Author Correction: Jawsamycin exhibits in vivo antifungal properties by inhibiting Spt14/Gpi3-mediated biosynthesis of glycosylphosphatidylinositol.

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Jawsamycin exhibits in vivo antifungal properties by inhibiting Spt14/Gpi3-mediated biosynthesis of glycosylphosphatidylinositol.

Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.

Dark chemical matter as a promising starting point for drug lead discovery.

High-throughput screening (HTS) is an integral part of early drug discovery. Herein, we focused on those small molecules in a screening collection that have never shown biological activity despite having been exhaustively tested in HTS assays. These compounds are referred to as 'dark chemical matter' (DCM). We quantified DCM, validated it in quality control experiments, described its physicochemical properties and mapped it into chemical space. Through analysis of prospective reporter-gene assay, gene expression and yeast chemogenomics experiments, we evaluated the potential of DCM to show biological activity in future screens. We demonstrated that, despite the apparent lack of activity, occasionally these compounds can result in potent hits with unique activity and clean safety profiles, which makes them valuable starting points for lead optimization efforts. Among the identified DCM hits was a new antifungal chemotype with strong activity against the pathogen Cryptococcus neoformans but little activity at targets relevant to human safety.

FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p.

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae--Erg26p, Homo sapiens--NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound.

Use of principal components analysis and protein microarray to explore the association of HIV-1-specific IgG responses with disease progression.

The role of HIV-1-specific antibody responses in HIV disease progression is complex and would benefit from analysis techniques that examine clusterings of responses. Protein microarray platforms facilitate the simultaneous evaluation of numerous protein-specific antibody responses, though excessive data are cumbersome in analyses. Principal components analysis (PCA) reduces data dimensionality by generating fewer composite variables that maximally account for variance in a dataset. To identify clusters of antibody responses involved in disease control, we investigated the association of HIV-1-specific antibody responses by protein microarray, and assessed their association with disease progression using PCA in a nested cohort design. Associations observed among collections of antibody responses paralleled protein-specific responses. At baseline, greater antibody responses to the transmembrane glycoprotein (TM) and reverse transcriptase (RT) were associated with higher viral loads, while responses to the surface glycoprotein (SU), capsid (CA), matrix (MA), and integrase (IN) proteins were associated with lower viral loads. Over 12 months greater antibody responses were associated with smaller decreases in CD4 count (CA, MA, IN), and reduced likelihood of disease progression (CA, IN). PCA and protein microarray analyses highlighted a collection of HIV-specific antibody responses that together were associated with reduced disease progression, and may not have been identified by examining individual antibody responses. This technique may be useful to explore multifaceted host-disease interactions, such as HIV coinfections.

Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.