Progress 01/01/16 to 05/31/19
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 more sustainable 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 #1-2 were targeted at a scientific meeting (2018 Gordon Research Conference on Cyclic Nucleotide Phosphodiesterases, June, 2018) at whcih the work was presented in a poster presentation. Approximately 150 attended the meeting. Groups #1-2 were targeted at a poster presentation during the 2018 UNH Graduate Research Conference at which 200 people attended. Groups #1-4 were targeted in press releases associated with the publication of a journal article in PLOS One in 2019. Number of people targeted is not known. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two undergraduate students conducting senior theses, twograduate students conducting doctoral research, one postdoctoral research associate, and one research tehcnician have been trained during the project period in the design, conduct, and interpretation of these scientific experiments. The knowledge and skills acquired will advance these individuals in theirchosen careers as researchscientists. How have the results been disseminated to communities of interest?The results were recently published in a peer-reviewed scientific journal:Schuster, K.D., Mohammadi, M., Cahill, K.B., Matte, S.L., Maillet, A.D., Vashisth, H., and Cote, R.H. (2019). Pharmacological and molecular dynamics analyses of differences in inhibitor binding to human and nematode PDE4: implications for management of parasitic nematodes. PLOS One 14, e0214554. In the 4.5 months since publication, this journal article has been viewed 795 times at the publisher's web site. A press release was widely distributed to the media:https://www.unh.edu/unhtoday/news/release/2019/03/27/unh-researchers-advance-effort-manage-parasitic-roundworms-patent-pending. Posting of the citation on ResearchGate has generated 51 views of the article to date. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: Plant-parasitic nematodes are a major cause of reduced agricultural productivity ($80-$100 billion in crop damage annually). Our goal was to identify novel compounds that effectively target plant parasitic nematodes without adverse consequences to plants or other animals. We identified potential nematicidal targets as members of the phosphodiesterase (PDE) enzyme family that degrade cyclic nucleotides involved in cellular signaling pathways important for growth and development, sensory perception (especially chemosensation), and locomotion. Bioinformatic analyses of nematode genomes identified six of the eleven vertebrate PDE families; these six PDE families are found in all nematode species for which data is available, including plant and animal parasitic nematodes. Relying on PDE inhibitor compounds already developed for therapeutic uses in humans, we determined that nematode PDEs can be inhibited by these same compounds, although with lower potency than for human PDEs. Using atomic-level simulations of the binding of human and nematode PDEs to specific inhibitor compounds allowed us to detect differences in the inhibitor binding pocket. This work is an important step in the design of next-generation nematicides that target plant parasitic nematodes without adverse effects on plants or animals. Such compounds will benefit U.S. agriculture by reducing crop damage, improving the sustainability of farming operations, enhancing food security, and eliminating adverse environmental and public health impacts associated with traditional nematicides. Objective 1: Characterize the pharmacological properties of nematode PDE catalytic domains. 1A. Clone and express the catalytic domains of selected PDEs fromM. hapla, C. elegans, and H. sapiens. We conducted an extensive bioinformatic analysis of PDE genes in the nematode phylum (including free-living as well as plant and animal parasitic nematodes), and found that nematode orthologs of vertebrate PDE1, PDE2, PDE3, PDE4, PDE8, and PDE10 are present throughout the phylum. The observation that nematode PDE protein sequences diverge significantly from vertebrate PDE orthologs support the hypothesis that compounds can be identified that bind specifically to nematode PDEs, but not to vertebrate PDEs--a necessary prerequisite for developing nematicides that are non-toxic to humans and other vertebrate animals. We then cloned, expressed, and purified recombinant catalytic domains of human and nematode PDE4. This PDE family was chosen based on the scientific literature as well as relying on experiments in which nematodes exposed to PDE4 inhibitors displayed altered behavior. 1B. Determine the kinetic and pharmacological properties of nematode PDEs. The substrate specificity of nematode PDE4 was examined and we determined that the kinetic properties were in very close agreement with the human PDE4 ortholog: both nematode and human PDE4 were cyclic AMP-specific enzymes with negligible activity toward cyclic GMP, and the Michaelis constant for cyclic AMP was identical (within experimental error) for human and nematode PDE4. Investigations of the pharmacological sensitivity of nematode PDE4 to inhibitors originally designed to target human PDE4 was carried out. We observed that roflumilast (a human PDE4-selective inhibitor) and zardaverine (selective for human PDE3 and PDE4) were 159- and 77-fold less potent, respectively, in inhibiting nematode PDE4. The non-specific PDE inhibitor, isobutyl methyl xanthine (IBMX), had similar affinity for nematode and human PDE4. Objective 2: Identification of novel compounds that bind to and inhibit the catalytic activity of nematode (M. hapla or C. elegans) PDEs. 2A. Homology modeling of nematode PDE catalytic domains based on human PDE templates. Amino acid sequence alignment of human and nematode PDE4, in conjunction with published x-ray crystal structures for human PDE4 complexed with the same three inhibitor compounds described above, resulted in structural homology models of nematode PDE4 with each inhibitor bound to