Exo1: A new chemical inhibitor of the exocytic pathway

Abstract

A phenotypic screen was used to search for drug-like molecules that can interfere with specific steps in membrane traffic. 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo1), identified in this screen, induces a rapid collapse of the Golgi to the endoplasmic reticulum, thus acutely inhibiting the traffic emanating from the endoplasmic reticulum. Like Brefeldin A (BFA), Exo1 induces the rapid release of ADP-ribosylation factor (ARF) 1 from Golgi membranes but has less effect on the organization of the trans-Golgi network. Our data indicate that Exo1 acts by a different mechanism from BFA. Unlike BFA, Exo1 does not induce the ADP-ribosylation of CtBP͞Bars50 and does not interfere with the activity of guanine nucleotide exchange factors specific for Golgibased ARFs. Thus, Exo1 allows the fatty acid exchange activity of Bars50 to be distinguished from ARF1 activity in the control of Golgi tubulation.Golgi ͉ ADP-ribosylation factor ͉ endoplasmic reticulum (ER) ͉ imaging-based screen ͉ Bars50

Synthesis of a 10,000-Membered Library of Molecules Resembling Carpanone and Discovery of Vesicular Traffic Inhibitors

“…Utilizing an image-based phenotypic screen that we developed previously, 50 we screened the carpanone-based library for molecules that perturb exocytic traffic of membrane proteins from the ER to the plasma membrane. The screen utilizes the temperature-sensitive vesicular stomatitis virus glycoprotein fused to green fluorescent protein (VSVG ts -GFP).…”

Using High-Throughput Screening Data To Discriminate Compounds with Single-Target Effects from Those with Side Effects

“…This diverse collection of assays includes target-based fluorescence polarization assays, 27 growth-based assays, 8,9 and phenotypic assays measuring microtubule assembly, actin polymerization, endocytosis, and acetylation among others. [10][11][12][13][14][15][16][17][18][19][20][21][22][23] Given the large number of assays screened and the large number of targets that can induce a single phenotype, there is a good chance that chemical action on a single biological target will induce more than one assay phenotype.…”

“…This list was supplemented with compound classes containing 66 compounds published as bioactive in various assays of this library. [8][9][10][11][12][13][14][15][16][17][18][19][20][21] To identify compound classes with side effects (having multiple targets), compound classes scoring positively in two or more pure protein assays or scoring positively in an assay for fluorescence and another assay employing a nonfluorescent readout were identified. The compounds identified as bioactive and the classes containing them generally only had a single reported target and were assumed to be specific since many of these compounds were previously evaluated for side effects prior to their publication or were optimized structurally: this does not preclude the possibility that secondary targets of these compounds may be observed at higher compound concentrations or in other biological assays not tested; but for the sake of this comparison, they will be labeled as "single target" classes.…”

“…Substantially the same collection of phenotypic assay data has previously been shown to contain information useful in describing the biological activity of small molecules 41 and has led to identification of numerous biologically active molecules. [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]26 To annotate each structural class with assay data from the 48 ICCB high-throughput screens, screening results for each compound were converted to binary designations ('hit' or 'nonhit') such that the top or bottom 4% of screened compounds were considered hits in a given assay if it employed a numeric measurement (visually assessed phenotypes were textual and did not use thresholds.) This choice of thresholds was based on previous results showing that top-4% and bottom-4% thresholds yield statistically significant concentrations of hits in compound classes obtained by clustering, 41 and these measurements were filtered statistically by class scoring (described below) before their use.…”

“…In a forward chemical-genetics experiment, a collection of compound leads identified by a high-throughput phenotypic assay is studied in subsequent rounds of follow-up assays until the biological targets of those compounds can be identified. [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] Because these primary assays survey phenotypes, rather than binding to a single protein, many different biological targets are possible. By contrast, in a reverse chemical genetics experiment, a compound target is known, but the small-molecule-induced phenotype is not wellcharacterized: 27 this also presents experimental challenges requiring numerous rounds of follow-up phenotypic assays and the possible discovery of side effects.…”