5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum

Huang, Wenlin; Choi, Ryan; Hulverson, Matthew A.; Zhang, Zhongsheng; McCloskey, Molly C.; Schaefer, Deborah A.; Whitman, Grant R.; Barrett, Lynn K.; Vidadala, Rama Subba Rao; Riggs, Michael W.; Maly, Dustin J.; Voorhis, Wesley C. Van; Ojo, Kayode K.; Fan, Erkang

doi:10.1128/aac.00020-17

Cited by 17 publications

(26 citation statements)

References 14 publications

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“…However, CpCDPK1 has been shown to be involved in C. parvum invasion rather than growth (Castellanos-Gonzalez et al, 2016;Kuhlenschmidt et al, 2016). Although a recent study has shown that some candidate CpCDPK1 inhibitors could potently inhibit C. parvum growth in vitro, there was no apparent correlation between anti-CpCDPK1 activities and C. parvum growth inhibition (Huang et al, 2017). In contrast, data generated with candidate inhibitors in the present study suggest that CpCDPK3 is involved in C. parvum growth but not invasion.…”

Section: Discussioncontrasting

confidence: 89%

“…Most previous studies of CDPKs of C. parvum (CpCDPKs) are on CpCDPK1, which is expressed in all life cycle stages, and believed to play an important role in the invasion (Castellanos-Gonzalez et al, 2016;Kuhlenschmidt et al, 2016) and possibly growth of C. parvum (Huang et al, 2017). In comparison, the function of CpCDPK3, which shares high structural homology with CpCDPK1, is still poorly understood.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Calcium-Dependent Protein Kinases 3, a Protein Involved in Growth of Cryptosporidium parvum

Zhang

Guo

et al. 2020

Front. Microbiol.

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Calcium-dependent protein kinases (CDPKs) are considered promising targets for pharmaceutical intervention of cryptosporidiosis. Whole-genome sequencing has revealed the presence of several CDPKs (CpCDPKs) in Cryptosporidium parvum. In this study, we expressed recombinant CpCDPK3 encoded by the cgd5_820 gene in Escherichia coli. The biologic characteristics and functions of CpCDPK3 were examined using qRT-PCR, immunofluorescence microscopy, and in vitro neutralization assay. The expression of the cgd5_820 gene peaked in merozoites during in vitro culture while the CpCDPK3 protein was expressed in both sporozoites and merozoites. Polyclonal antibodies against CpCDPK3 showed no significant inhibitory effects on host invasion by the parasites. We assessed the inhibitory effects of 46 candidate compounds from molecular docking of CpCDPK3 on both C. parvum development and CpCDPK3 enzyme activities. One compound was identified to be effective. Results of these analyses suggest that CpCDPK3 might play an important role in the growth of C. parvum.

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Section: Discussioncontrasting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Characterization of Calcium-Dependent Protein Kinases 3, a Protein Involved in Growth of Cryptosporidium parvum

Zhang

Guo

et al. 2020

Front. Microbiol.

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“…11–12 Solubility tests for all inhibitors were determined at pH 2 and pH 6.5 as previously described. 10…”

Section: Methodsmentioning

confidence: 99%

“…6–8 Selective targeting of Cryptosporidium parvum CDPK1 ( Cp CDPK1) with “bumped kinase inhibitors” (BKIs) based on 1 H -pyrazolo[3,4- d ]pyrimidin-4-amine (pyrazolopyrimidine, PP) and 5-aminopyrazole-4-carboxamide (AC) chemical scaffolds blocks parasite cell proliferation in both in vitro and in vivo models of cryptosporidiosis. 6–10 Partial knockdown of Cp CDPK1 with small interfering RNAs (siRNAs) led to significantly decreased C. parvum parasite invasion and growth. Moreover, partial Cp CDPK1 siRNA knockdown in combination with treatment of either BKIs 1294 (PP) or 1517 (AC) produced further inhibitory effects on the proliferation of C. parvum parasites.…”

