title: Advantages and Challenges of Phenotypic Screens: The Identification of Two Novel Antifungal Geranylgeranyltransferase I Inhibitors.
abstract: Phenotypic screens are effective starting points to identify compounds with desirable activities. To find novel antifungals, we conducted a phenotypic screen in Saccharomyces cerevisiae and identified two discrete scaffolds with good growth inhibitory characteristics. Lack of broad-spectrum activity against pathogenic fungi called for directed chemical compound optimization requiring knowledge of the molecular target. Chemogenomic profiling identified effects on geranylgeranyltransferase I (GGTase I), an essential enzyme that prenylates proteins involved in cell signaling, such as Cdc42p and Rho1p. Selection of resistant mutants against both compounds confirmed the target hypothesis and enabled mapping of the compound binding site to the substrate binding pocket. Differential resistance-conferring mutations and selective substrate competition demonstrate distinct binding modes for the two chemotypes. Exchange of the S. cerevisiae GGTase I subunits with those of Candida albicans resulted in an absence of growth inhibition for both compounds, thus confirming the identified target as well as the narrow antifungal spectrum of activity. This prenylation pathway is reported to be nonessential in pathogenic species and challenges the therapeutic value of these leads while demonstrating the importance of an integrated target identification platform following a phenotypic screen.
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Verena
authors.initials: V
authors.lastname: Pries
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Simona
authors.initials: S
authors.lastname: Cotesta
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Ralph
authors.initials: R
authors.lastname: Riedl
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Aust
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Sven
authors.initials: S
authors.lastname: Schuierer
authors.affiliation: Novartis Institutes for BioMedical Research, Cambridge, MA, USA.
authors.forename: Jianshi
authors.initials: J
authors.lastname: Tao
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Ireos
authors.initials: I
authors.lastname: Filipuzzi
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland dominic.hoepfner@novartis.com.
authors.forename: Dominic
authors.initials: D
authors.lastname: Hoepfner
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D055808:Drug Discovery / Q000379:methods*; D025141:Drug Resistance, Fungal; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D020869:Gene Expression Profiling; D055432:Metabolomics / Q000379:methods; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics
chemical_list: D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I
keywords: HIP; HOP; functional variomics; geranylgeranyltransferase; target identification