Progress 09/15/05 to 09/14/07
Outputs
OUTPUTS: In order to build upon the potency of the scaffolds from Phase I studies while circumventing their drawbacks, Divergence has entered into an exclusive relationship with a cheminformatics company, Cresset BioMolecular Discovery Ltd. As a result of this collaboration, over 600 new compounds have been tested against C. elegans, with a significant percentage demonstrating activity comparable to the azobenze series. The search, therefore, successfully identified a valuable collection of structurally diverse broad spectrum anthelmintic compounds likely to work by a common and novel mode of action. Divergence has established a highly productive collaboration effort with a large animal health company to perform larval migration assays against H. contortus using the compounds identified from our in-house screening effort. We have tested over 200 compounds as part of this effort. In addition, we have initiated testing of lead compounds against H. contortus and T. circumcincta in a
rodent model of infection.
PARTICIPANTS: Divergence Team Members: Dr. Michelle Coutu Hresko, Principal Investigator - Dr. Hresko served as the PI on this project and was responsible for coordination of all efforts, including experimental design, implementation of protocols and communication with all project team members. Dr. Michael Crawford was responsible for all in vitro assays and development and oversight of the implementation of the biochemical assays. Dr. Matthew Dimmic, Dr. James McCarter and Dr. Jeremy Williams took responsibility for assay development, bioinformatic and cheminformatic searches for the selection and acquisiton of additional chemical candidates, and managed the relationships with Opus Organics, Nanosyn and Cresset. Dr. Robert Schenkel managed the relationship with the external animal health company. Mr. William P. Haakenson, Jr. and Mr. Andrew Martens served as technicians on this project under the direction of Dr. Hresko. Bomi Framroze - Opus Organics P. Ltd. and Nanosyn, Inc.
performed all compound synthesis for Divergence. Cresset Biomolecular Discovery, LTD. partners with Divergence on biomolecular discovery.
TARGET AUDIENCES: Divergence's strategy for the commercialization of anthelmintic compounds is to capture value through an alliance with one or more of the major global animal health companies. These companies already have in place the infrastructure, from both a Research/Development and commercial perspective, which will allow them to rapidly bring to market a major new anthelmintic. Divergence has negotiated arrangements with five of these companies to test the chemistry developed in Phase I and Phase II activities. In addition, Divergence has a strong patent estate in the class of chemistry used in the Phase I and Phase II activities. Divergence is committed to an aggressive pursuit of intellectual property, which strengthens the opportunity to be commercially successful.
PROJECT MODIFICATIONS: The project proceeded along the proposed objectives with additional work performed as success was met throughout the project.
Impacts
During the course of this USDA-funded project, Divergence has been able to improve upon both the anthelmintic potency as well as the toxicology profile of the original azobenze, stilbene, and chalcone scaffolds. Based on the establishment of several productive collaborations in cheminformatics, chemical synthesis, and testing, greater than 600 compounds have been purchased commercially or synthesized, with a hit rate far exceeding other anthelmintic screening efforts. Current areas of emphasis include improving the potency in the animal model of infection as well as determining the mode of action of these promising compounds, which would be valuable not only for increasing intrinsic potency but also for resistance management, an emerging threat to anthelmintic therapy in the animal health industry.
Publications
- Palavalli LH, Brendza KM, Haakenson W, Cahoon RE, McLaird M, Hicks LM, McCarter JP, Williams DJ, Hresko MC, Jez JM. (2006) Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis. Biochemistry. 45(19):6056-65.
- Brendza KM, Haakenson W, Cahoon RE, Hicks LM, Palavalli LH, Chiapelli BJ, McLaird M, McCarter JP, Williams DJ, Hresko MC, Jez JM. (2007) Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans. Biochem J. 404(3):439-48.
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Progress 09/15/05 to 09/14/06
Outputs
Aim I:We established a relationship with a veterinary division of a major pharmaceutical company to perform larval migration assays against H. contortus. 9 of our compounds demonstrated effects at or below the IC50 of levamisole.The in vitro efficacy of 23 compounds was sufficient for advancement into a small rodent model for H. contortus infection. 10 of the compounds have been evaluated with 2 demonstrating partial control of H. contortus infection at 100mg/kg. The other compounds did not significantly control infection. Metabolic stability assays have been initiated for advancement prioritization. We continue to send compounds active against C. elegans to our collaborator for testing.We are developing a larval migration assay following the protocol of Kotze et. al., 2006 to give flexibility with assay methodology and allow us to share data with USDA and other potential partners. Aim II:Using our previously developed biochemical assay of recombinant C. elegans PMT-1
and PMT-2 expressed in bacteria (Palavalli et al., 2006.), we tested the ability of selected compounds effective against C. elegans to inhibit PMT-1 or PMT-2 in vitro. None of our lead compounds significantly inhibit the PMT enzymes, making it unlikely that the nematicidal properties of these compounds are due to PMT inhibition. We are collaborating with cheminformatic companies to identify new compounds for testing and will use the data to refine new SAR models (Aim III). We also plan to further characterize the PMT enzymes, including solving the crystal structure. Aim III:3 active scaffolds were identified through work with Tripos.We have tested additional azobenzene derivatives and over 300 compounds related to the other two scaffolds.The additional azobenzenes were designed by our scientists to be non-mutagenic and were synthesized by Opus Organics.Using SAR models to help guide molecule selection, we chose to synthesize four novel azobenzenes (4296, 4299, 4350, 4356).These were
tested for mutagenicity using the Ames test; the molecules were not Ames positive.When tested in our C. elegans assay, 2 showed no effect and 2 showed effects that were weaker than other derivatives.Through work with a 2nd cheminformatic company we have tested over 600 compounds in the past 5 months against C. elegans. We have identified 78 compounds that show effects within 24 hours of exposure to L4 larvae at concentrations at or below 12.5 ppm, a hit rate of 12.5 percent. 28 of these compounds were tested by our collaborating animal health company and 13 of those 28 progressed into in vivo screens. Aim IV:Cheminformatic approaches predicting potential mutagenicity of compounds are being performed on c.elegans active compounds to prioritize the lead scaffolds. In vitro metabolic stability assays are performed with mammalian microsomes on active compounds to provide an indication of degradation by the host organism. We are using a cell-based toxicity assay that measures cellular ATP
content (Cell Titer-Glo by Promega) to provide an early indication of scaffold toxicity against mammalian cells. We tested 44 compounds as part of our Phase II studies and 31 were found to be non-toxic at or below 75mM.
Impacts
Nematode control is essential to the economic viability of livestock producers and is an obligatory part of veterinary care of companion animals. Heavy use of existing anthelmintics has led to the evolution of drug-resistant nematodes. No new families of nematicidal chemistry have entered the market in the last 20 years. The development of a novel chemistry would represent a significant advancement in the treatment of nematode infections of animals and a significant potential economic opportunity.
Publications
- No publications reported this period
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