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 μM, cytotoxicity (TC₅₀) values from 6.31 to >100 μm] and their safety margins. Among them, 11 compounds were derived from plants with EC₅₀ values from 0.267 to 3.940 μM [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.

Keywords: Cryptosporidium parvum; apicomplexan; drug discovery; high-throughput screening; natural products.

FIGURE 1

Scatter plot of the primary screening of 800 natural products (10 μM) against the growth of Cryptosporidium parvum in vitro. Blue triangles represent data from the positive control compound paromomycin (150 μM). Green squares represent the 16 top hits selected for determining their antiparasitic half maximal effective concentration (EC₅₀) values and cytotoxicity shown in Table 1 and Figure 2. Each plate included 0.5% dimethyl sulfoxide diluent only as negative control (6 wells/plate). Bars show the standard error of the mean (N ≥ 3).

FIGURE 2

Chemical structures of the 11 top hits derived from plants and their in vitro EC₅₀ and safety interval (SI) values.

FIGURE 3

Effects of cedrelone (1.0 μM), Deox B 7,4 (deoxysappanone B 7,4′-dimethyl ether) (2.0 μM) and baicalein (3.3 μM) on various developmental stage of Cryptosporidium parvum in vitro. (A) The effects were expressed as relative parasite loads in bar chart. (B) The effects were shown as percentage inhibitions on the parasite growth. The data included the effects on the excystation and invasion of sporozoites (0-3 hpi treatment group), early developmental stages representing first generation and some second generation of merogony (3–10 hpi and 3–22 hpi), and second generation of merogony and gametogenesis stage (22–44 hpi). Intracellular parasites receiving a full course of treatment were used for comparison (3–44 hpi). Diluent [dimethyl sulfoxide (DMSO) at 0.5%] only was used as a negative control. Paromomycin (PRM; 150 μM) was used as a positive control. In this assay, parasite loads were determined at the end of each treatment. hpi, hours post-infection time. Bars show the standard error of the mean (N = 6 or 8).

FIGURE 4

Drug withdrawal assay to evaluate the reversibility of the inhibition by cedrelone (1.0 μM), Deox B 7,4 (deoxysappanone B 7,4′-dimethyl ether) (2.0 μM), and baicalein (3.3 μM) on the growth of C. parvum in vitro. (A) Data were expressed as relative parasite loads in bar chart. (B) Data were shown as percentage inhibition on the parasite growth. (C) Illustration of the assay. In this assay, the parasites received treatments by individual compounds from 3 to 22 hpi time points, followed by the removal of compounds and continuous growth for up to 44 hpi time point. Intracellular parasites receiving a full course of treatment were used for comparison (3–44 hpi). Diluent (dimethyl sulfoxide at 0.5%) only was used as a negative control. Paromomycin (PRM; 150 μM) was used as a positive control. In this assay, parasite loads were determined at 44 hpi. hpi, hours post-infection time. Bars show the standard error of the mean (N = 4 or 8).