the enzyme active site. 2B. In silico docking of inhibitor-like fragments to nematode PDE catalytic domains. Initial efforts to conduct in silico docking experiments with PDE inhibitors were unsuccessful when using available computational methods. Instead, we used the structural homology models of nematode and human PDE4 complexed with roflumilast, zardaverine, and IBMX as a template for atomistic molecular dynamics (MD) simulations of the binding interactions of each inhibitor with the PDE4 catalytic site. Simulations of the unliganded catalytic domains of human and nematode PDE4 demonstrated the stability of our structural models, validating these models for subsequent analyses of inhibitor binding to the enzyme active site. MD simulations with both human and nematode PDE4 binding to the three selected inhibitors identified 32 amino acid residues with 5 Å of the ligand binding site. These 32 amino acid residues represent the ligand binding pocket and were examined for their individual contributions to the binding of an inhibitor to the enzyme. Of these 32 amino acid residues, five displayed significant differences in non-bonded interaction energies that could account for the differential binding affinities of roflumilast and zardaverine. One site (Phe506 in the human PDE4 amino acid sequence corresponding to Tyr253 in nematode PDE4) is predicted to alter the binding conformation of roflumilast and zardaverine (but not IBMX) into a less energetically favorable state for the nematode enzyme. The pharmacological differences in sensitivity to PDE4 inhibitors in conjunction with differences in the amino acids comprising the inhibitor binding sites of human and nematode PDE4 catalytic domains together support the feasibility of designing the next generation of nematicides (and anthelmintics targeting animal parasites of agricultural importance) that could selectively bind to nematode PDEs. 2C. Pharmacological screening of candidate fragment compounds for differential inhibition of catalysis of nematode and human PDEs. The final sub-aim was, in retrospect, was overly ambitious and so we restricted efforts to characterization of available PDE inhibitor compounds for testing (as described above).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Schuster, K.D., Mohammadi, M., Cahill, K.B., Matte, S.L., Maillet, A.D., Vashisth, H., and Cote, R.H. (2019). Pharmacological and molecular dynamics analyses of differences in inhibitor binding to human and nematode PDE4: implications for management of parasitic nematodes. PLOS One 14, e0214554.
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Progress 01/01/16 to 12/31/16
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 more sustainabletools 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 (2016 Society of Nemtologists and Organization of Nematologists of Tropical America annual meetingsin June, 2016at 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, 2016at which 15 people were present. *Groups #1-2 were targeted at a poster presentation during the UNH Graduate Research Conference in Mrch 2016 at which 150were present. Changes/Problems:Identifying and hiring personnel to work on the project was slower than anticipated and delayed the initiation of the research and the expenditure of funds. This issue has been solved. The difficulties with obtaining catalytically active, recombinantly expressed PDE catalytic domains described in the Accomplishments section were anticipated and will be addressed as described in the original Project Narrative. Limitations in the ability of available molecular docking programs (e.g., AutoDock) to accurately predict the binding affinity of inhibitors to the PDE catalytic site will be addressed by evaluating other approaches (e.g., molecular dynamics) for predicting which compounds may have high affinity for the various human and nematode PDE families. What opportunities for training and professional development has the project provided?One undergraduate student conducting a senior thesis, one graduate student conducting doctoral research, and one postdoctoral research associate have been trained during the project period in the design, conduct, and interpretationof scientific experiments. The knowledge and skills acquired will advance these individuals in teh chosen careers as research scientists. How have the results been disseminated to communities of interest?This work was presented as part of a scientific poster at the University of New Hampshire Graduate Research Conference in March, 2016, at which 150 people from across all academic disciplines were present. Aspects of this work were also presented at the2016 Society of Nemtologists and Organization of Nematologists of Tropical America annual meetingin Montreal (July, 2016) at which100people attended the posterpresentation. Meeting attendees includedexperts in the field of nematology, scientific directors from major agrichemical comanies, and local media. What do you plan to do during the next reporting period to accomplish the goals?In the second and final year of the project period, we will undertake the following work: Objective 1: Continuation of Activities A and B will proceed in parallel. Unexpected difficulties in recombinant expression of PDE catalytic domains will be addressed as described in the "Pitfalls" section of the Project Narrative, namely reliance on a eukaryotic cell expression system that has a greater likelihood of expressing functionally active, purifiedPDE catalytic domain proteins. Objective 2: With Activity A essentially completed, we can now undertake Activities B and C as planned. Due to limitations of the molecular docking program AutoDock to accurately predict the binding affinity of inhibitor compounds for their binding sites in PDE catalytic domains, we are exploring a molecular dyanmics approach to evaluating the ability of test compounds to bind to the catalytic pocket of different human and nematode PDE family members.
Impacts
What was accomplished under these goals?
