A small molecule discrimination map of the antibiotic resistance kinome

Authors: Shakya T, Stogios PJ, Waglechner N, Evdokimova E, Ejim L, Blanchard JE, McArthur AG, Savchenko A, Wright GD

Chemistry & Biology 2011 Dec 23;18(12):1591-601

Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes share a common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases in vitro and in vivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2″)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.

Nrf2b: novel zebrafish paralog of the oxidant-responsive transcription factor NF-E2-related factor 2 (NRF2)

Authors: Timme-Laragy AR, Karchner SI, Franks DG, Jenny MJ, Harbeitner RC, Goldstone JV, McArthur AG, Hahn ME

Journal of Biological Chemistry 2012 Feb 10;287(7):4609-27.

NF-E2-related factor 2 (NRF2, also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a), but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide (tBOOH) but not tert-butyl hydroquinone (tBHQ), whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes including p53, cyclin g1, and heme oxygenase 1 in embryos. Nrf2a and Nrf2b exhibited different mechanisms of crosstalk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.

Current and Recent Projects

Dr. Gerry Wright - construction of a Comprehensive Antibiotic Resistance Database, http://arpcard.mcmaster.ca. Michael G. Institute for Infectious Disease Research, McMaster University, Canada

Dr. John Brennan - construction of a Database of Biointerface Interactions. Biointerfaces Institute, McMaster University, Canada

Dr. Joanna Wilson - phylogenetics of P450 proteins, microarray analysis, environmental toxicology, zebrafish. Department of Biology, McMaster University, Canada

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Dr. Matthew Jenny - microarray analysis, ChIP-Seq analysis, environmental toxicology, developing zebrafish. Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, USA

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Dr. John Stegeman - microarray analysis, environmental toxicology, developing zebrafish. Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA, USA

Dr. Mark Hahn - microarray analysis, ChIP-Seq analysis, cis-regulatory element prediction, environmental toxicology, developing zebrafish. Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA, USA

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Dr. Vanessa Allen - whole genome shotgun (454) genome assembly and SNP analysis of Salmonella enteritidis. Ontario Agency for Health Protection and Promotion, Ontario, Canada

Generic Model Organism Database Meeting, Ontario Institute for Cancer Research, Toronto, Canada

A.G. McArthur, N. Waglechner, F. Nizam, M.A. Azad, K. Bhullar, M.J. Canova, G. De Pascale, L. Ejim, L. Kalan, A.M. King, K. Koteva, M. Morar, J.S. O’Brien, A.C. Pawlowski, P. Spanogiannopoulos, A.D. Sutherland, I. Tang, P.L. Taylor, M. Thaker, W. Wang, M. Yan, T. Yu, & G.D. Wright. 2011. Towards a Comprehensive Antibiotic Resistance Database (CARD). Oral presentation at the Generic Model Organism Database Meeting, Ontario Institute for Cancer Research, Toronto, Canada.

Addressing the challenge of antibiotic resistance requires the combined efforts of researchers that span inquiry at the molecular, patient, and population levels. Yet, despite the common objective of finding solutions to drug resistance, practitioners of these research areas do not often integrate their research findings. This reality reflects the disparate investigative tools, timelines and specific project aims of these disciplines. Nevertheless, resistance genes and their products are the common elements that bridge antibiotic resistance research from bench to clinic to population. What is lacking in the field is a comprehensive database of resistance genes that includes molecular, clinical, and surveillance data that can serve to unify research. The Comprehensive Antibiotic Resistance Database (CARD) is a bioinformatic database of resistance genes, their products and associated phenotypes designed to address this need by combining a Drupal front-end, an ontological-centric design, GMOD's Chado schema, and the Gbrowse genome browser.

http://arpcard.mcmaster.ca

16th International Symposium on Pollutant Response in Marine Organisms

Smith, E.M., A.G. McArthur, M. Galus, S. Higgins, N. Kirischian, J. Jeyaranjaan, & J.Y. Wilson. 2011. Transcriptional responses of zebrafish to chronic, low-dose pharmaceutical exposure. Oral presentation at the 16th International Symposium on Pollutant Response in Marine Organisms, Long Beach, California.

