Novel Approach for Drug Development against Protozoan

Mucosal infections with anaerobic parasitic protozoa, including Giardia lamblia, Entamoeba histolytica and Trichomonas vaginalis, occur in a billion people worldwide and have significant impact medical and economic impact. G. lamblia and E. histolytica are intestinal pathogens. CDC classifies G. lamblia as a category B agent of bioterrorism and E. histolytica can be fatal if left untreated. T. vaginalis is a sexually transmitted infection and known to be associated with preterm delivery, low birth weight, and increased fetal mortality, and predisposes to HIV infection, AIDS, and cervical cancer. Metronidazole (Mz) has been used for more than 50 years against anaerobic protozoa, is listed by the WHO as an essential medicine, and is easily available worldwide. Antimicrobial therapy with Mz is usually effective, but Mz treatment failures and resistance occur in all target protozoa. Few drug alternatives exist for E. histolytica and T. vaginalis, so new drugs that can overcome Mz resistance are needed. In our studies, we have focused on two drug classes, 5-nitroimidazoles (5-NI) and organic gold compounds as a starting point for developing new treatment strategies against pathogenic protozoa. The general approach involves three major steps: 1. Structural diversification of existing lead compounds to generate new compound libraries;  2. Evaluation of activity against the target protozoa and toxicity against mammalian cells in vitro; 3. Evaluation of efficacy and toxicity in vivo. Compounds that meet defined functional requirements constitute promising new leads for further drug development. For the group 5-NI compounds, which includes the reference drug Mz, diversification was achieved by applying click chemistry to the 1’ or 2’ position of the base imidazole ring. This synthetic strategy enables a modular approach to ready library expansion, allowing us to generate >1,000 5-NI compounds. Several promising compounds were identified with excellent in vitro activity and the ability to overcome Mz resistance. Several of them were subsequently shown to be active in murine models of G. lamblia and trichomonas infections. 5-NIs are prodrugs that act in a two-step fashion. As an initial step, they are selectively reduced and activated by reductases in the target microbes. In a second step, the activated drugs react with target molecules to form adducts, leading to target inactivation. While the reduction steps have been explored extensively, much less is not known about the ultimate adduction targets. Using a modified 5-NI compound and click chemistry, we have identified a spectrum of adducted targets in target protozoa and have explored the mechanism of adduction-induced target inactivation. These data not only contribute to our understanding of the mechanisms of 5-NI action but also reveal new targets for drugs independent of 5-NI prodrug activation. In another set of studies, we have focused on organic gold(I) compounds as alternatives to 5-NI treatment. The reference compound in his group, auranofin (AF), was developed for rheumatoid treatment, but then shown by us to be active against several anaerobic protozoa. Structural diversification of AF led to the identification of two new gold(I) compounds with excellent activity against T. vaginalis and improved selectivity over human cells. Exploration of potential targets of these compounds revealed that the improved gold(I) compounds inhibited thioredoxin reductase, but that other targets are likely to exist and be more important for mediating the cidal activity in T. vaginalis. Overall, our efforts have identified several new drug candidates with improved activity against a range of pathogenic protozoa, but have also advanced our understanding of drug action mechanisms as a step towards novel target-centered efforts for alternative drug development.