Micro-NMR and Nanoparticle Amplification                              for Botulinum Toxin                                              Diagnostics

Recombinant Envelope Protein Domain III as a                              Candidate Subunit Dengue                               Vaccine

A Highly Sensitive, Low-labor Pathogen Detector                              Based on Retroreflector-                              linked Immunosorbent                              Assay

Genetic Screens to Identify the Ebola Virus Receptor

High-throughput Assay Development Against                              Cryptosporidium Glycotlytic                              Enzymes

Model for Oral Ingestion of Ricin Toxin

High-throughput Assay Development Against Cryptosporidium Glycolytic Enzymes

Institution: Texas A&M University, College Station, Texas

Principal Investigator: Guan Zhu, Ph.D.

Expected Product: Therapeutics for cryptosporidiosis that target specific parasite enzymes.

Description: Cryptosporidium parvum is a unicellular pathogen that can cause severe watery diarrhea in humans and animals. Because the infectious oocysts are highly resistant to chemical stresses including chlorine treatment applied to the community water supplies, C. parvum is a significant water- and food-borne pathogen, and is listed as one of the Category B priority pathogens in the NIH and CDC biodefense research programs. Currently, only a single drug (nitazoxanide) has been approved for treating cryptosporidiosis in Central and South America, or cryptosporidiosis in immunocompetent (but not immunocompromised) patients in the United States. Therefore, there is an urgent need to develop new anti-Cryptosporidium drugs. Drug development against cryptosporidiosis has been a slow process, which is primarily due to the poor understanding of the basic metabolic pathways in the parasite. Many well-defined or promising drug targets found in other apicomplexans are either absent or highly divergent in C. parvum. However, on the positive side, recent advancements in genome sequencing and biochemical studies have revealed many unique proteins that may serve as drug targets in this parasite, which include bacterial-type lactate dehydrogenase (CpLDH1) and two distinct alcohol dehydrogenases (CpADH-E and CpADH2).

Our long-term goal is to develop drugs that target the C. parvum CpLDH1, CpADH-E and CpADH2 enzymes. Because C. parvum relies solely on glycolysis for its energy, we hypothesize that the glycolytic enzymes may serve as rational drug targets in this parasite. As a first step to reach the long-term goal and to test the hypothesis, we plan to perform experiments to achieve the following two specific aims:
Aim 1: To characterize the molecular features of CpLDH1, CpADH-E, and CpADH2, which includes validating whether these enzymes could serve as drug targets, analyzing expression patterns of C. parvum LDH and MDH genes, and detecting these enzymes in the parasite’s complex life cycle.
Aim 2: To develop assays for high-throughput screening (HTS) of compounds inhibiting these glycolytic enzymes, which include expressing C. parvum LDH and ADH enzymes as recombinant proteins, determining their enzymatic and inhibitory kinetics, developing assays in HTS format, and screening inhibitors from compound libraries.

Completion of these aims will not only deepen our understanding on the energy metabolism of C. parvum, but also has a great potential for the development of new drugs against this medically important parasite.