factor of 0.84 (Suppl. Fig. S1B). DMSO tolerance was also tested, and no significant decrease in ATP consumption was observed up to 2% DMSO. For the HTS, pyrophosphatase was decreased from 10 to 0.1 U/mL with no significant decrease in ATP consumption. The assay was miniaturized to a 384-well format at the Scripps Research Institute (La Jolla, CA), and a pilot screen of 5120 Maybridge compounds tested at 1 µM was completed. The pilot screen had excellent Z′ factors of 0.75 to 0.89. Finally, the assay was further miniaturized to a 1536-well format as discussed in the Results section. A stepwise protocol is depicted in Supplemental Table S1. During the primary luminescence-based screen, test compounds were analyzed in singlicate at a final nominal concentration of 12 µM using the automated Kalypsys/GNF platform (GNF Systems, San Diego, CA) at the Scripps Research Institute Molecular Screening Center (SRIMSC, Jupiter, FL). The cutoff used to qualify active compounds was calculated as the average percent inhibition of all the compounds tested plus three times their standard deviation.16 The confirmation assay and the orthogonal FP-based counterscreen tested each compound in triplicate at a single concentration (~12 µM). The primary assay hit cutoff was applied to the confirmation assay. The counterscreen employed the same algorithm to determine its hit cutoff. Actives were progressed forward to determine their concentration-response curves (CRCs) (Table 1). CRC experiments were also performed as above, testing the compounds in triplicate as a
Downloaded from jbx.sagepub.com at PORTLAND STATE UNIV on September 15, 2014
3
Pedró-Rosa et al. Table 1. Ultra-High-Throughput Screening Campaign Summary and Results. Assay Statistics
Step 1 2a 2b 2c 3a 3b 3c
Screen type
Target
Primary MetRS Confirmation MetRS Cytotoxicity Cell growth Orthogonal MetRS Titration MetRS Cytotoxicity Cell growth Orthogonal MetRS
No. of No. of Compounds Replicates/ Tested Concentrations 364,131 1370 1370 1370 249 249 249
1/1 3/1 3/1 3/1 3/10 3/10 3/10
No. of Selected Compounds PubChem (Hits) AIDa
Selection Criteria %inh> 5.61% %inh> 5.61% %inh> 14.97% %inh> 16.67% IC50
Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening Laura Pedró-Rosa, Frederick S. Buckner, Ranae M. Ranade, Christina Eberhart, Franck Madoux, J. Robert Gillespie, Cho Yeow Koh, Steven Brown, Jacqueline Lohse, Christophe L. M. Verlinde, Erkang Fan, Thomas Bannister, Louis Scampavia, Wim G. J. Hol, Timothy Spicer and Peter Hodder J Biomol Screen published online 27 August 2014 DOI: 10.1177/1087057114548832 The online version of this article can be found at: http://jbx.sagepub.com/content/early/2014/08/26/1087057114548832
Published by: http://www.sagepublications.com
On behalf of:
Journal of Biomolecular Screening
Additional services and information for Journal of Biomolecular Screening can be found at: Email Alerts: http://jbx.sagepub.com/cgi/alerts Subscriptions: http://jbx.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> OnlineFirst Version of Record - Aug 27, 2014 What is This?
Downloaded from jbx.sagepub.com at PORTLAND STATE UNIV on September 15, 2014
548832
research-article2014
JBXXXX10.1177/1087057114548832Journal of Biomolecular ScreeningPedró-Rosa et al.
