DISCOVERY AND DEVELOPMENT OF INSECTICIDES WITH NOVEL MECHANISMS OF ACTION FOR MOSQUITO CONTROL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013867
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Entomology

Non Technical Summary
Mosquitoes are 'the most dangerous animals on earth', because they transmit deadly pathogens to humans, including protozoans (e.g., malaria parasites), arboviruses (e.g., Zika virus), and filarial nematodes (e.g., filariasis). The yellow fever mosquito Aedes aegypti is the primary vector of the arboviruses that cause dengue, chikungunya , and Zika in humans, which are three important diseases that are emerging and reemerging around the globe. Effective vaccines and therapeutics for these viruses are not widely available or are years away from development/deployment. Thus, the primary strategy for controlling these diseases is to prevent the mosquito vector (Ae. aegypti) from biting humans, which typically involves the spraying of insecticides and/or the use of insecticide-treated materials. Unfortunately, only a limited number of insecticides are available for public health use, which has led to the evolution of insecticide resistance in Ae. aegypti. Thus, to overcome the challenge of insecticide resistance in mosquitoes and control the spread of emerging mosquito-borne arboviruses, there is a need for new control agents with novel mechanisms of action. In response to this need, the goal of the present Hatch proposal is to facilitate the development of a new generation of insecticides that will kill mosquitoes via mechanisms not previously exploited by existing insecticides.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3110	1020	20%
721	3110	1180	40%
721	3110	1150	40%

Knowledge Area
305 - Animal Physiological Processes; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3110 - Insects;

Field Of Science
1180 - Pharmacology; 1020 - Physiology; 1150 - Toxicology;

Keywords

insecticide resistance

mosquito

arbovirus

medicinal chemistry

natural products

small molecule

Goals / Objectives
Objective 1: Development of small-molecule inhibitors of Ae. aegypti Kir channels with mosquitocidal activity We discovered, in a high-throughput screen, 3 chemically-diverse, small-molecule inhibitors of Ae. aegypti Kir1 (AeKir1) that are toxic to adult female Ae. aegypti when applied to their cuticle. At least one of these inhibitors (VU041) is equally efficacious against pyrethroid-susceptible and pyrethroid-resistant strains of Ae. aegypti. In Objective 1, medicinal chemistry will be utilized to establish the structure-activity relationships of these lead compounds in a series of established, high-throughput in vitro screening assays. Specifically, we will aim to develop chemical analogs of our lead compounds that 1) potently inhibit AeKir1 and 2) do not inhibit a panel of human Kir channels. In addition, we will establish a similar in vitro assay for the Ap. mellifera Kir1 channel (ApKir1) to identify analogs that do not inhibit Kir1 of honey bees. We will then characterize the dose-toxicity relationship of the analogs when applied to the cuticle of representative pyrethroid-susceptible and pyrethroid-resistant strains of adult female Ae. aegypti. Analogs exhibiting similar toxicity against both strains of mosquitoes will be applied to the cuticle of adult honey bees (Ap. mellifera) to evaluate toxic effects on a representative beneficial insect. Analogs showing potent toxicity against mosquitoes, but not honey bees, will then be tested in vitro with radioligand displacement assays against a panel of 87 high-priority mammalian off targets (e.g., ion channels, G protein-coupled receptors) to assess potential mammalian toxicity.Objective 2: Discovery and development of natural plant compounds with mosquitocidal activity against Ae. aegypti Objective 2 will utilize three parallel approaches to construct a chemical library of CDIAL analogs. First, naturally-occurring analogs of CDIAL (i.e., drimane sesquiterpenes) will be isolated from three species of Cinnamosma that are endemic to Madagascar. Second, biotransformation reactions will be performed on CDIAL and its dimeric form (capsicodendrin) using a Penicillium sp., which is a fungus capable of structurally modifying sesquiterpene natural products, to generate natural derivatives. Third, synthetic analogs of CDIAL will be developed via medicinal chemistry. The resulting library of natural and synthetic analogs will be evaluated for toxicity against larval and adult female Ae. aegypti mosquitoes from representative pyrethroid-susceptible and pyrethroid-resistant strains. The results will elucidate the structure-toxicity relationship of CDIAL against Ae. aegypti mosquitoes and identify analogs of CDIAL with improved toxicity against adult female and/or larval Ae. aegypti mosquitoes. The most potent mosquitocidal analogs of CDIAL will be evaluated for potential mammalian/human safety by assessing their cytotoxicity against a normal cell line (Vero/African green monkey kidney cells).