Human pharmaceuticals have been well documented in receiving waters yet their impacts on aquatic species are not clear. We have exposed adult zebrafish for 6 weeks to waterborne acetaminophen, gemfibrozil, venlafaxine, and carbamazepine at two doses (0.5 and 10 μg L-1) and assessed hepatic transcriptional responses using a commercial microarray. These four pharmaceuticals have been frequently found in wastewater effluent and receiving waters. For all compounds, reproduction was significantly reduced and histopathological changes induced in kidney with at least the 10 μg L-1 exposure. Livers were pooled to provide sufficient RNA for microarray and qPCR analyses. Gene expression was determined with a modified Agilent 44K zebrafish microarry using a single channel approach. Significantly different probes were identified with a 2-way ANOVA (sex and treatment) and rank product analyses with a 10% false discovery rate. Transcriptional responses were particularly marked with acetaminophen exposure and there was broad overlap in the significant probes found between doses and across gender for this compound. 52 probes were at least 20 fold up- or down- regulated in acetaminophen exposed fish; 3 probes (LIST) had 100 fold up- or down- regulated. Venlafaxine responses were few in males and carbamazepine impacted hepatic transcription the least in both sexes. There was a strong sex dependant response to both venlafaxine and carbamazepine. Unique probes were identified for all exposures suggesting a unique transcriptional response may occur for each pharmaceutical. The biological relevance of these exposures is being explored through analysis of enriched GO terms, metabolic pathways and interactome networks.

Phylogenetic and functional analysis of the vertebrate cytochrome p450 2 family

Authors: Kirischian N, McArthur AG, Jesuthasan C, Krattenmacher B, Wilson JY.

J Mol Evol. 2011 Jan;72(1):56-71.

Cytochrome P450 (CYP) proteins compose a highly diverse superfamily found in all domains of life. These proteins are enzymes involved in metabolism of endogenous and exogenous compounds. In vertebrates, the CYP2 family is one of the largest, most diverse and plays an important role in mammalian drug metabolism. However, there are more than 20 vertebrate CYP2 subfamilies with uncertain evolution and fairly discrete subfamily composition within vertebrate classes, hindering extrapolation of knowledge across subfamilies. To better understand CYP2 diversity, a phylogenetic analysis of 196 CYP2 protein sequences from 16 species was performed using a maximum likelihood approach and Bayesian inference. The analyses included the CYP2 compliment from human, fugu, zebrafish, stickleback, medaka, cow, and dog genomes. Additional sequences were included from rabbit, marsupial, platypus, chicken, frog, and salmonid species. Three CYP2 sequences from the tunicate Ciona intestinalis were utilized as the outgroup. Results indicate a single ancestral vertebrate CYP2 gene and monophyly of all CYP2 subfamilies. Two subfamilies (CYP2R and CYP2U) pre-date vertebrate diversification, allowing direct comparison across vertebrate classes, while all other subfamilies originated during vertebrate diversification, often within specific vertebrate lineages. Analysis of site-specific evolution indicates that some substrate recognition sites (SRS) previously proposed for CYP genes do not have elevated rates of evolution, suggesting that these regions of the protein are not necessarily important in recognition of CYP2 substrates. Type II functional divergence analysis identified multiple residues in the active site of CYP2F, CYP2A, and CYP2B proteins that have undergone radical biochemical changes and may be functionally important.

Identification and developmental expression of the full complement of cytochrome P450 genes in zebrafish

Authors: Goldstone, J.V., A.G. McArthur, A. Kubota, J. Zanette, T. Parente, M. Jönsson, D.R. Nelson, & J.J. Stegeman

BMC Genomics 2010, 11: 643.

Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.

