Project : USDA ARS

ARS Home » Southeast Area » Oxford, Mississippi » Natural Products Utilization Research » Research » Research Project #428996

Research Project: Discovery and Development of Natural Products for Pharmaceutical and Agrochemical Applications II

Location: Natural Products Utilization Research

2019 Annual Report

Objectives
Objective 1: Discover new leads from plant-based natural resources with anti-infective and anti-cancer, immunomodulator and anti-inflammatory activities using cell-based screens and mechanistic assays using molecular target-based approaches. Sub-objective 1A: Source novel natural resources from terrestrial plants, marine organisms and microbes from around the world for biological testing. Sub-objective 1B: Prepare, maintain and manage Natural Products Repository and Laboratory Information Management System (LIMS). Sub-objective 1C: Evaluate natural product extracts and natural product derived pure compounds for potential anticancer, anti-infective, anti-inflammatory, neuroprotective and anti-diabetic properties, and for utility in metabolic and immune disorders. Sub-objective 1D: Isolation and structural elucidation of lead compounds. Objective 2: For the best candidates, characterize mechanisms of action, selectivity, toxicity, functional activity in secondary assays and in animal models of plant-based anti-infective and anti-cancer, immunomodulator and anti-inflammatory compounds. Sub-objective 2A: Characterization of mechanisms of action and functional activity of leads. Sub-objective 2B: Characterization of selectivity and toxicity of lead compounds. Objective 3: Develop methods for analysis of bioactive or medicinally important plants and quality control of their derived products. Objective 4: Assess selected medicinal or aromatic plants for cultivation, harvest and processing to optimize yields of biomass and active principles.

Approach
The approach includes a program of: (1) Using cell-based screening and mechanistic assays to discover new pharmaceutical and agrochemical leads from natural sources; (2) Using secondary assays and animal models to characterize mechanisms of action, selectivity, toxicity and functional activity of the best candidate compounds having anti-infective, anti-cancer, immunomodulatory, or anti-inflammatory properties; (3) Selection, agronomics and analysis of bioactive or medicinally important plants and their derived products.

Progress Report
Researchers at the National Center for Natural Products Research (NCNPR) at the University of Mississippi, Oxford, Mississippi maintained basic discovery operations, with emphasis on the discovery of antifungals, anticancer, anti-inflammatory agents and immunomodulating agents. The researchers continued to source plant materials for screening from our own plant collections and from numerous collaborators. We added 955 plant samples to our inventory this year and screened over 6700 natural product crude extracts, semi-purified fractions and purified compounds for biological activities against specific molecular targets and whole cell systems. As part of our continuing effort in the search for anti-infective, cancer chemopreventive, and immunomodulator/anti-inflammatory leads from natural sources, more than 100 compounds were isolated. In addition, 20 compounds were synthesized. Of the compounds tested, 21 were found to be biologically active for potential agricultural or medical uses. Many showed potent phytotoxic, antifungal, antibacterial, or antimalarial activities. In order to explore the antidiabetic potential of medicinal plants, about 30 plant extracts were screened for peroxisome proliferator-activated receptor (PPAR) agonistic activity. About 20 pure compounds were explored for antidiabetic potential. The actives were selected and were followed further in secondary assays such as adipogenic effect, antiadipogenic effect, and the agonistic effect towards liver X receptor (LXR), which regulates lipid metabolism. In addition to these basic operations, we have selected a number of these compounds for more advanced study, whether for characterizing mechanisms of action, determining suitability for further pharmaceutical development, evaluation in disease models in preclinical studies, or in field applications. About 125 plant extracts were screened for anti-inflammatory activities through target-based cellular assays including Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB), Inducible Nitric Oxide Synthase (iNOS), and oxidative stress and about 50 pure compounds were screened for anti-inflammatory activities through these targets. About 25 extracts of medicinal plants and 20 isolated pure constituents from medicinal plants were also screened for their potential of causing drug interaction in terms of pregnane X receptor (PXR) activation and Cytochrome P405s (CYPs) and permeability glycoprotein (P-gp) inhibitions. In collaboration with ElSohly Laboratories, Inc., continued development of a product shown to be effective in animal models for desensitization to poison ivy dermatitis. The lead compound in the poison ivy project is now being evaluated in stage 1 clinical trials. The Medicinal Plant Garden at the University of Mississippi continues to expand its renowned collection of living medicinal plants. New demonstration beds and field plots were developed. Here NCNPR cultivates and processes medicinal plants to be used in the discovery program. The new research wing will expand and enhance the research capabilities of NCNPR with a second plant specimen repository, herbarium, and laboratories for plant tissue cultures, cellular cultures, scale-up isolation and synthetic chemistry.

Accomplishments
1. Researchers continued with the development of agents for the prevention/treatment of poison ivy dermatitis through cooperative agreement with ARS scientists in Oxford, Mississippi. Scientists at the University of Mississippi, Oxford, Mississippi, in collaboration with ElSohly Laboratories, Inc., are developing preventive treatments for poison ivy dermatitis. Development continued on the compound shown to be effective in animal models for desensitization to poison ivy dermatitis and shown to have desirable bioavailability and toxicological properties. This product has attained investigational new drug (IND) status by the U.S. Food and Drug Administration.

