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Discovery of Novel Anti-cryptosporidial Activities From Natural Products by in vitro High-Throughput Phenotypic Screening.

ABSTRACT: Cryptosporidium parvum is a globally distributed zoonotic protozoan parasite of both medical and veterinary importance. Nitazoxanide is the only FDA-approved drug to treat cryptosporidiosis in immunocompetent people, but it is not fully effective. There is no drug approved by FDA for use in immunocompromised patients or in animals. In the present study, we conducted phenotypic screening of 800 nature products with defined chemical structures for potential novel activity against the growth of C. parvum in vitro. We identified a large number of compounds showing low to sub-micromolar anti-cryptosporidial activity, and fully characterized 16 top hits for anti-parasitic efficacies in vitro [EC50 values from 0.122 to 3.940 ?M, cytotoxicity (TC50) values from 6.31 to >100 ?m] and their safety margins. Among them, 11 compounds were derived from plants with EC50 values from 0.267 to 3.940 ?M [i.e., cedrelone, deoxysappanone B 7,4'-dimethyl ether (Deox B 7,4), tanshinone IIA, baicalein, deoxysappanone B 7,3'-dimethyl ether acetate, daunorubicin, dihydrogambogic acid, deacetylgedunin, deacetoxy-7-oxogedunin, dihydrotanshinone I, 2,3,4'-trihydroxy-4-methoxybenzophenone, and 3-deoxo-3beta-hydroxy-mexicanolide 16-enol ether]. Three compounds with sub-micromolar EC50 values (i.e., cedrelone, Deox B 7,4, and baicalein) were further investigated for their effectiveness on various parasite developmental stages in vitro. Cedrelone and baicalein were more effective than Dexo B 7,4 when treating parasite for shorter periods of time, but all three compounds could kill the parasite irreversibly. These findings provide us a large selection of new structures derived from natural products to be explored for developing anti-cryptosporidial therapeutics.

SUBMITTER: Jin Z

PROVIDER: S-EPMC6736568 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Discovery of Novel Anti-cryptosporidial Activities From Natural Products by <i>in vitro</i> High-Throughput Phenotypic Screening.

Jin Zi Z Ma Jingbo J Zhu Guan G Zhang Haili H

Frontiers in microbiology 20190829

<i>Cryptosporidium parvum</i> is a globally distributed zoonotic protozoan parasite of both medical and veterinary importance. Nitazoxanide is the only FDA-approved drug to treat cryptosporidiosis in immunocompetent people, but it is not fully effective. There is no drug approved by FDA for use in immunocompromised patients or in animals. In the present study, we conducted phenotypic screening of 800 nature products with defined chemical structures for potential novel activity against the growth ...[more]

PMID: 31551955

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Project description:BackgroundIn-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides (HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.MethodsTo identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3D4/31 macrophages against a Gram-negative bacterium (enterotoxigenic E. coli) and a Gram-positive bacterium (Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds.ResultsA total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening, with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment. Xanthohumol and deoxyshikonin were further found to be the most potent in inducing pBD3 mRNA expression, showing a minimum 10-fold increase in IPEC-J2, 3D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants, but not 3D4/31 cells. In addition to pBD3, other porcine HDP genes such as pBD2, PG1-5, and pEP2C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene- and cell type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3D4/31 macrophages (P < 0.05) against both Gram-negative and Gram-positive bacteria.ConclusionsOur results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.

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Dataset Information

Discovery of Novel Anti-cryptosporidial Activities From Natural Products by in vitro High-Throughput Phenotypic Screening.

Publications

Discovery of Novel Anti-cryptosporidial Activities From Natural Products by <i>in vitro</i> High-Throughput Phenotypic Screening.

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