Project Methods
Specific methods associated with this proposal are medicinal chemistry (lead compound optimization), natural product chemistry (biotransformation), heterolgous expression of ion channels, electrophysiology, adult and larval mosquito toxicological assays, honey bee toxicological assays, radioligand dispalcement assays, and cytotoxicity assays.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Target audiences include academics, medical and veterinary professionals, vector-management specialists, and industry who are faced with the challenge of controlling disease-spreading mosquitoes that are resistant to conventional insecticides. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for the individuals listed below who are trainees of the principal investigator (Dr. Peter M. Piermarini): Postdoctoral trainees: Dr. Megha Kalsi Masters student: Ms. Edna Alfaro Inocente Visiting Scholars: Mr. Anton Walter, Mr. Erick Martinez Rodriguez Research assistants: Ms. Nuris Acosta How have the results been disseminated to communities of interest?Results of our work have been disseminated through peer-reviewed journals, invited seminars at other universities, and invited and submitted presentations at local, regional, national, and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue our pursuit of the research goals as outlined in the Hatch proposal.

Impacts
What was accomplished under these goals? In collaboration with researchers at Vanderbilt University and the University of Nebraska, we have discovered several small molecule inhibitors of mosquito Kir1 channels (lead compounds) that possess insecticidal activity against larval and adult female mosquitoes. We are currently developing analogs of the lead compounds to improve their insecticidal potency and selectivity.In collaboration with researchers in the OSU College of Pharmacy, we have demonstrated that an endemic medicinal plant from Madagascar produces chemicals that are toxic and repellent to mosquitoes. Notably, one of the active plant compounds (OSU-1) is similarly toxic against insecticide-susceptible and insecticide-resistant strains of mosquitoes. Moreover, OSU-1 outperforms DEET, an industry standard, at repelling mosquitoes. Current efforts are identifying the chemical features of OSU-1 responsible for its insecticidal and repellent activity and understanding the mode of action of OSU-1 against mosquitoes to facilitate the development of improved insecticides and repellents for mosquito control.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Inocente EA, Nguyen B, Manwill PK, Benatrehina A, Kweka E, Wu S, Cheng X, Rakotondraibe LH, Piermarini PM (2019) Insecticidal and antifeedant activities of Malagasy medicinal plant (Cinnamosma sp.) extracts and drimane-type sesquiterpenes against Aedes aegypti mosquitoes. Insects 10: 373.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Aretz C, Kharade SV, Chronister K, Trigueros RR, Rodriguez EM, Piermarini PM, Denton JS, Hopkins CR (2020) Further SAR on the (phenylsulfonyl)piperazine scaffold as inhibitors of the Aedes aegypti Kir1 (AeKir) channel and larvicides. ChemMedChem. doi:10.1002/cmdc.202000598.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Manwill PK, Kalsi M, Wu S, Martinez Rodriguez EJ, Cheng X, Piermarini PM, Rakotondraibe HL (2020) Semi-synthetic cinnamodial analogues: Structural insights into the insecticidal and antifeedant activities of drimane sesquiterpenes against the mosquito Aedes aegypti. PLoS Neglected Tropical Diseases 14: e0008073. doi:10.1371/journal.pntd.0008073.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Kalsi M, Walter A, Lee B, DeLaat A, Rusconi Trigueros R, Happel K, Sepesy R, Nguyen B, Manwill PK, Rakotondraibe HL, Piermarini PM (2020) Stop the crop: insights into the insecticidal mode of action of cinnamodial against mosquitoes. bioRxiv: 2020.2005.2030.125203. doi:10.1101/2020.05.30.125203.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Target audiences include academics, medical and veterinary professionals, vector-management specialists, and industrywho are faced with the challenge of controlling disease-spreading mosquitoes that are resistant to conventional insecticides. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for the individuals listed below who are trainees of the principal investigator (Dr. Peter M. Piermarini): Postdoctoral trainees: Dr. Megha Kalsi Masters student: Ms. Edna Alfaro Inocente Undergraduate students: Mr. Andrew DeLaat, Mr. Jacob Newsome, Ms. Grace Lee Visiting Scholars: Mr. Anton Walter, Ms. Renata Rusconi Trigueros, Mr. Erick Martinez Rodriguez Research assistants: Ms. Nuris Acosta, Ms. Rose Sepesy How have the results been disseminated to communities of interest?Results of our work have been disseminated through peer-reviewed journals, invited seminars at other universities, and invited and submitted presentations at local, regional, national, and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue our pursuit of the research goals as outlined in the Hatch proposal.

