Composite synthetic lethal identification of membrane traffic inhibitors

Abstract

Small molecule inhibitors provide powerful tools to characterize highly dynamic and complex eukaryotic cell pathways such as those mediating membrane traffic. However, a lack of easy and generalizable assays has constrained identification of novel inhibitors despite availability of diverse chemical libraries. Here, we report a facile growth-based strategy in yeast to screen for pathway-specific inhibitors. The approach uses well characterized synthetic genetic growth defects to guide design of cells genetically sensitized for inhibition of chosen pathways. With this strategy, we identified a family of piperazinyl phenylethanone compounds as inhibitors of traffic between the trans-Golgi network (TGN) and endosomes that depends on the clathrin adaptor complex AP-1. The compounds did not significantly alter other trafficking pathways involving the TGN or endosomes, indicating specificity. Compound treatment also altered localization of AP-1 in mammalian cells. These previously uncharacterized inhibitors will be useful for future studies of clathrin-mediated transport in yeast, and potentially in other organisms. Furthermore, the easily automated technology should be adaptable for identification of inhibitors of other cellular processes.clathrin ͉ small molecule ͉ AP-1 ͉ GGA ͉ yeast

Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation

“…To generate a suitable strain for screening, genes for the multidrug resistance pumps PDR5 and SNQ2 were disrupted to increase the steady-state intracellular concentration of the drugs in yeast (19). The tim10-1 mutant grew similar to the parental strain (designated TIM10) at 25°C but failed to grow at the restrictive temperature of 37°C (Fig.…”

“…We exploited a large collection of temperature-sensitive mutants for the TIM22 import pathway (10,(16)(17)(18) and developed a composite synthetic lethal screen to identify small molecule inhibitors that blocked the TIM22 import pathway (19). The tim10-1 mutant was used as the starting strain (16); the strains used in this study are described in Table S1.…”

“…Here we report the development of a small molecule screening approach to identify inhibitors of the TIM22 import pathway. Taking advantage of our large collection of temperature-sensitive mutants for the TIM22 import pathway, we conducted a chemical-genetic screen with a tim10-1 mutant to identify small molecules that caused a synthetic lethality at the permissive temperature of 25°C (16)(17)(18)(19). Our results indicate that a new set of tools for mechanistic studies in protein translocation can be developed and may be useful for characterizing protein translocation in mammalian mitochondria, where tools are lacking.…”

Vesicle trafficking plays a novel role in erythroblast enucleation

“…21 Compound A5 is a piperazinyl phenylethanone derivative that specifically inhibits trafficking between TGN and endosomes that depend on AP-1-mediated, clathrin-coated vesicle formation. 22 Sucrose effectively inhibits clathrin-coated pit formation and thus blocks endocytosis. 23 BFA is a fungal metabolite that interacts with the sec7 domain of the large guanine nucleotide exchange factors (GEFs) GBF1 (Golgiassociated BFA-resistant), BIG1 (BFA-inhibited1), and BIG2 (BFAinhibited2) and inhibits their action on Arf1-3 (class I ADP ribosylation factor) GTPases.…”

Mimicking tricks from nature with sensory organic–inorganic hybrid materials

“…The latter feature, however, can also be used deliberately to inhibit certain cellular trafficking pathways. 5 In living organisms, the majority of these sensory elements does not exist as dissolved molecules, but is bound to a more or less flexible bio(in)organic skeletal backbone. The organizing support and supramolecular (bio)recognition element thus present a hybrid ensemble, often exerting synergistic effects and being implemented in signal transduction cascades.…”