High-throughput screening (HTS) hits include compounds with undesirable properties. Many filters have been described to identify such hits. Notably, pan-assay interference compounds (PAINS) has been adopted by the community as the standard term to refer to such filters, and very useful guidelines have been adopted by the American Chemical Society (ACS) and subsequently triggered a healthy scientific debate about the pitfalls of draconian use of filters. Using an inhibitory frequency index, we have analyzed in detail the promiscuity profile of the whole GlaxoSmithKline (GSK) HTS collection comprising more than 2 million unique compounds that have been tested in hundreds of screening assays. We provide a comprehensive analysis of many previously published filters and newly described classes of nuisance structures that may serve as a useful source of empirical information to guide the design or growth of HTS collections and hit triaging strategies.

New drugs are critically needed to treat Cryptosporidium infections, particularly for malnourished children under 2 years old in the developing world and persons with immunodeficiencies. Bioactive compounds from the Tres-Cantos GSK library that have activity against other pathogens were screened for possible repurposing against Cryptosporidium parvum growth. Nineteen compounds grouped into nine structural clusters were identified using an iterative process to remove excessively toxic compounds and screen related compounds from the Tres-Cantos GSK library. Representatives of four different clusters were advanced to a mouse model of C. parvum infection, but only one compound, an imidazole-pyrimidine, led to significant clearance of infection. This imidazole-pyrimidine compound had a number of favorable safety and pharmacokinetic properties and was maximally active in the mouse model down to 30 mg/kg given daily. Though the mechanism of action against C. parvum was not definitively established, this imidazole-pyrimidine compound inhibits the known C. parvum drug target, calcium-dependent protein kinase 1, with a 50% inhibitory concentration of 2 nM. This compound, and related imidazole-pyrimidine molecules, should be further examined as potential leads for Cryptosporidium therapeutics.

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