Dark chemical matter – could “inactive” compounds be good starting points for lead discovery?

High-throughput screening (HTS) has become an integral part of early drug discovery in the pharmaceutical industry and academia and has enabled the discovery and insight into the biological actions of numerous reagents. Here, we focus on those small molecules in a screening collection that have shown no biological activity despite having been exhaustively tested in numerous HTS assays. These compounds are referred to as “dark chemical matter” (DCM). For the first time, we quantify DCM, validate it in quality control experiments, describe its physicochemical properties, and map it into the chemical space relative to the other compounds in the screening collection. Through analysis of prospective reporter gene assay, gene expression, and yeast chemogenomics experiments, we evaluate the potential of dark matter compounds to show biological activity in future screens. We demonstrate that despite the apparent lack of activity of DCM, occasionally these compounds can result in potent hits with unique activity profiles. Among the identified DCM hits is a new anti-fungal chemotype with strong activity against the pathogen Cryptococcus neoformans, but little activity at human host targets. Testing DCM for biological activity may be a widely applicable strategy to finding hits with reduced promiscuity and clean safety profiles, which makes these compounds valuable starting points for small molecule lead optimization efforts

Data from: Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents

High-throughput phenotypic screening against yeast Saccharomyces cerevisiae revealed a series of triazolo-pyrimidine-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, catalyzes the first common step in branched chain amino acid biosynthesis) as a possible target. High correlation to 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 the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity and it was possible to dock the triazolo-pyrimidine-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 triazolo-pyrimidine-sulfonamide as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo

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

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper

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

Supplemental_Data.zip

The compressed Supplemental_Data.zip archive contains two files: 1. Supplemental Data_S3, the complete HIP HOP profiles are provided as tab delimited .txt files. The sensitivity and z-scores for all HIP strains present in the pool across all tested compounds are listed in the HIP-exp-scores-annotation.txt file, scores for all HOP strains present in the pool are listed in the HOP-exp-scores-annotation.txt file. To reproduce the plots depicted in the article the sensitivity can be plotted on the y-axis, the z-score on the x-axis. Each gene is annotated with systematic name, common name, description, GO category etc. 2. The Supplemental_Data_S4.zip file containing the in silico docking solution. It is provided as a standard Protein Data Bank file named 1N0H_compound1_docked.pdb. UNK1 refers to the docked compound 1, all other structures are labeled as in the original 1N0H.pdb file downloaded from RCSB protein databank www.pdb.org

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

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

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