mentioning

confidence: 99%

7H-Pyrrolo[2,3-d]pyrimidin-4-amine-Based Inhibitors of Calcium-Dependent Protein Kinase 1 Have Distinct Inhibitory and Oral Pharmacokinetic Characteristics Compared with 1H-Pyrazolo[3,4-d]pyrimidin-4-amine-Based Inhibitors

et al. 2018

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Selective inhibitors of Cryptosporidium calcium-dependent protein kinase 1 ( CpCDPK1) based on the 1 H-pyrazolo[3,4- d]pyrimidin-4-amine (pyrazolopyrimidine, PP) scaffold are effective in both in vitro and in vivo models of cryptosporidiosis. However, the search for distinct safety and pharmacokinetic (PK) properties has motivated our exploration of alternative scaffolds. Here, we describe a series of 7 H-pyrrolo[2,3- d]pyrimidin-4-amine (pyrrolopyrimidine, PrP)-based analogs of PP CpCDPK1 inhibitors. Most of the PrP-based inhibitors described potently inhibit the CpCDPK1 enzyme, demonstrate no toxicity against mammalian cells, and block proliferation of the C. parvum parasite in the low micromolar range. Interestingly, certain substituents that show reduced CpCDPK1 potency when displayed from a PP scaffold provided notably enhanced efficacy in the context of a PrP scaffold. PK studies on these paired compounds show that some PrP analogs have distinct physiochemical properties compared with their PP counterparts. These results demonstrate that inhibitors based on a PrP scaffold are distinct therapeutic alternatives to previously developed PP inhibitors.

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“…These efforts have led to the identification of numerous promising lead compounds and two pre-clinical candidates that are poised for human studies (i.e. a Novartis phosphatidyl-inositol-4-kinase inhibitor and the 6-carboxamide benzoxaborole AN7973) (12)(13)(14)(15)(16)(17)(18)(19)(20). There is the potential to realize an enormous public health benefit with intelligent deployment of improved anticryptosporidial drugs.…”

Section: Introductionmentioning

confidence: 99%

Spontaneous selection ofCryptosporidiumdrug resistance in a calf model of infection

Hasan

Stebbins

Choy

et al. 2021

Preprint

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The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years with numerous lead compounds identified including several tRNA synthetase inhibitors. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC50S of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.ImportanceCryptosporidium is a leading protozoan cause of diarrhea in young children with no reliable treatment. We report results of a dairy calf drug efficacy trial and the spontaneous emergence of drug resistance. Cryptosporidium parvum infected calves initially improved with drug treatment, but infection relapsed in two animals. Parasites shed by each recrudescent calf had mutations in the gene encoding the drug target that altered its amino acid sequence. Recapitulation of the drug target mutations by CRISPR/Cas9 genome editing resulted in highly drug-resistant parasites, and recombinant mutant enzymes were resistant to inhibition. This is the first report of naturally emerging Cryptosporidium drug resistance. There is a currently a great opportunity to impact public health with new drugs to treat cryptosporidiosis, and this report highlights the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence in order to realize their full potential.One-sentence summaryDrug-target point mutations mediating anticryptosporidial resistance spontaneously arose in the dairy calf C. parvum infection model.

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5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum

Cited by 17 publications

References 14 publications

Characterization of Calcium-Dependent Protein Kinases 3, a Protein Involved in Growth of Cryptosporidium parvum

Characterization of Calcium-Dependent Protein Kinases 3, a Protein Involved in Growth of Cryptosporidium parvum

7H-Pyrrolo[2,3-d]pyrimidin-4-amine-Based Inhibitors of Calcium-Dependent Protein Kinase 1 Have Distinct Inhibitory and Oral Pharmacokinetic Characteristics Compared with 1H-Pyrazolo[3,4-d]pyrimidin-4-amine-Based Inhibitors

Spontaneous selection ofCryptosporidiumdrug resistance in a calf model of infection

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