An atypical proprotein convertase in Giardia lamblia differentiation

Authors: Davids, B.J., M.A. Gilbert, Q. Liu, D.S. Reiner, A.J. Smith, T. Lauwaet, C. Lee, A.G. McArthur, & F.D. Gillin

Molecular and Biochemical Parasitology 2011, 175(2):169-180.

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

About McArthur Bioinformatics Consulting

My principal training was in Ecology and Statistics (undergraduate) and in Evolutionary and Molecular Biology at the University of Victoria (Canada), the National Museum of Natural History (Smithsonian Institution), and Marine Biological Laboratory. At the Marine Biological Laboratory between 1998-2006, I established an active research program in Global Infectious Disease with funding from NIH/NIAID and the Ellison Medical Foundation. My examination of molecular approaches to difficult evolutionary questions led me to join the effort to sequence the entire 12 Mbp genome of the protistan parasite Giardia lamblia, hypothesized to be one of the oldest and most primitive eukaryotes alive. I was the lead computational biologist for the Giardia lamblia genome project, and principal investigator on NIH-funded Giardia lamblia, Trypanosoma brucei, and Schistosoma mansoni gene expression projects, which used Serial Analysis of Gene Expression (SAGE) to examine global gene expression in the context of parasite life cycles and the origins of virulence. I have experience in mathematical, computational, and evolutionary biology approaches to complex biological questions. My expertise in genomics and computational biology is highlighted by my key role as faculty in the Institute of Genome Research (TIGR) – Marine Biological Laboratory “Advances in Genome Technology and Bioinformatics” course, where I instructed advanced students (late graduate, postdoctoral, and junior faculty) in DNA sequencing informatics, construction of gene expression libraries, Perl programming, gene finding / genome annotation, and analysis of digital gene expression profiling data.

In 2006, I left academia to act as a consultant in computational biology and bioinformatics for researchers in academia and government. With researchers at the Woods Hole Oceanographic Institution (Woods Hole, MA), McMaster University (Hamilton, Canada), and the University of Alabama (Tuscaloosa, AL), I have performed research in environmental toxicology, with emphasis on microarray and genomic investigation of the molecular response of adult and developing zebrafish to metal (e.g. metal stress transcription factor MTF-1), organic (e.g. tBHQ, TCDD), and pharmaceutical (acetaminophen, gemfibrozil, carbamazapine and venlafaxine) pollutants. These efforts have recently been expanded to include both computational and ChIP-Seq investigation of the involved regulatory mechanisms. Another major research area is in the construction of the Comprehensive Antibiotic Resistance Database with a consortium of academic and government researchers in Canada and the United Kingdom, based at McMaster University. This project involves sequencing and assembly of novel bacterial pathogen genomes using both 454 and Illumina technologies, and subsequent annotation of possible antibiotic resistance genes and mechanisms, with implications for clinical treatment. Overall, I have extensive experience in comparative genomics, genome sequencing and assembly, gene expression profiling using microarrays, examination of cis-regulatory regions using both computational and experimental methods, and construction of biological databases.

Downloadable Seminars

McArthur, A.G. Introduction to Phylogenetics. Invited seminar for the Graduate Student Association of the Department of Biochemistry, McMaster University, Hamilton, Canada. November 2011.

McArthur A.G., N. Waglechner, F. Nizam, M.A. Azad, K. Bhullar, M.J. Canova, G. De Pascale, L. Ejim, L. Kalan, A.M. King, K. Koteva, M. Morar, J.S. O'Brien, A.C. Pawlowski, P. Spanogiannopoulos, A.D. Sutherland, I. Tang, P.L. Taylor, M. Thaker, W. Wang, M. Yan, T. Yu, & G.D. Wright. 2011. Towards a Comprehensive Antibiotic Resistance Database (CARD). Oral presentation at the Generic Model Organism Database Meeting, Ontario Institute for Cancer Research, Toronto, Canada.