The folate biosynthetic (FOL) pathway has been targeted with enormous success in the development of anticancer, antibacterial as well as antiprotozoal drugs. Methotrexate and trimethoprim are potent inhibitors of mammalian and bacterial dihydrofolate reductase (DHFR) respectively, while a collection of ?sulfa drugs? perturb dihydropteroate synthase function in some bacterial as well as protozoan parasites. Additional antiprotozoal drugs that target this pathway include the DHFR inhibitors pyrimethamine, proguanil and chlorproguanil, as well as the DHPS inhibitor dapsone. However, these conventional antifolate drugs have little or no activity upon the major human fungal pathogens, either because of divergence of the fungal enzymes structure, or permeability issues that prevent them from entering fungal cells. Furthermore, efforts to adapt conventional antifolate scaffolds have failed to yield derivatives with the requisite properties of a viable antifungal drug and have focused almost exclusively upon DHFR, with the remaining enzymes almost completely uncharacterized in any pathogenic species. We propose that efforts to exploit this pathway for antifungal development should focus upon the FOL biosynthetic enzymes that have not yet been the subject of significant investigation and that are completely absent from mammals. In addition, they should seek novel antifolate scaffolds that are active upon whole fungal cells. The objective of this proposal is to substantiate the validity and feasibility of targeting fungal Fol1p and Fol3p, which together possess four FOL enzyme activities that are entirely absent from mammals.
In aim 1 we will confirm the essentiality of the Fol1p and Fol3p proteins in two of the most prevalent human fungal pathogens, the yeast Candida albicans and the infectious mold Aspergillus fumigatus, and establish the potential antifungal efficacy that can be achieved in targeting these enzymes using mouse models of invasive fungal infection.
In aim 2 we will establish and validate high-throughput compatible cell-based and biochemical assays that can be applied to identify small molecules inhibitors of these enzymes activity. Collectively, these studies will determine if FOL enzymes that are absent from mammals can provide chemically tractable and efficacious targets to devise new antifungal therapies and potentially yield lead compounds that can form the basis of such medications.
The number of deaths caused by invasive fungal infections (IFIs) continues to rise in large part due to the inadequacies of current treatment options and the emergence of resistant fungi. This study will foster the development of new and improved therapies to cure IFIs, counteract the increasing incidence of antifungal drug resistance, and therefore improve patient outcomes.