Progress 10/01/11 to 09/30/15
Outputs
Target Audience:Target groups include: (1) Researchers studying the molecular basis of host-pathogen interactions, particularly the cell signaling pathways in plant parasitic nematodes that may be amenable to disruption with pathway- and organism-specific chemical agents. (2) Scientists and educators interested in promoting sustainable agricultural practices that reduce/eliminate reliance on environmentally dangerous pesticides that are toxic to humans, birds, fish, and other non-target organisms. (3) Educated laypeople interested in using genomic tools to improve agricultural practices. (4) Agrochemical companies seeking novel approaches to parasitic nematode control, particularly companies with small-molecule, nematicide discovery programs. Audiences targeted during the project period: Groups #1-4 were targeted at national/internationalscientific meetings (2013Experimental Biology, 2014 Gordon Research Conference on Cyclic Nucleotide Phosphodiesterases, 2015Experimental Biology) Groups #1-2 were targeted at oral presentations to students, faculty, staff at the University of New Hampshire that occurred each year during (1)new graduate student orientation sessions(2)graduate student research seminars; (3) graduate student research conferences hosted by the UNH Graduate School. Changes/Problems:The major problems that impeded progress in completing the objectives were: (1) the preliminary state of the genome assembly for plant parasitic nematode species that were the focus of this project; (2) difficulties in the heterologous recombinant expression of nemaotde PDE catalytic domains in a properly folded and catalytically active form. What opportunities for training and professional development has the project provided?One Ph.D. student has been trained in this project. How have the results been disseminated to communities of interest?This work has resulted in multiple presentations at local andnational/international scientific meetings, the filing of a patent application, and inclusion on the laboratory's web site (www.cotelab.org). Presentations: The work was presented as a scientific poster at the Experimental Biology 2013 meeting. Meeting participants included academic and industrial research scientists, students, journalists, educators, and interested lay people. It is estimated that 250 people examined the poster, and 50 interacted with the poster presenter. The citation is:K. D. Schuster, K. B. Cahill, K. Morris, W. K. Thomas, and R. H. Cote. PDE inhibitors as potential pesticides targeting parasitic nematodes. Experimental Biology 2013 meeting C434 1052.6 (2013). The work was also presented as an invited talk at the 2014 Gordon Research Conference on Cyclic Nucleotide Phosphodiesterases in June, 2014. Meeting participants included academic and pharmaceutical company research scientists, students, educators, and interested lay people. It is estimated that 150 people attended the oral presentation, and 25 people interacted the presenter regarding the work. The work was also presented as a scientific poster at the Experimental Biology 2015 meeting. Meeting participants included academic and industrial research scientists, students, journalists, educators, and interested lay people. It is estimated that 150 people examined the poster, and 30 interacted with the poster presenter. The citation is:Schuster, K. D., Danquah, W. B., Williamson, V. M., and Cote, R. H. (2015). Exposure to phosphodiesterase inhibitors impairs physiological processes in juvenile nematodes. Experimental Biology 2015 meeting. A296, 721.41. The work was presented in several venues and formats to members of the University of New Hampshire community by the graduate student. Annual oral research presentations occurred in the context of the department graduate student seminar at which ~25 graduate students and faculty attended each time. In addition, the graduate student presented a poster at the UNH Graduate Student Research Conference at which 100 faculty, staff, students, and administrators attended. Patent: This work also resulted in filing a patent application in 2014 by the project director, the post-doctoral research associate, and graduate student. Cote, R. H., Cahill, K. B., and Schuster, K. D. (2014). Methods of identification and use of nematicide compounds. PCT Patent Application No. PCT/US14/29910. Available at:http://www.google.com/patents/WO2014145189A1?cl=en Web site: Public dissemination of the project has been posted on the web: http://www.cotelab.org/pdes-in-nematodes.html What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The issue is that current methods to control plant parasitic nematodes that infect agricultural crops are harmful to the environment and have deleterious consequences on non-target organisms and host plants. Farmers, agricultural businesses, and consumers are united in seeking new nematicides that combine efficacy, specificity, and food safety. The long-term goal of the work is to evaluate a class of enzymes called cyclic nucleotide phosphodiesterases (PDEs) as potential molecular targets for development of nematicidal agents. Using a bioinformatic approach, we first identified the occurrence of six families of PDEs in all nematodes (parasitic and non-parasitic) for which genomic information was available. Using atomic-level structural modeling, we then predicted that