CRYPTOSPORIDIUM PARVUM FERMENTATION ENZYMES AS DRUG TARGETS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1002549
• Project Start Date: Feb 26, 2014
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Veterinary Pathobiology

Non Technical Summary
Cryptosporidium parasites may cause severe or deadly infections in humans and animals. However, options to treat crpytosporidiosis are limited. In fact, there is no FDA-approved drugs to treat cryptosporidiosis in animals. This project is part of our long-term effort in discovery anti-cryptosporidial drugs by understanding the uniquemetabolic features in the parasite. More specifically, we will characterize some fermentative enzymes fromthe parasites and explore them as potential drug targets for developing new therapeutics.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4050	1110	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4050 - Protozoa;

Field Of Science
1110 - Parasitology;

Keywords

parasite

cryptosporidium parvum

hexokinase

lactate dehydrogenase

alcohol dehydrogenase

inhibitors

Goals / Objectives
Our long-term goal is to dissect the unique energy metabolic pathway including enzymes involved in glycolysis and fermentation in C. parvum as novel targets for drug development. In this project, we will test our hypothesis by focusing on two bacterial-type fermentation enzymes in the parasite, i.e., lactate dehydrogenase (CpLDH) and alcohol dehydrogenase (CpADH). We will perform experiments to achieve the following three objectives: 1) To identify potential hits against CpLDH from 1280 FDA-approved known drugs; 2) To characterize the biochemical features of CpADH1; and 3) To test potential synergistic anti-cryptosporidial efficacy by targeting both glycolysis (i.e. CpHK) and fermentation (i.e., CpLDH and/or CpADH1).

Project Methods
Parasite enzymes will be expressed as recombinant proteins to study their biochemical features; The actions of inhibitors on recombinant parasite proteins will be assayed biochemically; Contributions of various inhibitors on their anti-cryptosporidial activities will be assayed using an in vitro cell cultivation system.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student and a postdoctoral fellow have participiated this project and received training opportunity. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue experiments towards achieving the proposed objectives.

Impacts
What was accomplished under these goals? Major accomplishments include the identification of serveral inhibitors of Cryptosporidium parvum phosphoglucose isomerase (CpPGI) and determination of the inhibitory kinetics offluorodeoxyglucose (FDG) on C. parvum hexokinase (CpHK), which allowed us to further investigate their anti-cryptosporidialefficacy in vitro. Experiments are ongoing to identify new CpHK inhibitors and to study the action of CpPGI and CpHK inhibitors on the parasite growth in vitro and theircytotoxicity to host cells. Some of the data produced in this project were presented in a published manuscript (i.e.,Yu et all, 2014. A unique hexokinase in Cryptosporidium parvum, an apicomplexan pathogen lacking the Krebs cycle and oxidative phosphorylation. Protist. 165(5):701-14).

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yu Y, Zhang H, Guo F, Sun M, Zhu G (2014). A unique hexokinase in Cryptosporidium parvum, an apicomplexan pathogen lacking the Krebs cycle and oxidative phosphorylation. Protist. 165(5):701-14. PMID: 25216472; PMCID: PMC4252602.

Progress 02/26/14 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD graduate student participated in this project and received research training. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the past 6 months, we made key progress critical to the next phase of study. More specifically, we cloned the Cryptosporidium parvumphosphoglucose isomerase (CpPGI; aka glucose-6-phosphate isomerase, CpGPI) gene, andexpressed the CpPGIprotein as recombinant fusion protein. The recombinantCpPGI was enzymatically active. Experiments to biochemically characterize the properties were ongoing. We also determined that FDG was also capable of inhibiting CpHK enzyme activity as well as the growth of parasite. These data enabled us to continue this project into next phase of study as proposed.

Publications