We propose to develop an assay for high throughput screening (HTS) of small molecules for compounds that disrupt the fungal cell wall based on the fact that lysed fungal cells release adenylate kinase (AK) into the culture medium. AK activity can be directly measured in the medium using luminescence-based detection assays. In order to increase the sensitivity of the AK lysis assay, we further propose a chemical genetic approach in which fungi with mutations that affect cell wall integrity will be used to increase the susceptibility of the fungi to small molecules that affect cell wall biosynthesis. Our preliminary data in Saccharomyces cerevisiae indicate that the AK lysis assay: (1) is more sensitive than traditional growth assays and specific for fungicidal molecules, (2) is compatible with 384-well format and HTS and (3) applicable to the most common pathogenic yeast in humans, Candida albicans. The features of our AK lysis assay based approach include: a simple, """"""""add and read"""""""" AK assay protocol, increased sensitivity relative to growth assays, and potential applicability to any cultivatable fungi. In order to develop a systematic screening program for fungal cell wall-directed small molecules based on the AK lysis assay, we propose the following specific aims: (1) Identify and optimize cell wall gene mutants for chemical genetic screening using the AK lysis assay in S. cerevisiae;(2) adapt the optimized AK lysis assay to pathogenic fungi including Candida albicans;(3) develop secondary screening assays to distinguish cell wall-targeted molecules from those with other mechanisms of action.
The ultimate goal of the proposed project is to identify small molecules that disrupt fungal cell wall integrity as an approach to the development of mechanistic probes of fungal cell wall biosynthesis and new antifungal drugs with low human toxicity. Invasive, life threatening-fungal infections are an increasingly common complication of extreme prematurity, cancer chemotherapy, organ and stem cell transplantation, and primary or acquired immunodeficiency. Current treatment options for invasive fungal infections are limited and have significant side effects. The chemical genetic screening strategies we propose to develop will facilitate the identification of new antifungal drug candidates and, hopefully, improve the treatment of life threatening fungal infections in immunocompomised hosts.