Original Research
Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening
Journal of Biomolecular Screening 1–9 © 2014 Society for Laboratory Automation and Screening DOI: 10.1177/1087057114548832 jbx.sagepub.com
Laura Pedró-Rosa1*, Frederick S. Buckner2*, Ranae M. Ranade2*, Christina Eberhart1, Franck Madoux1, J. Robert Gillespie2, Cho Yeow Koh3, Steven Brown4, Jacqueline Lohse4, Christophe L. M. Verlinde3, Erkang Fan3, Thomas Bannister1, Louis Scampavia1, Wim G. J. Hol3, Timothy Spicer1, and Peter Hodder1†
Abstract 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 aminoacyltRNA 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. Keywords aminoacyl-tRNA synthetases, high-throughput screening, human African trypanosomiasis, orthogonal screening, Trypanosoma brucei
Introduction
1
Aminoacyl-tRNA synthases (aaRS) are essential in protein synthesis, catalyzing the synthesis of aminoacyl-tRNAs. The catalyzed reaction of aaRS usually proceeds via two steps: first the recognition of a specific amino acid and adenosine triphosphate (ATP) to form the aminoacyl-adenylate intermediate and, second, the recognition of associated tRNA and the transfer of the aminoacyl group to the terminal adenosine of the tRNA. Inhibition of either step results in disruption of protein biosynthesis, attenuating cell growth and compromising its viability. aaRSs are thus excellent drug targets for inhibition, delivering selective cytotoxicity versus pathogens. This has been demonstrated by the development of the drug mupirocin, which selectively inhibits isoleucyl-tRNA synthetase (IleRS) from archaea and eubacteria but does not inhibit eukaryotic IleRS.1 Other aaRSs have been targeted in high-throughput
The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA 2 Department of Medicine, University of Washington, Seattle, WA, USA 3 Department of Biochemistry, University of Washington, Seattle, WA, USA 4 Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA *These authors contributed equally to this work. † Current affiliation: Amgen, Inc., Thousand Oaks, CA. Received May 21, 2014, and in revised form July 25, 2014. Accepted for publication Aug 4, 2014. Supplementary material for this article is available on the Journal of Biomolecular Screening Web site at http://jbx.sagepub.com/supplemental. Corresponding Author: Timothy Spicer, Scripps Florida, 130 Scripps Way #1A1, Jupiter, FL 33458, USA. Email: [email protected]
Downloaded from jbx.sagepub.com at PORTLAND STATE UNIV on September 15, 2014
2
Journal of Biomolecular Screening
screening (HTS) efforts, such as phenylalanyl tRNA synthetase (PheRS) and methionyl-tRNA synthetase (MetRS) from Staphylococcus aureus.2 Herein we describe an HTS program targeting MetRS from the protozoan parasite Trypanosoma brucei, including an orthogonal counterscreen. T. brucei is the causative agent of the neglected tropical disease human African trypanosomiasis (HAT), which threatens ~60 million people in sub-Saharan Africa.3 When left untreated, HAT is fatal, and current treatment outcomes are unsatisfactory due to high toxicity, difficult administration regimes, and poor efficacy of existing drugs.4 Hence, new drugs that are effective, nontoxic, and affordable are needed. Early studies using RNA interference (RNAi) demonstrated that MetRS from T. brucei is essential for parasite survival, making MetRS inhibition an attractive strategy for treating HAT.5–7 Potent aminoquinolone-based compounds, such as 2-((3-((3,5-dichlorobenzyl)amino)propyl)amino) quinolin-4(1H)-one (CID 18353708), have been identified but present poor bioavailability.8 Moderately potent ureabased compounds and oxazolone-dipeptides have been reported.9–11 The goal of the HTS campaign presented here is to identify selective, potent, and drug-like small-molecule T. brucei MetRS inhibitors, with the ultimate potential to become novel therapeutics for HAT. To minimize false leads, a key aspect of our effort was to use two orthogonal screens early in the program, using different assay technologies to identify novel inhibitors of the MetRS catalyzed reaction in vitro. This reaction is monitored by measuring the amount of residual ATP or adenosine monophosphate (AMP) from the MetRS reaction via luminescence and fluorescence polarization (FP), respectively. Excellent performance of both assays in the 1536-well plate format was achieved throughout the screen of the Molecular Libraries Small Molecule Repository (MLSMR) collection of 364,131 compounds. More than a dozen tractable compounds with sub-micromolar potency were identified using these assays.