Impacts
What was accomplished under these goals? In collaboration with researchers at Vanderbilty University and the University of Nebraska, we have discovered several small molecule inhibitors of mosquito Kir1 channels (lead compounds) that possess insecticidal activity against larval and adult female mosquitoes. We are currently developing analogs of the lead compounds to improve their insecticidal potency and selectivity.In collaboration with researchers in the OSU College of Pharmacy, we have demonstrated that an endemic medicinal plant from Madagascar produces chemicals that are toxic and repellent to mosquitoes. Notably, one of the active plant compounds (OSU-1) is similarly toxic against insecticide-susceptible and insecticide-resistant strains of mosquitoes. Moreover, OSU-1 outperforms DEET, an industry standard, at repelling mosquitoes. Current efforts are identifying the chemical features of OSU-1 responsible for its insecticidal and repellent activity and understanding the mode of action of OSU-1 against mosquitoes to facilitate the development of improved insecticides and repellents for mosquito control.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Piermarini PM, Inocente EA, Acosta N, Hopkins CR, Denton JS, Michel AP (2018) Inward rectifier potassium (Kir) channels in the soybean aphid Aphis glycines: Functional characterization, pharmacology, and toxicology. J Insect Physiol 110: 57-65. doi:https://doi.org/10.1016/j.jinsphys.2018.09.001
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Rusconi Trigueros R, Hopkins CR, Denton JS, Piermarini PM (2018) Pharmacological inhibition of inward rectifier potassium channels induces lethality in larval Aedes aegypti. Insects 9. doi:10.3390/insects9040163
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Aretz C, Morwitzer MJ, Sanford A, Hogan AM, Portillo M, Kharade SV, Kramer M, McArthey J, Trigueros RR, Piermarini PM, Denton JS, Hopkins CR (2019) Discovery and characterization of 2-nitro-5-(4-(phenylsulfonyl)piperazin-1-yl)-N-(pyridin-4-ylmethyl)anilines as novel inhibitors of the Aedes aegypti Kir1 (AeKir1) channel. ACS Infect Dis. doi:10.1021/acsinfecdis.8b00368.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kalsi M, Gillen C, Piermarini PM (2019) Heterologous expression of Aedes aegypti cation chloride cotransporter 2 (aeCCC2) in Xenopus laevis oocytes induces an enigmatic Na+/Li+ conductance. Insects 10: 71.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Montes de Oca-Aguilar AC, De Luna E, Rebollar-T�llez EA, Piermarini PM, Ib��ez-Bernal S (2019) Morphological discontinuous variation and disparity in Lutzomyia (Tricholateralis) cruciata Coquillett, 1907 are not related to contrasting environmental factors in two biogeographical provinces. Zoomorphology. doi:10.1007/s00435-019-00450-8.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Edna Alfaro Inocente, Harinantenaina Liva Rakotondraibe, and Peter M. Piermarini (2018) Toxic, antifeedant, and repellent activity of drimane sesquiterpenes from the medicinal plant Cinnamosma fragrans against Aedes aegypti mosquitoes. American Society of Tropical Medicine and Hygeine Annual Meeting, New Orleans, LA. Submitted Poster.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Christopher M. Gillen, Grace F. Riley, John C. Crow, Adrienne C. DeBrosse, Mary Sawyer, Megha Kalsi, Peter M. Piermarini (2018) Sequence analysis, expression, and preliminary functional characterization of Aedes aegypti sodium-dependent cation-chloride cotransporters. 