Utilizing Ayurvedic literature for the identification of novel phytochemical inhibitors of botulinum neurotoxin A

Authors: YALAMANCHILI, CHINNI - University Of Mississippi; MANDA, VAMSHI - University Of Mississippi; CHITTIBOYINA, AMAR - University Of Mississippi; GUERNIERI, REBECCA - Us Army Medical Research Institute Of Infectious Diseases; HARRELL, JR., WILLIAM - Us Army Medical Research Institute Of Infectious Diseases; WEBB, ROBERT - Us Army Medical Research Institute Of Infectious Diseases; SMITH, LEONARD - Us Army Medical Research Institute Of Infectious Diseases; KHAN, IKHLAS - University Of Mississippi

Submitted to: Journal of Ethnopharmacology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/25/2016
Publication Date: 7/26/2016
Citation: Yalamanchili, C., Manda, V.K., Chittiboyina, A.G., Guernieri, R.L., Harrell, Jr., W.A., Webb, R.P., Smith, L.A., Khan, I.A. 2016. Utilizing Ayurvedic literature for the identification of novel phytochemical inhibitors of botulinum neurotoxin A. Journal of Ethnopharmacology. 197:211-217.

Interpretive Summary: Ayurveda, an ancient holistic system of health care practiced on the Indian subcontinent, utilizes a number of multi-plant formulations and is considered as a potential source for novel treatments, as well as the identification of new drugs. Our aim is to identify novel phytochemicals for the inhibition of bacterial exotoxin, Botulinum neurotoxin A (BoNT A) based on Ayurvedic literature. Fifteen plants were selected from the book ‘Diagnosis and treatment of diseases in Ayurveda’ by Vaidya Bhagwan Dash and Lalitesh Kashyap, based on their frequency of use in the formulations used for the treatment of six diseases with neuromuscular symptoms similar to that to botulism. Phytochemicals from these plants were screened using in silico, in vitro and ex vivo methods. Structures of 850 reported phytochemicals from 15 plants were docked inside six reported BoNT A crystal structures using ensemble docking module in Maestro (Schrodinger, LLE). From the docking scores, and structural diversity, 30 compounds were screened in vitro using BoNT A Light Chain (LC) HPLC/UPLC based protease assay. Few compounds showed 60 to 70% inhibition of BoNT A LC when compared to positive controls NSC 84094 and CB7967495 (80 to 95%). Based on the in vitro screening results, seven compounds were further tested using ex vivo mouse phrenic nerve-hemidiaphragm assay. At 20 µM, acoric acid showed marginal protection by inhibiting the loss of twitch tension in the hemidiaphragm in the presence of BoNT A. The identification of acoric acid, a novel scaffold against BoNT A, exemplifies the utility of Ayurvedic literature for the discovery of novel drugs.

Technical Abstract: Ethnopharmacological relevance: Ayurveda, an ancient holistic system of health care practiced on the Indian subcontinent, utilizes a number of multi-plant formulations and is considered by many as a potential source for novel treatments, as well as the identification of new drugs. Our aim is to identify novel phytochemicals for the inhibition of bacterial exotoxin, botulinum neurotoxin A (BoNT/A) based on Ayurvedic literature. BoNT/A is released by Clostridium species, which when ingested, inhibits the release of acetylcholine by concentrating at the neuromuscular junction and causes flaccid paralysis, resulting in a condition termed as botulism, and may also lead to death due to respiratory arrest. Methods: Fifteen plants were selected from the book ‘Diagnosis and treatment of diseases in Ayurveda’ by Vaidya Bhagwan Dash and Lalitesh Kashyap, based on their frequency of use in the formulations used for the treatment of six diseases with neuromuscular symptoms similar to botulism. Phytochemicals from these plants were screened using in silico, and in vitro methods. Structures of 570 reported phytochemicals from 14 plants were docked inside six reported BoNT/A light chain crystal structures using ensemble docking module in Maestro (Schrödinger, LLE). Results: From the docking scores and structural diversity, nine compounds including acoric acid 1, three flavonoids, three coumarins derivatives, one kava lactone were selected and screened using an in vitro HPLC-based protease assay. The bioassay results showed that several compounds possess BoNT/A LC inhibition of 50–60% when compared to positive controls NSC 84094 and CB7967495 (80–95%). Conclusion: Further testing of the active compounds identified from Ayurvedic literature and structure-activity studies of acoric acid 1 using more sensitive bioassays is under way. The identification of acoric acid 1, a novel scaffold against BoNT/A, exemplifies the utility of Ayurvedic literature for the discovery of novel drug leads.