A Screening Pipeline for Antiparasitic Agents Targeting Cryptosporidium Inosine Monophosphate Dehydrogenase

Experimental Parasitology

Elankumaran

Hijjawi

et al. 2015

• Scanning electron microscopy (SEM) characterisation of life cycle stages in cell-free culture.• Comparison of gene expression in cell culture and cell-free culture.• Gene expression cell-free culture was delayed and was lower.• Data provide support for cell-free culture of Cryptosporidium. A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. G R A P H I C A L A B S T R A C T

Section: Introductionmentioning

confidence: 99%

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies

Experimental Parasitology

Elankumaran

Hijjawi

et al. 2015

“…One compound, P131, was designed to be retained in the gastrointestinal tract and therefore exceeded the recommended TPSA (TPSA of Յ140 Å 2 ). Compounds were evaluated for enzyme inhibition and antiparasitic activity against a reporter Toxoplasma gondii strain (T. gondii/ CpIMPDH) engineered to rely on CpIMPDH for the production of guanine nucleotides (54). Compounds that performed well in these two assays, with 50% inhibitory concentrations (IC 50 ) of Յ20 nM and EC 50 s of Յ2 M, were candidates for testing in the IL-12 knockout mouse model of acute cryptosporidiosis.…”

Section: Resultsmentioning

confidence: 99%

“…The excystation mixture was diluted with Ultraculture medium (BioWhittaker Inc., Walkersville, MD), and approximately 1 ϫ 10 5 oocysts and sporozoites were allowed to infect confluent human ileocecal adenocarcinoma epithelial cells (HCT-8) or Madin-Darby canine kidney cells (MDCK). The monolayer was washed with PBS after 3 h and incubated with fresh Ultraculture medium with or without test compounds, inhibitor and media were refreshed after 24 h, and the parasites were cultured for a total of 48 h. Cultures were fixed and counted using an anti-C. parvum fluoresceinlabeled monoclonal antibody (C3C3-fluorescein isothiocyanate [FITC]) or a high-content imaging assay (54). The 50% effective concentration (EC 50 ) values were calculated using the Hill-Slope model using Prism v5 (GraphPad Software Inc., La Jolla, CA): % growth ϭ (maximum Ϫ minimum)/{1 ϩ (EC 50 …”

Section: Methodsmentioning

confidence: 99%

Validation of IMP Dehydrogenase Inhibitors in a Mouse Model of Cryptosporidiosis

Gorla

McNair

Antimicrob Agents Chemother

et al. 2014

Self Cite

f Cryptosporidium parasites are a major cause of diarrhea and malnutrition in the developing world, a frequent cause of waterborne disease in the developed world, and a potential bioterrorism agent. Currently, available treatment is limited, and Cryptosporidium drug discovery remains largely unsuccessful. As a result, the pharmacokinetic properties required for in vivo efficacy have not been established. We have been engaged in a Cryptosporidium drug discovery program targeting IMP dehydrogenase (CpIMPDH). Here, we report the activity of eight potent and selective inhibitors of CpIMPDH in the interleukin-12 (IL-12) knockout mouse model, which mimics acute human cryptosporidiosis. Two compounds displayed significant antiparasitic activity, validating CpIMPDH as a drug target. The best compound, P131 (250 mg/kg of body weight/day), performed equivalently to paromomycin (2,000 mg/kg/day) when administered in a single dose and better than paromomycin when administered in three daily doses. One compound, A110, appeared to promote Cryptosporidium infection. The pharmacokinetic, uptake, and permeability properties of the eight compounds were measured. P131 had the lowest systemic distribution but accumulated to high concentrations within intestinal cells. A110 had the highest systemic distribution. These observations suggest that systemic distribution is not required, and may be a liability, for in vivo antiparasitic activity. Intriguingly, A110 caused specific alterations in fecal microbiota that were not observed with P131 or vehicle alone. Such changes may explain how A110 promotes parasitemia. Collectively, these observations suggest a blueprint for the development of anticryptosporidial therapy.

“…Thus, the exclusive reliance on the salvage pathway by Cryptosporidium and its high metabolic demand for nucleotides due to the parasite's complicated lifecycle make IMPDH a potential drug target candidate. This hypothesis is supported by the recent discovery of several Cryptosporidium IMPDH inhibitors Johnson et al, 2013;Maurya et al, 2009;Sharling et al, 2010;Umejiego et al, 2008).…”

Section: Introductionmentioning

confidence: 80%

“…Despite this, efforts to develop new and more effective treatments for this disease have been hindered by (1) lack of suitable targets and (2) lack of target validation and transfection tools. The completion of the Cryptosporidium genome (Abrahamsen et al, 2004;Xu et al, 2004), which resulted in a range of potential drug targets being identified, has overcome the first hindrance and IMPDH is now a well recognised and important drug target Johnson et al, 2013;Maurya et al, 2009;Sharling et al, 2010;Umejiego et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer® peptides

Jefferies

Experimental Parasitology

Woh

et al. 2015

• Yielded a relatively high functional hit rate (~17% i.e. 2/12 Phylomer ® peptides tested).• Successfully shown that TAT can deliver therapeutic Phylomer cargoes inside cells.• Opens up new therapeutic opportunities. Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function. G R A P H I C A L A B S T R A C T