Materials and Methods Protein Expression and Purification The full-length gene for T. brucei MetRS (Tb927.10.1500) was amplified from T. brucei brucei strain 427, expressed in Escherichia coli, and purified as previously described.8 The N-terminal 6-His tag was retained. The human mitochondrial MetRS enzyme was expressed, as previously reported, with a plasmid provided by Dr. L. Spremulli.12
Optimization and Miniaturization of MetRS Inhibition Assays The assay for HTS required a robust, nonradiometric methodology. An ATP depletion assay based on a chemiluminescence was developed for the initial screen.13,14 Optimization
with the Kinase-Glo reagent (Promega, Madison, WI) was initially done in a 96-well format using buffer conditions previously used for the radiometric aminoacylation assay8: 10 mM MgCl2, 25 mM HEPES-KOH (pH 7.9), 50 mM KCl, 2.5 mM dithiothreitol, 0.1 mg/mL bovine serum albumin, 0.2 mM spermine, and 10 U/mL pyrophosphatase. To achieve separation between the high controls (wells not containing cold L-methionine and inhibitors) and the low controls (wells not containing inhibitors), different variables (incubation time, substrate concentration, and enzyme concentration) were manipulated independently while all other conditions were held constant. Optimized substrate concentrations were as follows: L-methionine, 32 µM; bulk E. coli tRNA (Sigma-Aldrich, St. Louis, MO), 200 µg/mL; and ATP, 100 nM. The L-methionine is in the range of the reported Km of 54 ± 42 µM,15 whereas the concentration of ATP is lower than the Km reported for the related MetRS enzyme of E. coli (20 µM).12 It was necessary to use a low concentration of ATP to achieve optimal separation between the high and low controls. Once the substrate concentrations were established, enzyme reaction progress curves (Suppl. Fig. S1A) were completed to determine the optimal enzyme concentration and incubation time, 50 nM and 120 min, respectively. Under these conditions, the amount of ATP consumed was around 85% and resulted in excellent assay reproducibility and statistics with an average Z′ factor of 0.84 (Suppl. Fig. S1B). DMSO tolerance was also tested, and no significant decrease in ATP consumption was observed up to 2% DMSO. For the HTS, pyrophosphatase was decreased from 10 to 0.1 U/mL with no significant decrease in ATP consumption. The assay was miniaturized to a 384-well format at the Scripps Research Institute (La Jolla, CA), and a pilot screen of 5120 Maybridge compounds tested at 1 µM was completed. The pilot screen had excellent Z′ factors of 0.75 to 0.89. Finally, the assay was further miniaturized to a 1536-well format as discussed in the Results section. A stepwise protocol is depicted in Supplemental Table S1. During the primary luminescence-based screen, test compounds were analyzed in singlicate at a final nominal concentration of 12 µM using the automated Kalypsys/GNF platform (GNF Systems, San Diego, CA) at the Scripps Research Institute Molecular Screening Center (SRIMSC, Jupiter, FL). The cutoff used to qualify active compounds was calculated as the average percent inhibition of all the compounds tested plus three times their standard deviation.16 The confirmation assay and the orthogonal FP-based counterscreen tested each compound in triplicate at a single concentration (~12 µM). The primary assay hit cutoff was applied to the confirmation assay. The counterscreen employed the same algorithm to determine its hit cutoff. Actives were progressed forward to determine their concentration-response curves (CRCs) (Table 1). CRC experiments were also performed as above, testing the compounds in triplicate as a
Downloaded from jbx.sagepub.com at PORTLAND STATE UNIV on September 15, 2014
3
Pedró-Rosa et al. Table 1. Ultra-High-Throughput Screening Campaign Summary and Results. Assay Statistics
Step 1 2a 2b 2c 3a 3b 3c
Screen type
Target
Primary MetRS Confirmation MetRS Cytotoxicity Cell growth Orthogonal MetRS Titration MetRS Cytotoxicity Cell growth Orthogonal MetRS
No. of No. of Compounds Replicates/ Tested Concentrations 364,131 1370 1370 1370 249 249 249
1/1 3/1 3/1 3/1 3/10 3/10 3/10
No. of Selected Compounds PubChem (Hits) AIDa
Selection Criteria %inh> 5.61% %inh> 5.61% %inh> 14.97% %inh> 16.67% IC50