2018 APS Intersociety Meeting - Comparative Physiology, New Orleans, LA. Submitted Poster.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: James McCarthey, Christopher D. Aretz, Melody J. Morwitzer, Austin G. Sanford, Alicia M. Hogan, Madelene V. Portillo, Sujay V. Kharade, Meghan Kramer, Renata Rusconi Trigueros, Peter M. Piermarini, Jerod S. Denton, Corey R. Hopkins (2019) Discovery and characterization of 2-nitro-5-(4-(phenylsulfonyl)piperazin-1-yl)-N-(pyridin-4-ylmethyl)anilines as novel inhibitors of the Aedes aegypti Kir1 (AeKir1) potassium channel. Experimental Biology 2019 Annual Meeting. Submitted Poster.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Peter M. Piermarini, Xiaolin Cheng, and Harinantenaina Liva Rakotondraibe (2019) Take your medicine: developing novel insecticides and repellents for mosquito control from medicinal plants.EMBO Conference: Molecular and population biology of mosquitoes and other disease vectors. Chania, Greece. Invited talk.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Piermarini PM, Kalsi M, Rakotondraibe LH (2019) STOP THE CROP: TOWARDS ELUCIDATING THE MODE AND MECHANISM OF ACTION OF A NATURAL PRODUCT INSECTICIDE. Insect Biotechnology Conference, Niagara on the Lake, Canada. Submitted Talk.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Erick Martinez Rodriguez, Megha Kalsi, Noah Rosenblatt, Morgan Stanley, Peter M. Piermarini (2019) Toxicity of carbon-based nanoparticles on pyrethroid-susceptible and pyrethroid-resistant larvae of the yellow fever mosquito Aedes aegypti. 7th Symposium of Zamorano in the USA, Raleigh, NC. Submitted Poster.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yang L, Turo KJ, Riley CB, Inocente EA, Tian J, Hoekstra NC, Piermarini PM, Gardiner MM (2019) Can urban greening increase vector abundance in cities? The impact of mowing, local vegetation, and landscape composition on adult mosquito populations. Urban Ecosystems. doi:10.1007/s11252-019-00857-7.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Manwill P, Kalsi M, Wu S, Cheng X, Piermarini PM, Rakotondraibe HL (2019) Semi-synthetic cinnamodial analogues: structural Insights into the insecticidal and antifeedant activities of drimane sesquiterpenes against the mosquito Aedes aegypti. bioRxiv: 536961. doi:10.1101/536961.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Target audiences include academics, medical and veterinary professionals, vector-management specialists, and industry who are faced with the challenge of controlling disease-spreading mosquitoes that are resistant to traditional insecticides. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has providing training and professional development opportunities for the individuals listed below who are trainees of the principal investigator (Dr. Peter M. Piermarini): Postdoctoral trainees: Dr. Megha Kalsi Masters student: Ms. Edna Alfaro Inocente Undergraduate students: Mr. Andrew DeLaat, Ms. Bao Nguyen, Mr. Yamil Miranda Negron, Ms. Katharina Happel Visiting Scholars: Ms. Renata Rusconi Trigueros Research assistants: Ms. Nuris Acosta How have the results been disseminated to communities of interest?Results of our work have been disseminated through peer-reviewed journals, invited seminars at other universities, and invited and submitted presentations at local, regional, national, and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue our pursuit of the research goals as outlined in the Hatch proposal.