Impact: Plant parasitic nematodes are the most destructive of plant pathogens worldwide. Traditional nematicides for plant parasitic nematodes (e.g., methyl bromide, carbamates) are indiscriminate in their toxic effectson other animals. Development of novel nematicides precisely targeting phytoparasitic nematodes is needed to enhance agricultural crop productivity and sustainability by reducing damage to plants from biotic stresses. Cyclic nucleotides are universal intracellular second messengers, and disruption of their metabolism is known to have large physiological effects on the physiology and lifecycle of nematodes. Targeting the phosphodiesterase enzyme that degrades cyclic nucleotides using highly specific inhibitors may lead to development of "next-generation" nematicides targeting plant parasitic nematodeswhich lack adverse effects on plants, vertebrate animals, and the environment and thus enhance yields of agriculturally important crops. Objective 1 Activity A:We have subcloned and expressed H. sapiens PDE3, PDE4, and PDE10 catalytic domains in a recombinant bacterial expression system. The human PDE4 catalytic domain has been affinity purified and shown to retain enzymatic properties consistent with previous literature reports. Human PDE3 and PDE10 catalytic domains have been expressed in bacteria, but only PDE10 is able to hydrolyze cyclic AMP whereas purified PDE3 lacks significant catalytic activity. Nematode PDE3, PDE4, and PDE10 catalytic domains were also subcloned into bacterial expression vectors with a variety of fusion partners, using synthetically prepared DNA fragments that were codon-optimized for bacterial expression. We obtained low-level expression of functionally active Caenorhabditis elegans (C. elegans) PDE4 catalytic domain, but C. elegans PDE3 and PDE10 as well as Meloidogyne hapla PDE4 failed to generate soluble, catalytically active protein when expressed as either a glutathione-S-transferase (GST) fusion protein or with a maltose binding protein fusion partner. To address this issue, we have subcloned several nematode PDE catalytic domains into an eukaryotic expression vector (pIEx/Bac) which has been used for transient transfection into cultured eukaryotic Sf9 insect cells. Activity B: Having obtained purified, catalytically active human and C. elegans PDE4 catalytic domains, we have carried out side-by-side comparisons of the relative binding affinity of these two enzymes with several PDE inhibitor compounds. The nonspecific inhibitor, IBMX, inhibits human and nematode PDE4 similarly, with less than a 2-fold difference in inhibitory potency. Zardaverine, a selective inhibitor of human PDE3 and PDE4 family members, inhibits human PDE4 60-fold more potently than C. elegans PDE4. Roflumilast (a potent and specific inhibitor of human PDE4) requires 200-fold higher concentrations to inhibit C. elegans PDE4. We observed a good correlation between the number of inhibitor binding interactions (identified by structural determinations of the human PDE complexed with these inhibitors, and compared with the structural homology model for C. elegans PDE4 catalytic domain) and the selectivity of these inhibitors for human over nematode PDE4. Objective 2 Activity A: As the first step in predicting the structure of the catalytic domains of Class I cyclic nucleotide phosphodiesterases (PDEs) found in nematodes, we created an alignment of amino acid sequences for all known human PDEs plus previously determined sequences for Caenorhabditis elegans (C. elegans) PDEs available in WormBase. Using these sequences as queries in BLAST searches, we identified putative PDEs in several parasitic nematode species, including Brugia malayi, Pristionchus pacificus, and Trichinella spiralis. For Meloidogyne hapla, putative PDE2, PDE3, PDE4, and PDE8 sequences were identified either from the annotated genome or from prediction of the open reading frames of putative PDE sequences using MAKER to define intron-exon boundaries. In the case of PDE1 and PDE10 sequences in M. hapla, MAKER failed to provide reliable sequence predictions, so these were manually predicted based on available information of nematode intron and exon boundary sequences. To determine orthology of the nematode PDE sequences, a bioinformatics pipeline was created in which PDE sequences for organisms where the organismal phylogenetic relationship has been established were combined with the nematode sequences. The results supported the presence of orthologs of vertebrate PDE1, PDE2, PDE3, PDE4, PDE8 (referred to as PDE-6 in C. elegans gene nomenclature), and PDE10 (PDE-5 in C. elegans) in the tested nematode species. Structural homology modeling (using SWISS-MODEL) of the C. elegans and M. hapla PDE4 orthologs of human PDE4B reveal conservation of the three-dimensional structure of the catalytic domains (Pfam PF00233) of human and the two nematode PDEs. When these homology models for nematode PDEs were tested with Autodock to compare the predicted conformation of PDE inhibitors (including 3-isobutyl-1-methylxanthine (IBMX) and roflumilast) bound to the catalytic domains of human versus nematode PDE4, we observed altered binding conformations for the PDE4 inhibitor roflumilast but not for the non-specific inhibitor, IBMX. These in silico results support the hypothesis that differences in the amino acid sequences of orthologous vertebrate and nematode PDEs will result in differential susceptibility to enzyme inhibition. Creating homology models for nematode PDE catalytic domains was a prerequisite for conducting Activities B and C. In summary, these results advance the overall objective of identifying novel, potent nematode PDE inhibitors that can discriminate nematode from vertebrate PDEs, hence serving as nematicides lacking adverse effects on vertebrates.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Schuster, K. D., et al. (2016). Nematode phosphodiesterases are promising targets for novel nematicides. Society of Nematology Annual Meeting. Montreal, Quebec, Canada: 116.
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