Prodrug Activation by Cryptosporidium Thymidine Kinase

Sun, Xin; Sharling, Lisa; Muthalagi, Mani; Mudeppa, Devaraja G.; Pankiewicz, Krzysztof W.; Felczak, Krzysztof; Rathod, Pradipsinh K.; Mead, Jan R.; Striepen, Boris; Hedstrom, Lizbeth

doi:10.1074/jbc.m110.101543

Cited by 20 publications

(13 citation statements)

References 49 publications

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“…A previous study demonstrated the ability of C. parvum thymidine kinase to activate pyrimidine analogs including floxuridine. Additionally, trifluoromethyl thymidine (TFT; another fluoropyrimidine) exhibited efficacy in a mouse model of acute C. parvum infection (22). Furthermore, widespread use of nucleoside analogs in the prevention of motherto-child transmission of HIV (23) suggests that certain formulations of the chemical family are safe for use in pediatric populations.…”

Section: Resultsmentioning

confidence: 99%

“…Fifteen of the 21 confirmed compounds identified by our screen are currently FDA approved for use in patients, but only five of these compounds, floxuridine (22), clofazimine (46), indomethacin (47), minocycline (48,49), and doxycycline (50), are described in the literature as having activity against Cryptosporidium species in vitro or in vivo (animal models or human patients), suggesting that the list is rich in new repurposing candidates. While we chose to pursue the HMG-CoA reductase inhibitor itavastatin, we have made the screening results of all 727 compounds available as supplemental data, which we hope will inform additional studies aimed at repurposing drugs for cryptosporidiosis and other apicomplexan infections as well as elucidate apicomplexan biology and novel host-microbe interactions.…”

Section: Discussionmentioning

confidence: 99%

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Drug Repurposing Screen Reveals FDA-Approved Inhibitors of Human HMG-CoA Reductase and Isoprenoid Synthesis That Block Cryptosporidium parvum Growth

Bessoff

Sateriale

Lee

et al. 2013

Antimicrob Agents Chemother

120

118

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dCryptosporidiosis, a diarrheal disease usually caused by Cryptosporidium parvum or Cryptosporidium hominis in humans, can result in fulminant diarrhea and death in AIDS patients and chronic infection and stunting in children. Nitazoxanide, the current standard of care, has limited efficacy in children and is no more effective than placebo in patients with advanced AIDS. Unfortunately, the lack of financial incentives and the technical difficulties associated with working with Cryptosporidium parasites have crippled efforts to develop effective treatments. In order to address these obstacles, we developed and validated (Z= score ‫؍‬ 0.21 to 0.47) a cell-based high-throughput assay and screened a library of drug repurposing candidates (the NIH Clinical Collections), with the hopes of identifying safe, FDA-approved drugs to treat cryptosporidiosis. Our screen yielded 21 compounds with confirmed activity against C. parvum growth at concentrations of <10 M, many of which had well-defined mechanisms of action, making them useful tools to study basic biology in addition to being potential therapeutics. Additional work, including structure-activity relationship studies, identified the human 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor itavastatin as a potent inhibitor of C. parvum growth (50% inhibitory concentration [IC 50 ] ‫؍‬ 0.62 M). Bioinformatic analysis of the Cryptosporidium genomes indicated that the parasites lack all known enzymes required for the synthesis of isoprenoid precursors. Additionally, itavastatin-induced growth inhibition of C. parvum was partially reversed by the addition of exogenous isopentenyl pyrophosphate, suggesting that itavastatin reduces Cryptosporidium growth via on-target inhibition of host HMG-CoA reductase and that the parasite is dependent on the host cell for synthesis of isoprenoid precursors.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Drug Repurposing Screen Reveals FDA-Approved Inhibitors of Human HMG-CoA Reductase and Isoprenoid Synthesis That Block Cryptosporidium parvum Growth

Bessoff

Sateriale

Lee

et al. 2013

Antimicrob Agents Chemother

120

118

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“…We have found that the inactive compounds A110 and P83 displayed the best plasma pharmacokinetic behavior, while the active compounds P96 and P131 have relatively poor systemic exposure. In vivo anticryptosporidial activity has also been reported for several other compounds with poor systemic bioavailability, most notably paromomycin, and also including pyrvinium pamoate, dication carbazoles, and trifluoromethylthymidine (15,20,25). While nitazoxanide exhibits good systemic exposure, its active metabolite tizoxanide is efficiently recycled to the intestine via glucuronidation (12).…”

Section: Discussionmentioning

confidence: 94%

“…Paromomycin is effective in rodents and has also been used to treat cryptosporidiosis, though a significant benefit has not been demonstrated in humans (13). Approximately 20 additional compounds have displayed at least some anticryptosporidial activity in animal models (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Unfortunately, as with nitazoxanide, the targets of most of these compounds have not been identified, which makes further development difficult.…”

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confidence: 99%

Validation of IMP Dehydrogenase Inhibitors in a Mouse Model of Cryptosporidiosis

Gorla

McNair

Yang

et al. 2014

Antimicrob Agents Chemother

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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.

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“…Among the available options, the wheat germ system is robust, it quickly produces high yields, and it is scalable and cost-effective. Previously, we demonstrated the successful cell-free expression of very challenging parasite enzymes in fully functional form: P. falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) and Cryptosporidium parvum thymidine kinase (20,21). The cell-freeproduced DHFR-TS had all three known functions of native protein: DHFR catalytic activity, TS catalytic activity, and autologous RNA binding (20).…”

mentioning

confidence: 99%

Expression of Functional Plasmodium falciparum Enzymes Using a Wheat Germ Cell-Free System

Mudeppa

Rathod

2013

Eukaryot Cell

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One decade after the sequencing of the Plasmodium falciparum genome, 95% of malaria proteins in the genome cannot be expressed in traditional cell-based expression systems, and the targets of the best new leads for antimalarial drug discovery are either not known or not available in functional form. For a disease that kills up to 1 million people per year, routine expression of recombinant malaria proteins in functional form is needed both for the discovery of new therapeutics and for identification of targets of new drugs. We tested the general utility of cell-free systems for expressing malaria enzymes. Thirteen test enzyme sequences were reverse amplified from total RNA, cloned into a plant-like expression vector, and subjected to cell-free expression in a wheat germ system. Protein electrophoresis and autoradiography confirmed the synthesis of products of expected molecular masses. In rare problematic cases, truncated products were avoided by using synthetic genes carrying wheat codons. Scaled-up production generated 39 to 354 g of soluble protein per 10 mg of translation lysate. Compared to rare proteins where cell-based systems do produce functional proteins, the cell-free yields are comparable or better. All 13 test products were enzymatically active, without failure. This general path to produce functional malaria proteins should now allow the community to access new tools, such as biologically active protein arrays, and lead to the discovery of new chemical functions, structures, and inhibitors of previously inaccessible malaria gene products.

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Prodrug Activation by Cryptosporidium Thymidine Kinase

Cited by 20 publications

References 49 publications

Drug Repurposing Screen Reveals FDA-Approved Inhibitors of Human HMG-CoA Reductase and Isoprenoid Synthesis That Block Cryptosporidium parvum Growth

Drug Repurposing Screen Reveals FDA-Approved Inhibitors of Human HMG-CoA Reductase and Isoprenoid Synthesis That Block Cryptosporidium parvum Growth

Validation of IMP Dehydrogenase Inhibitors in a Mouse Model of Cryptosporidiosis

Expression of Functional Plasmodium falciparum Enzymes Using a Wheat Germ Cell-Free System

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