Impacts
What was accomplished under these goals? In collaboration with researchers at Vanderbilty University and the University of Nebraska, we have discovered several small molecule inhibitors of mosquito Kir1 channels (lead compounds) that possess insecticidal activity against larval and adult female mosquitoes. We are currently developing analogs of the lead compounds to improve their insecticidal potency and selectivity. In collaboration with researchers in the OSU College of Pharmacy, we have demonstrated that an endemic medicinal plant from Madagascar produces chemicals that are toxic and repellent to mosquitoes. Notably, one of the active plant compounds (OSU-1) is similarly toxic against insecticide-susceptible and insecticide-resistant strains of mosquitoes. Moreover, OSU-1 outperforms DEET, an industry standard, at repelling mosquitoes. Current efforts are identifying the chemical features of OSU-1 responsible for its insecticidal and repellent activity and understanding the mechanism of action of OSU-1 against mosquitoes to facilitate the development of improved insecticides and repellents for mosquito control.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Calkins T, Piermarini P (2017) A Blood Meal Enhances Innexin mRNA Expression in the Midgut, Malpighian Tubules, and Ovaries of the Yellow Fever Mosquito Aedes aegypti. Insects 8: 122.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Calkins TL, DeLaat A, Piermarini PM (2017) Physiological characterization and regulation of the contractile properties of the mosquito ventral diverticulum (crop). Journal of Insect Physiology 103: 98-106.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Calkins TL, Piermarini PM (2017) Mosquito Gap Junctions: Molecular Biology, Physiology, and Potential for Insecticide Development. Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest Control: American Chemical Society. pp. 91-110.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Piermarini PM, Akuma DC, Crow JC, Jamil TL, Kerkhoff WG, Viel KCMF, Gillen CM (2017) Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 214: 40-49.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Miller JR, Koren S, Dilley KA, Puri V, Brown DM, Harkins DM, Thibaud-Nissen F, Rosen B, Chen X-G, Tu Z, Sharakhov IV, Sharakhova MV, Sebra R, Stockwell TB, Bergman NH, Sutton GG, Phillippy AM, Piermarini PM, Shabman RS (2018) Analysis of the�Aedes albopictus�C6/36 genome provides insight into cell line utility for viral propagation. GigaScience: gix135-gix135.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Piermarini PM, Inocente E, Shaya M, and Rakotondraibe LR Toxicity and repellency of a Madagascan medicinal plant (Cinnamosma fragrans) and its major secondary metabolite against mosquitoes Annual Meeting of the Entomological Society of America, Denver, CO (2017) PBT Section SymposiumInsecticide Science in the 21st Century: The Age of Increased Targets, Resistance, and Biorational Insecticides. Invited National Symposium Talk.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Calkins TL and Piermarini PM Gap junctions and crop contraction of Aedes aegypti Annual Meeting of the Entomological Society of America, Denver, CO (2017) Submitted National Poster.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Inocente EA, Shaya M, Acosta N, Rakotondraibe LH, Piermarini PM (2018) A natural agonist of mosquito TRPA1 from the medicinal plant Cinnamosma fragrans that is toxic, antifeedant, and repellent to the yellow fever mosquito Aedes aegypti. PLoS Negl Trop Dis 12: e0006265.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kolosov D, Piermarini PM, O'Donnell MJ (2018) Malpighian tubules of Trichoplusia ni: recycling ions via gap junctions and switching between secretion and reabsorption of Na(+) and K(+) in the distal ileac plexus. J Exp Biol. 221, jeb172296.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kolosov D, Tauqir M, Rajaruban S, Piermarini PM, Donini A, O'Donnell MJ (2018) Molecular mechanisms of bi-directional ion transport in the Malpighian tubules of a lepidopteran crop pest, Trichoplusia ni. J Insect Physiol 109: 55-68.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Alfaro E, Shaya M, Acosta N, Rakotondraibe HL, and Piermarini PM Insecticidal and repellent activities of natural drimane sesquiterpenes from Madagascan medicinal plants (Cinnamosma sp.) against Aedes aegypti mosquitoes Annual Meeting of the Ohio Valley Entomological Association, Columbus, OH (2017) Submitted Talk.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Piermarini PM "Discovery of Natural and Synthetic Small Molecule Mosquitocides With Novel Mechanisms of Action" Annual Meeting of the Michigan Mosquito Control Association, Lansing, MI (2018) Invited Talk.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Piermarini PM "Discovery of Natural and Synthetic Small Molecule Mosquitocides With Novel Mechanisms of Action" Paul A. Dahm Memorial Lecture, Department of Entomology, Iowa State University (2018) Invited Seminar.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: DeLaat AR, Rakotondraibe L, Piermarini PM "The Effect of Natural Products on Digestive Muscle Contractions in Female Aedes aegypti Mosquitoes" Experimental Biology 2018 Annual Conference, San Diego, CA (2018) Submitted poster.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Manwill PK, Kalsi M, Piermarini PM, Rakotondraibe HL "The Structure-Activity Relationship of Natural and Semi-Synthetic Cinnamodial Analogues Against the Aedes aegypti Mosquito" American Society of Pharmacognosy Annual Conference, Lexington, KY (2018) Submitted poster.