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      Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

          Author summary

          AN11736 is a member of the benzoxaborole class identified as a development candidate for animal African trypanosomiasis, a deadly livestock disease with huge economic impact. As part of its early evaluation phase, we set to unravel the risk and mode of resistance to this new trypanocide. We discovered that AN11736 behaves as a prodrug that, once inside trypanosomes, is cleaved by the activity of specific serine carboxypeptidases. AN11736-resistant Trypanosoma brucei and T. congolense had deletions within the serine carboxypeptidase gene array, resulting in their being unable to efficiently process the parent drug. Other benzoxaboroles with a similar sub-structure are also substrates for the serine carboxypeptidases, hence our findings assume great importance in considering the future development and deployment of this class of compounds.

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          Most cited references 47

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          The Sequence Alignment/Map format and SAMtools

          Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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            Fast gapped-read alignment with Bowtie 2.

            As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
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              featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.

               Y. Liao,  G Smyth,  W Shi (2014)
              Next-generation sequencing technologies generate millions of short sequence reads, which are usually aligned to a reference genome. In many applications, the key information required for downstream analysis is the number of reads mapping to each genomic feature, for example to each exon or each gene. The process of counting reads is called read summarization. Read summarization is required for a great variety of genomic analyses but has so far received relatively little attention in the literature. We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. featureCounts implements highly efficient chromosome hashing and feature blocking techniques. It is considerably faster than existing methods (by an order of magnitude for gene-level summarization) and requires far less computer memory. It works with either single or paired-end reads and provides a wide range of options appropriate for different sequencing applications. featureCounts is available under GNU General Public License as part of the Subread (http://subread.sourceforge.net) or Rsubread (http://www.bioconductor.org) software packages.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: Project administrationRole: ValidationRole: Writing – original draft
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: SoftwareRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Validation
                Role: ConceptualizationRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: Funding acquisitionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Validation
                Role: Formal analysisRole: MethodologyRole: Software
                Role: Data curationRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: MethodologyRole: Validation
                Role: ConceptualizationRole: InvestigationRole: SupervisionRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                3 November 2020
                November 2020
                : 16
                : 11
                Affiliations
                [1 ] Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
                [2 ] Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United Kingdom
                [3 ] Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, United Kingdom
                [4 ] Anacor Pharmaceuticals, Inc., Palo Alto, California, United States of America
                [5 ] Global Alliance for Livestock and Veterinary Medicine, Pentlands Science Park, Penicuik, Edinburgh, United Kingdom
                [6 ] Glasgow Polyomics, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
                [7 ] Current address: Bioinformatics Group, Wageningen University, Wageningen, the Netherlands
                [8 ] Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic
                University of Geneva, SWITZERLAND
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PPATHOGENS-D-20-00932
                10.1371/journal.ppat.1008932
                7710103
                33141865
                © 2020 Giordani et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 5, Tables: 0, Pages: 20
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BB/S001034/1
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BB/S001034/1)
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 100320/Z/12/Z
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 203134/Z/16/Z
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 104111/Z/14/Z
                Award Recipient :
                This work was funded by a BBSRC grant (BB/S001034/1) and Global Alliance for Livestock Veterinary Medicine (GALVmed) with funding from BMGF and UKAID grant (OPP-1093639) to L.J.M. and M.P.B., a Wellcome Trust Senior Investigator Award to D.H. (100320/Z/ 12/Z), a Wellcome Trust Centre Award (203134/Z/ 16/Z) to Dundee and a core grant to the Wellcome Centre for Integrative Parasitology (104111/Z/14/ Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Trypanosoma
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Trypanosoma
                Trypanosoma Brucei
                Trypanosoma Brucei Gambiense
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Amino Acids
                Hydroxyl Amino Acids
                Serine
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Hydroxyl Amino Acids
                Serine
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Hydroxyl Amino Acids
                Serine
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Trypanosoma
                Trypanosoma Brucei
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Trypanosoma
                Trypanosoma Congolense
                Biology and Life Sciences
                Genetics
                Genomics
                Biology and life sciences
                Genetics
                Epigenetics
                RNA interference
                Biology and life sciences
                Genetics
                Gene expression
                RNA interference
                Biology and life sciences
                Genetics
                Genetic interference
                RNA interference
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                RNA interference
                Custom metadata
                vor-update-to-uncorrected-proof
                2020-12-02
                Metabolomics data are available on MetaboLights ( http://www.ebi.ac.uk/metabolights/) at accession numbers MTBLS1309 (T. congolense) and MTBLS1474 (T. brucei). Genome data are available on European Nucleotide Archive (ENA) and Sequence Read Archive (SRA) at accession number PRJEB34627.

                Infectious disease & Microbiology

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