An Integrated Approach for Identification and Target Validation of Antifungal Compounds Active against Erg11p

Abstract

bSystemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents

“…To identify novel compounds with antifungal activity, high-throughput screening was performed using the Novartis compound archive against S. cerevisiae cells in a miniaturized 1,536-well plate assay (9). Compound 1 showed complete growth inhibition at the screening concentration of 20 M. Using compound 1 as a lead tool molecule, similarity searches revealed 7 structurally similar substances of the triazolopyrimidine-sulfonamide class (Fig.…”

“…The primary compound screen against S. cerevisiae was performed as described previously (9). Antifungal susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution M27-A3 and M38-A2 (10,11).…”

Antifungal Drug Discovery: The Process and Outcomes

“…A similar approach was recently used to identify novel antifungal compounds from a Novartis compound archive using the S. cerevisiae haploinsufficiency profiling (HIP) assay [32]. Preliminary screens led to the identification of three compounds with MIC values of 0.25–24 µg/ml against C. albicans but with limited activity against A. fumigatus .…”

Genome-wide investigation of cellular targets and mode of action of the antifungal bacterial metabolite 2,4-diacetylphloroglucinol inSaccharomyces cerevisiae

“…In addition, the S. cerevisiae deletion libraries are useful and widely used tools for chemical screenings to perform drug sensitivityrelated genome studies. Commonly, toxicogenomic studies employ either the haploid gene deletion collections with no gene expression (Parsons et al, 2004;Arita et al, 2009;Zhou et al, 2009;Costanzo et al, 2010;Emadi et al, 2010;Fujii et al, 2010;Stefanini et al, 2010;Kwak et al, 2011) or the heterozygous diploid collection in which gene expression is reduced (also referred to as haploinsufficiency profiling; Giaever et al, 1999;Baetz et al, 2004;Hillenmeyer et al, 2008;Bendaha et al, 2011;Hoepfner et al, 2012). The aim of these approaches is to establish chemical-genetic profiles of specific compounds such as arsenic or naphthoquinones Emadi et al, 2010) or on a larger scale to develop genetic interaction maps (Parsons et al, 2004;Costanzo et al, 2010), both of which can lead to the identification of the compound's target pathways or proteins.…”

“…Genomic profiling can in some cases even identify the direct target of a drug as exemplified by tunicamycin and its target gene ALG7 (Giaever et al, 1999). Furthermore, a recent study illustrates the potential of genomic profiling to facilitate the discovery of novel antifungal drugs for a known target, in this case Erg11p (Hoepfner et al, 2012). In general, a major advantage of a chemical-genetic approach is that it is unbiased and may therefore point to novel and unexpected drug targets, side effects or modes of action by covering the whole genomic response.…”