2. Natural immunomodulators to suppress the pathogenic effects of influenza virus infections. Through cooperative agreement with ARS scientists in Oxford, Mississippi, scientists at the University of Mississippi, Oxford, Mississippi, have established a new animal model to study the effect of the plant-based immunomodulatory product(s) for effectiveness on minimizing the pathogenic effect of influenza virus infection. Pneumonic lesions in the lung after influenza virus infection in mice have been characterized. The influenza virus from the infected lung was successfully titrated and its pathogenicity in cell culture has been established.

3. Identify dietary or botanical products with cancer chemopreventive potential. Through cooperative agreement with ARS scientists in Oxford, Mississippi, this program takes the most promising lead natural product extracts/pure compounds, identified using our battery of 15 cancer signaling pathway luciferase assays, and determines their cytotoxic potential against both bulk and tumor stem-like cells isolated from various types of patient-derived tumor biopsies in vitro. As a result, a provisional patent has been filed on the most promising formulation, identified a possible molecular target and studied with mouse melanoma models to establish the in vivo efficacy. Both biological and analytical methods have been developed to evaluate the pharmacokinetic properties of this formulation with mouse models and might serve as a candidate for translational research and product development. To overcome the supply issue of the test article, an efficient synthetic strategy has been proposed and is being evaluated for feasibility. In addition, several herbal extracts/pure compounds synergistic interactions (about 20 combinations) were evaluated in various cancer cell lines (glioblastoma, triple-negative breast cancer, and melanoma). Effective combinations were screened further in bulk and tumor stem-like cells (isolated from various types of patient-derived tumor biopsies) in vitro.

4. Characterization and development of immunomodulatory natural products. Through cooperative agreement with ARS scientists in Oxford, Mississippi, for over 15 years, a major focus of the scientists in this research area at the National Center for Natural Products Research (NCNPR), University of Mississippi, has been identifying the components responsible for the immune-enhancing properties of botanicals. This research has resulted in the development of a novel theory that the naturally occurring bacterial communities within plants produce potent activators of pathogen recognition receptors that are principal contributors in the activation of innate immune cells. Building on this foundation, current efforts are directed towards characterization of the immune-enhancing properties of mushrooms. Data indicate that the macrophage stimulatory activity of edible mushrooms is due to the collaborative interaction of water-soluble Toll-like receptor agonists (derived from microbiome bacteria) and water-insoluble particulate beta glucans (derived from microbiome yeast). Continued efforts support the development of the patented extract, ImmulinaTM, that is commercially available as a dietary supplement for modulating innate immune function. This extract was discovered at the NCNPR and it is enriched with the active immunostimulatory compounds in Arthrospira (Spirulina) platensis, i.e., Braun-type lipoproteins (potent Toll-like receptor 2 agonists). A research service center has been established for the biological standardization of this botanical product. Screening efforts identified a bacterium exhibiting selective inhibition of the Toll-like receptor 4 (TLR4) signaling pathway. The compound responsible was isolated using bio-assay-guided fractionation and structural characterization is currently in progress. Inflammatory conditions and disease are potential therapeutic targets for TLR4 inhibitors.

5. New antifungal natural products for use in agriculture and medicine. Through cooperative agreement with ARS scientists in Oxford, Mississippi, scientists at the National Center for Natural Products Research (NCNPR), University of Mississippi, have continued on this research effort of the identification of compounds that can synergize with current antifungal drugs with the long-term goal of developing new combination therapies to treat fungal infections. First, we have evaluated ~2500 compounds for their ability to induce the fungal cell wall damage response pathway. Compounds identified will be expected to synergize with the cell wall-targeting antifungal drug caspofungin (CAS). Over 20 hits were identified and several were confirmed to potentiate CAS activity. Second, we have conducted dose-matrix assays to directly evaluate compounds for CAS-synergizing activity in a CAS-resistant pathogen. Over 300 compounds were evaluated, and 10 candidates showed synergistic activity. Follow-up mechanistic and animal studies are planned for the most promising compounds from both projects. We have also evaluated 14 compounds and 21 extracts for their ability to unmask beta-glucan from the cell walls of the fungal pathogen, Candida albicans. Samples identified will be expected to expose fungal cells to host immune cells, thereby allowing fungal clearance from the infection site. Given that Candida species inherently exist in the human gut, this strategy will also be useful in preventing fungal infections, most of which originate from the gut population. In addition, given the role gut-residing fungi play in irritable bowel disease (IBD), this strategy will be useful in the management of IBD. In our assay, 15 samples exhibited beta-glucan unmasking activity, and follow-up studies on them are ongoing. The most promising ones will be evaluated mechanistically, and in studies with animal models of fungal infections and IBD.

U.S. DEPARTMENT OF AGRICULTURE

Natural Products Utilization Research: Oxford, MS