Unique glycolytic pathway enzymes as molecular targets for novel anti-cryptosporidial drugs

Khan, Shahbaz Manzoor

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https://hdl.handle.net/2142/121928 Copy

Description

Title

Unique glycolytic pathway enzymes as molecular targets for novel anti-cryptosporidial drugs

Author(s)

Khan, Shahbaz Manzoor

Issue Date

2023-08-09

Director of Research (if dissertation) or Advisor (if thesis)

Witola, William H

Doctoral Committee Chair(s)

Witola, William H

Committee Member(s)

• Zhang, Weiping
• Zuckermann, Federico A
• Reddi, Prabhakara P

Department of Study

Pathobiology

Discipline

VMS - Pathobiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Cryptosporidium
• diarrhea
• glycolysis
• pyruvate kinase
• lactate dehydrogenase
• enzyme inhibitors
• molecular targets
• treatments
• drug development
• drug combinaions

Abstract

The intracellular protozoan parasite of the genus Cryptosporidium is among the leading causes of waterborne diarrheal disease outbreaks throughout the world. The parasite is transmitted by ingestion of infective oocysts that are highly stable in the environment and resistant to almost all conventional disinfection methods and water treatments. Control of the parasite infection is exceedingly difficult due to the excretion of large numbers of oocysts in the feces of infected individuals that contaminate the environment and serve as a source of infection for susceptible hosts including humans and animals. Drug development against the parasite is challenging owing to its limited genetic tractability, absence of conventional drug targets, unique intracellular location within the host, and the paucity of robust cell culture platforms for continuous parasite propagation. Despite the high prevalence of the parasite, the only United States Food and Drug Administration (FDA)-approved treatment for Cryptosporidium infections is nitazoxanide, which has shown moderate efficacy in immunocompetent patients. More importantly, no effective therapeutic drugs are available for treating severe, potentially life-threatening cryptosporidiosis in immunodeficient patients, young children, and neonatal livestock. Several compounds have been tested for both in vitro and in vivo efficacy against the disease. However, to date, only a few experimental compounds have been subjected to clinical trials in natural hosts, and among those none have proven efficacious. Thus, safe, inexpensive, and efficacious drugs are urgently required to reduce the ever-increasing global cryptosporidiosis burden especially in low-resource countries. The long-term goal of this study is to unveil validated lead-compound combinations for developing new effective drugs for treating C. parvum infection in humans and livestock. C. parvum possesses greatly simplified and curtailed metabolic and biochemical pathways in comparison to other apicomplexan parasites. Owing to the nonexistence of tricarboxylic acid cycle and ATP generating oxidative phosphorylation, C. parvum depends heavily on glycolysis for energy production. In the glycolytic pathway of C. parvum, the last two reaction steps that lead to the generation of metabolic energy (ATP) are catalyzed by a plant-type C. parvum pyruvate kinase (CpPyK), and a bacterial-type C. parvum lactate dehydrogenase (CpLDH). CpLDH and CpPyK are significantly different from their mammalian counterparts, making them ideal molecular targets for developing safe and efficacious anti-cryptosporidial drugs. In our previous studies, we have found that inhibitors of CpLDH enzyme can stop growth of the parasite and prevent disease in infected mouse models. In the present study, we developed a CpPyK enzyme-based assay and identified several compounds with in vitro inhibitory activity against the recombinant CpPyK enzyme. We used a series of in vitro cytotoxicity and parasite growth inhibition assays to study the safety and anti-cryptosporidial efficacy of these CpPyK-inhibitors. Safety and effectiveness of identified compounds was further validated in an immunocompromised mouse infection model to select non-toxic compounds, NSC234945 and NSC252172, with high in vivo efficacy against the parasite. Herein, we also investigated the anti-cryptosporidial synergistic activities of CpLDH- and CpPyK-inhibitors in C. parvum-infected mammalian cells using multiple synergism assessment methods: curve-shift analysis, combination-index method, dose-reduction index analysis, and isobologram analysis. Synergy analysis of in vitro dose-response data identified combinations of CpLDH- and CpPyK-inhibitors with strong synergistic effects against the growth and survival of C. parvum. The combinations that showed synergistic activity were evaluated for safety and efficacy against C. parvum using an in vivo mouse infection model. In immunocompromised mice, NSC303244+NSC158011, and NSC252172+NSC158011 compound combinations demonstrated improved effectiveness in reducing C. parvum oocyst numbers, and alleviated the intestinal damage caused by cryptosporidiosis at doses lower than those required for individual compounds. Notably, the NSC303244+NSC158011 combination proved to be effective in completely eradicating the infection in immunocompromised mice, without any subsequent relapse. Although combination therapy has proven effective in treating other related apicomplexan diseases, minimal efforts have been made to evaluate drug combinations against cryptosporidiosis. Our study has discovered combinations of compounds that effectively inhibit two crucial catalytic steps involved in metabolic energy production in C. parvum with enhanced efficacy against the parasite. Collectively, the results of this study will have an overall positive and significant impact in the challenging field of Cryptosporidium drug discovery by generating a new class of highly active and safe therapeutic leads for treating cryptosporidiosis in vulnerable human and animal populations.

Graduation Semester

2023-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Shahbaz Khan

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