APEs as novel drug targets in AIDS opportunistic pathogen Toxoplasma

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DESCRIPTION (Provided by Applicant): New drugs against parasitic protozoa of phylum Apicomplexa are urgently needed. Significant threats include Plasmodium (malaria), Cryptosporidium (diarrheal disease), and Toxoplasma (toxoplasmic encephalitis). The latter two are on the NIAID list of pathogens of interest for Biodefense, but are also serious opportunistic infections in AIDS. We have launched an investigation to study the therapeutic potential of Toxoplasma homologues of apurinic/apyrimidinic (AP) endonuclease, a key enzyme in the DNA base excision repair (BER) pathway. Toxoplasma possesses two distinct types of AP endonuclease that have attributes making them worthy candidates for drug development: TgAPE, which is divergent from its human homologue, and TgAPN, a type not found in mammals. We have found that a known inhibitor of human APE1, lucanthone, results in a significant reduction in Toxoplasma growth. However, because Toxoplasma has both APE and APN homologues, it will be necessary to establish the functional roles of these two enzymes in order to successfully develop therapeutic agents. There are three possibilities with regard to the functional roles of Toxoplasma APE and APN enzymes: (1) TgAPE is the primary AP endonuclease required for BER (2) TgAPN is the primary AP endonuclease, or (3) both enzymes play important roles in Toxoplasma. We propose to establish the contributions of the TgAPE and TgAPN enzymes by using chemical biology and conditional knockouts to address our hypothesis that Toxoplasma AP endonucleases will serve as good drug targets. Aim 1 will identify compounds that selectively inhibit TgAPE and TgAPN for target validation. To identify candidate inhibitors, we will pursue a high-throughput screen in our core facility. We have already generated recombinant TgAPE and TgAPN, and the assay for drug screening has been established, putting us in an excellent position to complete these studies. The specific inhibitors will permit a pharmacological approach to validate TgAPE and TgAPN as drug targets. Aim 2 will determine the impact of TgAPE and TgAPN on parasite physiology using conditional knockouts. These mutant lines will allow us to determine if TgAPE and/or TgAPN are essential for parasite viability. We will also be able to assess which of these AP endonucleases functions as the primary enzyme (or determine if both are equally important) by evaluating the conditional knockout's response to DNA damaging agents. It is in the interest of public health to investigate novel drugs and drug targets against Toxoplasma gondii, a serious opportunistic parasite of AIDS and other immunosuppressed patients. Toxoplasma is also listed by NIAID as a category B pathogen relevant to Biodefense research. Moreover, Toxoplasma can be informative as a model organism to study Plasmodium, the causative agent of malaria.