nematode PDEs should have enzymatic activities and pharmacological profiles that are similar--but not identical--to those found in other animals. We then used molecular biological and pharmacological approaches to first isolate and purify several nematode PDEs, then measure their enzymatic activity, and finally evaluate the ability of variousclases of PDE inhibitors to block enzyme activity of the nematode PDEs. The impact of this work is in demonstrating "proof of principle" that nematode PDEs are excellentcandidates as molecular targets for future development of "next-generation" nematicides capable of disrupting thenematode lifecycle with greatly reduced adverse effects on host plants and vertebrates (including humans). Objective 1: The first objectivewas to perform bioinformatics analyses of known plant-parasitic nematode genomes to identify putative phosphodiesterases and classify them in relation to the eleven PDE families (designated as PDE1-11) present in vertebrates. To examine this, a phylogenetically diverse set of amino acid sequences was collected that contained all 11 PDE families from organisms representative of mammals, birds, amphibians, reptiles, and fish. We also collected invertebrate PDE sequences which contain varying numbers of PDE families. Ultimately, we collected ~300 sequences from all phyla for analysis. The model nematode, Caenorhabditis elegans, contains six PDE families, which we were able to identify in several other members of the Nematoda phylum, including free-living, animal-, and plant-parasitic species. For the plant-parasitic species Meloidogyne hapla, M. incognita, and Heterodera glycines, the genomes were still in draft format and unannotated at the time of analysis. We therefore used C. elegans PDE sequences as The Basic Local Alignment Search Tool (BLAST) queries to identify the contig containing the PDE gene in each species. Using knowledge of nematode intron-exon boundaries, the protein coding sequence for each PDE was predicted. Once the protein sequences were collected, phylogenetic analysis permitted assignment of putative nematode PDEs into one of the 11 vertebrate PDE families. We found that nematodes contain orthologs of PDE1, 2, 3, 4, 8, and 10. Multiple sequence alignment and evolutionary trace analysis of the PDE catalytic domains permitted identification of unanimous sites and sites that were conserved within a PDE family but which differed in other PDE families. Analysis of known drug interaction sites of selective inhibitors of human PDE3 and PDE4 revealed that ~80% of the residues responsible for stabilizing the binding of PDE inhibitors to human PDEs are also present in M. hapla orthologs. Structural homology modelling of the nematode PDE catalytic domains demonstrated a high degree of sequence and structural conservation in the inhibitor binding region of the catalytic domain, consistent with the idea that exposure of nematodes to family-specific PDE inhibitors may disrupt the normal lifecycle of nematodes that depend on cyclic nucleotide signaling pathways. These analyses also revealed differences within the catalytic domains of nematode PDEs that may confer altered pharmacological sensitivity to PDE inhibitors compounds relative to their human orthologs. Objective #2: The second objective was to express the catalytic domains of specific nematode PDEs for pharmacological profiling. We cloned and expressed recombinant catalytic domains of C. elegans PDE3, PDE4, and PDE10 fused to an N-terminal six-histidine tag. Bacterial expression of nematode PDE catalytic domains and affinity purification resulted in partially purified protein of the predicted size. The ability of C. elegans PDE3 and PDE4 to hydrolyze cAMP was established, whereas PDE10 could only be expressed in a catalytically inactive form (to date). C. elegans PDE3 is inhibited by both cilostazol and milrinone (two family-specific inhibitors of human PDE3) at similar concentrations to that reported in the literature for the human enzyme. In contrast, marked differences in the pharmacological properties of C. elegans and human PDE4 have been observed. Whereas isobutylmethyl xanthine (IBMX, a pan-specific PDE inhibitor) showed a <2-fold difference in inhibitor binding affinity, the PDE3/4-specific inhibitor zardaverine exhibited a 20-fold greater affinity for human versus C. elegans PDE4, and the PDE4-specific inhibitor roflumilast inhibited human PDE4 with 200-fold greater potency than for the C. elegans enzyme. The relative potency of these three PDE inhibitors to inhibitor human and nematode PDE4 catalysis correlates well with structural determinations of human PDE4 in a complex with these inhibitors and the structural homology modeling studies of nematode PDEs described in the first aim.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Cote, R.H., Evolutionary and structural approaches to understanding PDE inhibitors. Gordon Research Conference on Cyclic Nucleotide Phosphodiesterases 2014 meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Schuster, K. D., Danquah, W. B., Williamson, V. M., and Cote, R. H. (2015). Exposure to phosphodiesterase inhibitors impairs physiological processes in juvenile nematodes. Experimental Biology 2015 meeting. A296, 721.41.