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. 2014 May;58(5):2731-9.
doi: 10.1128/AAC.02641-13. Epub 2014 Feb 24.

Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria box

Kovi Bessoff  1 , Thomas Spangenberg, Jenna E Foderaro, Rajiv S Jumani, Gary E Ward, Christopher D Huston
Affiliations

Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria box

Kovi Bessoff et al. Antimicrob Agents Chemother. 2014 May.

Abstract

The apicomplexan parasites Cryptosporidium parvum and Cryptosporidium hominis are major etiologic agents of human cryptosporidiosis. The infection is typically self-limited in immunocompetent adults, but it can cause chronic fulminant diarrhea in immunocompromised patients and malnutrition and stunting in children. Nitazoxanide, the current standard of care for cryptosporidiosis, is only partially efficacious for children and is no more effective than a placebo for AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidium biology impede the development of better drugs for treating cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture (MMV) Open Access Malaria Box for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited submicromolar potency against C. parvum. Potency was conserved in a subset of compounds from each scaffold with varied physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvum growth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of the compounds in the allopurinol-based series against T. gondii and C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvum and other apicomplexan parasites.

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Figures

FIG 1
FIG 1
Open Access Malaria Box screening hits with confirmed activity. MMV identifiers (IDs) and structures (converted from SMILES notation to structures using ACD/ChemSketch version 12.01) were provided by the MMV as part of the supporting information for the Open Access Malaria Box. The commercial source indicates the vendor from whom compounds were purchased for follow-up testing. Compound E-1 (MMV009085) was recovered from the MMV Malaria Box and was not purchased for follow-up (as indicated in the Series MMV_ID source and the comment column in Fig. 2). IC50s were determined by combining data from at least two biological replicates of dose-response experiments generated using a repurchased compound (except for E-1, for which the IC50s are from a single experiment using a cherry-picked compound). The common quinolin-8-ol chemical scaffold is highlighted in red for compounds A-1 to A-5.
FIG 2
FIG 2
A-6 (MMV666022) and its enantiomers exhibit different activities against C. parvum. During in vitro follow-up, the quinolin-8-ol A-6 (MMV666022) was purchased and its IC50 against C. parvum determined. The compound has a chiral center (identified by the asterisk), and its two enantiomers were separated by chiral high-performance liquid chromatography (HPLC) (see the supplemental material). The two enantiomers were found to vary in potency by almost a log. The IC50s are a combination of data from at least two biological replicates. The AlogP and molecular weight (MW) data are provided in the supporting information for the Open Access Malaria Box.
FIG 3
FIG 3
Determination of minimum effective exposure time. (A) Schematic describing the experimental design. Beginning 3 h after infection, the infected cells were treated with experimental compound or 0.25% DMSO (vehicle) for 3, 6, 12, or 24 h, at which point the growth medium containing the compound was replaced with compound containing 0.25% DMSO for the duration of the 72-h culture period. The infected cells treated with experimental compounds for 69 h provided a control for the anti-Cryptosporidium activity of the compounds. The cells were then fixed, stained, and imaged. (B) Parasites treated with nitazoxanide (NTZ) or 2,4-diamino-quinazoline (B-13) for 6 h exhibited full recovery, and parasites treated with these compounds exhibited partial recovery despite treatment for up to 24 h. Parasites treated with representative quinolin-8-ol (A-4) and allopurinol-based (C-1) compounds showed no parasite recovery following a shorter treatment interval. n = 3; error bars represent the standard deviation.
FIG 4
FIG 4
Correlation of the relative potencies of allopurinol-based and 2,4-diamino-quinazoline compounds against C. parvum and T. gondii. (A) IC50s against C. parvum and T. gondii were plotted for 21 compounds from the allopurinol-based chemical series (see Table S3 in the supplemental material). One compound exhibited an IC50 against C. parvum and T. gondii that was outside the assayed range (i.e., >5 μM) and was excluded from the analysis. The Spearman rank correlation coefficient (rs) was determined to be 0.67 (95% confidence interval [CI], 0.32 to 0.86; P = 0.0009), suggesting good correlation between the compound activities. (B) IC50s against C. parvum and T. gondii were plotted for 21 compounds in the 2,4-diamino-quinazoline chemical series (see Table S2 in the supplemental material). Five compounds exhibited IC50s against C. parvum and/or T. gondii that were outside the assayed range (i.e., >10 μM) and were excluded from the analysis. There appeared to be no correlation between the compound activities (rs = −0.21; 95% CI, −0.60 to 0.26; P = 0.36).
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