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The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

BMS906024, a γ-secretase inhibitor that blocks Notch signaling, was previously shown to inhibit Cryptosporidium parvum growth in vitro. A structure-activity relationship (SAR) analysis of BMS906024 reported herein demonstrates the importance of the stereochemistry of the C-3 benzodiazepine and the succinyl β-substituent. However, concomitant removal of the succinyl α-substituent and switching the primary amide with secondary amides was tolerated. For example, 32 (SH287) inhibited C. parvum growth in HCT-8 host cells with an EC 50  = 6.4 nM and an EC 90  = 16 nM; however, blocking C. parvum growth with BMS906024 derivatives was correlative with inhibition of Notch signaling, highlighting that additional SAR analysis will be needed to separate these two activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Infection with Cryptosporidium spp. can cause severe diarrhea, leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a phase 2a clinical trial in HIV-infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine's lack of efficacy in the phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using E max and logistic models, which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the phase 2a study participants were well below concentrations associated with anti- Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed. (This study has been registered in ClinicalTrials.gov under identifier NCT03341767.).

Background: We evaluated the efficacy, pharmacokinetics (PK), and safety of clofazimine (CFZ) in patients living with human immunodeficiency virus (HIV) with cryptosporidiosis., Methods: We performed a randomized, double-blind, placebo-controlled study. Primary outcomes in part A were reduction in Cryptosporidium shedding, safety, and PK. Primary analysis was according to protocol (ATP). Part B of the study compared CFZ PK in matched individuals living with HIV without cryptosporidiosis., Results: Twenty part A and 10 part B participants completed the study ATP. Almost all part A participants had high viral loads and low CD4 counts, consistent with failure of antiretroviral (ARV) therapy. At study entry, the part A CFZ group had higher Cryptosporidium shedding, total stool weight, and more diarrheal episodes compared with the placebo group. Over the inpatient period, compared with those who received placebo, the CFZ group Cryptosporidium shedding increased by 2.17 log2 Cryptosporidium per gram stool (95% upper confidence limit, 3.82), total stool weight decreased by 45.3 g (P = .37), and number of diarrheal episodes increased by 2.32 (P = .87). The most frequent solicited adverse effects were diarrhea, abdominal pain, and malaise. One placebo and 3 CFZ participants died during the study. Plasma levels of CFZ in participants with cryptosporidiosis were 2-fold lower than in part B controls., Conclusions: Our findings do not support the efficacy of CFZ for the treatment of cryptosporidiosis in a severely immunocompromised HIV population. However, this trial demonstrates a pathway to assess the therapeutic potential of drugs for cryptosporidiosis treatment. Screening persons living with HIV for diarrhea, and especially Cryptosporidium infection, may identify those failing ARV therapy., Clinical Trials Registration: NCT03341767., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

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.

Introduction : Two landmark epidemiological studies identified Cryptosporidium spp. as a significant cause of diarrheal disease in pediatric populations in resource-limited countries. Notably, nitazoxanide is the only approved drug for treatment of cryptosporidiosis but shows limited efficacy. As a result, many drug discovery efforts have commenced to find improved treatments. The unique biology of Cryptosporidium presents challenges for traditional drug discovery methods, which has inspired new assay platforms to study parasite biology and drug screening. Areas covered : The authors review historical advancements in phenotypic-based assays and techniques for Cryptosporidium drug discovery, as well as recent advances that will define future drug discovery. The reliance on phenotypic-based screens and repositioning of phenotypic hits from other pathogens has quickly created a robust pipeline of potential cryptosporidiosis therapeutics. The latest advances involve new in vitro culture methods for oocyst generation, continuous culturing capabilities, and more physiologically relevant assays for testing compounds. Expert opinion : Previous phenotypic screening techniques have laid the groundwork for recent cryptosporidiosis drug discovery efforts. The resulting improved methodologies characterize compound activity, identify, and validate drug targets, and prioritize new compounds for drug development. The most recent improvements in phenotypic assays are poised to help advance compounds into clinical development.

Cryptosporidium is a protozoan parasite and a leading cause of diarrheal disease and mortality in young children. Currently, there are no fully effective treatments available to cure infection with this diarrheal pathogen. In this study, we report a broad drug repositioning effort that led to the identification of bicyclic azetidines as a new anticryptosporidial series. Members of this series blocked growth in in vitro culture of three Cryptosporidium parvum isolates with EC 50 ' s in 1% serum of <0.4 to 96 nM, had comparable potencies against Cryptosporidium hominis and C. parvum , and was effective in three of four highly susceptible immunosuppressed mice with once-daily dosing administered for 4 days beginning 2 weeks after infection. Comprehensive genetic, biochemical, and chemical studies demonstrated inhibition of C. parvum phenylalanyl-tRNA synthetase ( Cp PheRS) as the mode of action of this new lead series. Introduction of mutations directly into the C. parvum pheRS gene by CRISPR-Cas9 genome editing resulted in parasites showing high degrees of compound resistance. In vitro, bicyclic azetidines potently inhibited the aminoacylation activity of recombinant Ch PheRS. Medicinal chemistry optimization led to the identification of an optimal pharmacokinetic/pharmacodynamic profile for this series. Collectively, these data demonstrate that bicyclic azetidines are a promising series for anticryptosporidial drug development and establish a broad framework to enable target-based drug discovery for this infectious disease., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (∼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)