Developing New Antifungals Against Sterolglycosidases

ABSTRACT Invasive fungal infections are a leading cause of death in immunocompromised patients. While much is known about the cellular processes required for the pathogenesis of these infections, translating understanding into tangible clinical benefit has been difficult because these fungal pathogens and their hosts have similar physiology. As a result, current antifungal agents have limited clinical efficacy, are poorly fungicidal in the host, are occasionally toxic, and are increasingly ineffective due to emerging resistance. Thus, innovative antifungal agents are needed. In previous studies, the steryl-glucosidase 1 (SGL1) gene was deleted in Cryptococcus neoformans (Cn) and its homolog (SGLA) gene in Aspergillus fumigatus and found that the resulting mutants (Cn ?sgl1 or Af ?sgla) accumulates steryl glucosides (SGs), are not able to grow at physiological media, and are not pathogenic in a mouse model of infection. Sterylglucosidases are enzymes found in plants and fungi but not in humans. Amazingly, mice receiving an intranasal administration of Cn ?sgl1 or Af ?sgla rapidly eliminate the respective mutant because these mutants are not viable at 5% CO2 and low O2 (5-10%). This indicated that the fungal sterylglucosidase is necessary for fungal pathogenicity and it is a promising novel drug target. Therefore, a high throughput screening (HTS) assay was developed, used to screen a ChemBridge DiverSet library and inhibitors to Sgl1/SglA enzymes were identified. The crystal structures of both Sgl1 and SglA alone and with the specific inhibitors were obtained. In addition to inhibiting sterylglucosidase activity in vitro, these compounds also decrease Sgl1/SglA activity in living fungal cells, accumulating SGs, and blocking fungal growth of wild-type strains in physiological media, recapitulating the phenotype observed with the Cn ?sgl1 or Af ?sgla mutant. Treatment with Sgl1 inhibitor protects mice from developing cryptococcal meningoencephalitis and treatment with SglA inhibitor significantly improved mice survival upon pulmonary aspergillosis. Therefore, by applying structural & computational biology and medicinal chemistry approaches to the rigorous/go-no-go investigations, a novel anti-Sgl1/SglA antifungal inhibitor(s) with no human cross-reactivity will be developed. To test this hypothesis, the following aims are proposed: Aim 1. Structure-based computer-aided drug design (CADD), synthesis and biochemical studies for hit-to-lead and lead optimization of novel Sgl1/SglA inhibitors; and Aim 2. PK, toxicology and antifungal activity. Impact: By coordinating studies across two major fungal pathogens and bridging academia and industry, this effort will accelerate discovery and development beyond what would be possible separately. In addition, by combining discovery with optimization, these novel inhibitors will be positioned for further development by pharmaceutical companies. The ongoing collaboration on antifungal drug development among the Institute of Chemical Biology and Drug Discovery, the New York Center for Biotechnology, and MicroRid Technologies Inc. brings a unique opportunity to accomplish this new antifungal program.