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Preselection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ~81,000 compounds in Saccharomyces cerevisiae and identified ~7500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. These data were used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes, we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ~7500 growth-inhibitory molecules have been made commercially available and the computational model and filter used are provided., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short "dead-end" intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Many pathogenic prokaryotes and eukaryotes possess the machinery required to assemble galactofuranose (Galf)-containing glycoconjugates; these glycoconjugates can be critical for virulence or viability. Accordingly, compounds that block Galf incorporation may serve as therapeutic leads or as probes of the function of Galf-containing glycoconjugates. The enzyme UDP-galactopyranose mutase (UGM) is the only known generator of UDP-galactofuranose, the precursor to Galf residues. We previously employed a high-throughput fluorescence polarization assay to investigate the Klebsiella pneumoniae UGM. We demonstrate the generality of this assay by extending it to UGM from Mycobacterium tuberculosis. To identify factors influencing binding, we synthesized a directed library containing a 5-arylidene-2-thioxo-4-thiazolidinone core, a structure possessing features common to ligands for both homologs. Our studies offer a blueprint for identifying inhibitors of the growing family of UGM homologs and provide insight into UGM inhibition.

Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene into pentacyclic compounds. It is the prokaryotic counterpart of the eukaryotic oxidosqualene cyclase (OSC) that catalyzes the steroid scaffold formation. Because of clear sequence homology, SHC can serve as a model for OSC, which is an attractive target for anticholesteremic drugs. We have established the crystal structure of SHC complexed with Ro48-8071, a potent inhibitor of OSC and therefore of cholesterol biosynthesis. Ro48-8071 is bound in the active-center cavity of SHC and extends into the channel that connects the cavity with the membrane. The binding site of Ro48-8071 is largely identical with the expected site of squalene; it differs from a previous model based on photoaffinity labeling. The knowledge of the inhibitor binding mode in SHC is likely to help develop more potent inhibitors for OSC.

Background: The squalene:hopene cyclases (SHCs) are bacterial enzymes that convert squalene into hopanoids, a function analogous to the action of oxidosqualene cyclases (OSCs) in eukaryotic steroid and triterpenoid biosynthesis. We have identified the binding site for a selective, potent, photoactivatable inhibitor of an SHC., Results: SHC from Alicyclobacillus acidocaldarius was specifically labeled by [3H]Ro48-8071, a benzophenone-containing hypocholesteremic drug. Edman degradation of a peptide fragment of covalently modified SHC confirmed that Ala44 was specifically modified. Molecular modeling, using X-ray-derived protein coordinates and a single point constraint for the inhibitor, suggested several geometries by which Ro48-8071 could occupy the active site., Conclusions: A covalent complex of a potent inhibitor with a squalene cyclase has been characterized. The amino acid modification and molecular modeling suggest that Ro48-8071 binds at the junction between the central cavity and substrate entry channel, therefore inhibiting access of the substrate to the active site.