A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors

Abstract

Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.

Identification of a potent benzoxaborole drug candidate for treating cryptosporidiosis

“…The anticryptosporidial activity of AN7973 was further examined using a panel of in vitro assays to determine its mode of action 38 . AN7973 had no activity in an assay for C. parvum invasion of HCT-8 host cells ( Supplementary Fig.…”

“…It predominantly affected intracellular Cryptosporidium development, since it arrested new DNA synthesis as reflected by inhibition of incorporation of the thymidine analog 5-ethynyl-2′deoxyuridine (EdU) into newly synthesized DNA (Fig. 2c) NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10687-y ARTICLE potency in reducing the percent of parasites expressing the meiotic recombination protein DMC1, which is a biomarker for Cryptosporidium sexual development 38 . Unlike some compounds, e.g., the piperazine-based lead MMV665917, that have equal potency against asexual and sexual stage parasites 38 , AN7973 inhibited asexual parasite growth four times more potently than asexual-stage to sexual-stage differentiation ( Fig.…”

“…2c) NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10687-y ARTICLE potency in reducing the percent of parasites expressing the meiotic recombination protein DMC1, which is a biomarker for Cryptosporidium sexual development 38 . Unlike some compounds, e.g., the piperazine-based lead MMV665917, that have equal potency against asexual and sexual stage parasites 38 , AN7973 inhibited asexual parasite growth four times more potently than asexual-stage to sexual-stage differentiation ( Fig. 2d), which was felt to be consistent with its effect on intracellular replication.…”

“…2c) results demonstrate only that AN7973 arrests intracellular Cryptosporidium development prior to DNA synthesis, and do not imply that the compound directly affects DNA synthesis. Many compounds that do not directly target DNA synthesis (e.g., several tRNA-synthetase inhibitors presumed to inhibit protein synthesis) look similar to AN7973 in this assay 38 . Recent studies of AN7973 demonstrated that its trypanicidal activity results from inhibition of trypanosome mRNA processing 45 .…”

“…Host cell invasion was assayed by allowing C. parvum to invade host cell monolayers in the presence of compound, and enumerating parasites and host cells after just 3 h (i.e., before completion of a parasite division cycle) 38 . HCT-8 cells were grown in RPMI media supplemented with 120 U per mL penicillin and 120 μg per mL streptomycin and 10% heat inactivated fetal bovine serum (complete media) and C. parvum Iowa isolate oocysts from Bunch Grass Farms (Deary, ID) were used for infection.…”