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target groups include: (1) Researchers studying the molecular basis of host-pathogen interactions, particularly the cell signaling pathways in plant parasitic nematodes that may be amenable to disruption with pathway- and organism-specific chemical agents. (2) Scientists and educators interested in promoting sustainable agricultural practices that reduce/eliminate reliance on environmentally dangerous pesticides that are toxic to humans, birds, fish, and other non-target organisms. (3) Educated laypeople interested in using genomic tools to improve agricultural practices. (4) Agrochemical companies seeking novel approaches to parasitic nematode control, particularly companies with small-molecule, nematicide discovery programs. Audiences targeted during the project period: *Groups #1-4 were targeted at a scientific meeting (2014 Gordon Research Conference on Cyclic Nucleotide Phosphodiesterases) in June, 2014 at which the work was presented. Approximately 150 people attended the oral presentation. *Groups #1-2 were targeted at a short oral presentation to new graduate students in August, 2014 at which 15 people were present. Changes/Problems: We will take the following approaches to optimize the expression of catalytically active, purified catalytic domains for C. elegans PDE3, PDE4, and PDE10 in bacteria as follows: (1) Replace the 6-histidine fusion partner with a either a maltose binding protein (MBP) or glutathione-S-transferase (GST) fusion partner by subcloning the PDE open reading frames into pMal or pGEX plasmids, respectively. (2) Vary the beginning and end of the open reading frame that is subcloned into the expression vector. (3) Introduce a C-terminal fusion partner to replace the N-terminal fusion proteins tested thus far. (4) As a last resort, we will change the host cell expressing the recombinant C. elegans PDE catalytic domains by introducing the C. elegans PDE catalytic domain sequence into a pFastBac vector, generating baculovirus, and infecting Sf9 insect cells to express recombinant protein, using methods previously published by our lab. What opportunities for training and professional development has the project provided? One undergraduate student researcher, one doctoral student, and one postdoctoral research associate have been trained during this past project period in the design and conduct of scientific experiments. For both students and the postdoctoral trainee, the knowledge and skills acquired will advance these individuals in their chosen careers as research scientists. How have the results been disseminated to communities of interest? Preliminary results of the work accomplished in the past project period were presented as an invited talk at the 2014 Gordon Research Conference on Cyclic Nucleotide Phosphodiesterases in June, 2014. Meeting participants included academic and industrial research scientists, students, educators, and interested lay people. It is estimated that 150 people attended the oral presentation, and 25 people interacted the presenter regarding the work. What do you plan to do during the next reporting period to accomplish the goals? The plan of work for the next project period is to express in a heterologous expression system the catalytic domains of three PDE genes from the model nematode, C. elegans, that we have shown to disrupt nematode motility in vivo. These three PDE catalytic domains will be characterized pharmacologically with available PDE inhibitor compounds to determine whether inhibitor specificity of nematode PDEs differs from human PDEs. The outcome of this work will be the identification of which PDEs found in nematodes are the best candidates for future efforts to develop potent and selective nematicides targeting plant parasites.
Impacts
What was accomplished under these goals?
We have expanded our investigation of the molecular evolution of the drug-binding catalytic domain of the PDE superfamily to include close to 300 sequences from all animal phyla. Because PDE3 and PDE4 inhibitors show particular promise for disrupting the physiology of nematodes C. elegans and Meloidogyne hapla (root knot nematode), we have closely examined the evolution of the PDE3 and PDE4 enzyme families. We observe that nematode PDE3 sequences form a clade that exists as an out-group of other metazoan phyla; similar results are seen for PDE4. Furthermore, within the Anthropoda phylum, PDE3 is absent in the Insecta species we examined and was present only in the Arachnida class. The evolutionary divergence of nematode PDEs and the absence of PDE3 in insects makes PDE3-selective inhibitor compounds particularly attractive targets for development of nematicides with reduced adverse effects on animals other than nematodes. To evaluate whether nematode PDEs exhibit different pharmacological properties that result from amino acid differences in the drug binding pocket of the catalytic domain, we cloned (into the pET47b vector) and expressed recombinant catalytic domains of C. elegans PDE3, PDE4, and PDE10 fused to an N-terminal six-histidine tag. The open reading frames were commercially synthessized and codon-optimized for expression in E. coli. Protein of the predicted size was obtained in all cases, but the affinity purification of the expressed proteins was poor and requires optimization. The ability of C. elegans PDE3 and PDE4 to hydrolyze cAMP has been established, whereas PDE10 was expressed in a catalytically inactive form. Preliminary experiments indicate that partially purified C. elegans PDE3 is inhibited by both cilostazol and milrinone (two family-specific inhibitors of human PDE3) at similar concentrations to that reported in the literature for the human enzyme.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Target groups include: (1) Researchers studying the molecular basis of host-pathogen interactions, particularly the cell signaling pathways in plant parasitic nematodes that may be amenable to disruption with pathway- and organism-specific chemical agents. (2) Scientists and educators interested in promoting sustainable agricultural practices that reduce/eliminate reliance on environmentally dangerous pesticides that are toxic to humans, birds, fish, and other non-target organisms. (3) Educated laypeople interested in using genomic tools to improve agricultural practices. (4) Agrochemical companies seeking novel approaches to parasitic nematode control, particularly companies with small-molecule, nematicide discovery programs. Audiences targeted during the project period: *Groups #1-4 were targeted at a scientific poster presentation of the research at Experimental Biology April, 2013. It is estimated that 200-300 people viewed the poster during the day in which it was presented. *Groups #1-2 were targeted at the Graduate Seminar (MCBS 997) at which 25 people were in attendance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One undergraduate student researcher and one doctoral student have been trained during this past project period in the design and conduct of scientific experiments. In addition to the project director, two collaborating scientists in the UNH Hubbard Center for Genome Studies were involved in training the graduate student in performing bioinformatic studies. For both students, the knowledge and skills acquired will advance these students in their chosen careers as research scientists. How have the results been disseminated to communities of interest? Preliminary results of the work accomplished in the past project period were presented as a scientific poster at the Experimental Biology 2013 meeting. Meeting participants included academic and industrial research scientists, students, journalists, educators, and interested lay people. It is estimated that 250 people examined the poster, and 50 intereracted with the poster presenter. The citation is: K. D. Schuster, K. B. Cahill, K. Morris, W. K. Thomas, and R. H. Cote. PDE inhibitors as potential pesticides targeting parasitic nematodes. Experimental Biology 2013 meeting C434 1052.6 (2013). What do you plan to do during the next reporting period to accomplish the goals? The plan of work for the next project period is to express in a heterologous expression system the catalytic domains of three PDE genes from the model nematode, C. elegans that are known to disrupt nematode motility in vivo. These three PDE catalytic domains will be characterized pharmacologically with available PDE inhibitor compounds to determine whether inhibitor specificity of nematode PDEs differs from human PDEs. The outcome of this work will be the identification of which PDEs found in nematodes are the best candidates for future efforts to develop potentn and selective nematicides targeting plant parasites.
Impacts
What was accomplished under these goals?
Current methods to control plant parasitic nematodes that infect agricultural crops are harmful to the environment and have deleterious consequences on non-target animals and host plants. Farmers, agricultural businesses, and consumers are united in seeking new nematicides that combine efficacy, specifcity, and food safety. The long-term goal of the work is to evaluate cyclic nucleotide phosphodiesterases (PDEs) as potential molecular targets for development of enzyme inhibitors that will serve as a nematicidal agent. During the past project period, an evolutionarily diverse set of ~100 vertebrate PDE sequences were collected to identify orthologs of Class I PDEs present in phytoparasitic nematodes. Using a draft version of the Meloidogyne hapla genome, we assembled the complete coding sequence for six PDE genes that are orthologous to PDEs present in four species of the genus Caenorhabditis and that correspond to vertebrate PDE families 1, 2, 3, 4, 8, and 10. Preliminary genomic analysis of M. incognita and Heterodera spp. reveals PDE genes closely related to C. elegans orthologs exist in other phytoparasitic nematodes. Thirteen amino acids in the catalytic domain are unanimously present in all sequences studied. In addition, there are multiple family-specific sites that distinguish the different PDE families. Structural homology modeling of nematode PDEs with reference to human PDEs whose structures are solved with drugs bound to the active site reveal that most of the known drug interaction sites in human PDEs are conserved in nematodes. However, six class-specific differences in drug interaction sites between nematode and vertebrate PDE3, PDE4, and PDE10 support the idea that nematode-specific inhibitor compounds targeting specific PDEs may be feasible.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
K. D. Schuster, K. B. Cahill, K. Morris, W. K. Thomas, and R. H. Cote. PDE inhibitors as potential pesticides targeting parasitic nematodes. Experimental Biology 2013 meeting C434 1052.6 (2013).
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