Non-specificity of Pitstop 2 in clathrin-mediated endocytosis

Abstract

Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. ''Pitstop'' compounds have been developed that block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrin's N-terminal domain.

Targeted polyethylene glycol gold nanoparticles for the treatment of pancreatic cancer: from synthesis to proof-of-concept in vitro studies

“…68 Inhibition of clathrin-dependent endocytosis was achieved in this study by Pitstop ® 2. Although limitations are associated to its use due to the reported nonspecificity of its mode of action, 69 Pitstop ® 2 causes profound inhibition of the clathrin-dependent endocytosis pathway. 69,70 Filipin III is known in cellular biology as an inhibitor of the caveolae-mediated endocytosis pathway in mammalian cells.…”

“…Although limitations are associated to its use due to the reported nonspecificity of its mode of action, 69 Pitstop ® 2 causes profound inhibition of the clathrin-dependent endocytosis pathway. 69,70 Filipin III is known in cellular biology as an inhibitor of the caveolae-mediated endocytosis pathway in mammalian cells. 71 Finally, Brefeldin A was used as an indirect inhibitor of the hERG1 K + channel maturation, as it hinders the anterograde protein transport from the ER to the Golgi apparatus.…”

Delivery of the CSF-1R to the lumen of macropinosomes promotes its destruction in macrophages

“…The correlation of impaired CSF-1R internalization with impaired transferrin internalization under both inhibitor and CHC RNAi is consistent with CME as the route of entry. However, impaired delivery of the CSF-1R to the cell surface during CHC knockdown and possible inhibitor specificity issues (Dutta et al, 2012;Park et al, 2013;Willox et al, 2014) suggest that other small vesicle endocytic routes, such as caveolin-mediated endocytosis might be possible. Nonetheless, small vesicle endocytosis was the predominant route of CSF-1R internalization.…”

Exploiting endocytic pathways to prevent bacterial toxin infection

“…The recently developed clathrin inhibitor, Pitstop 2, could inhibit the entry of HIV into cultured cells [5], suggesting it as a therapeutic strategy to prevent uptake of viruses, microbes, and bacterial toxins. However, recent studies have highlighted some off-target effects of Pitstop 2 by demonstrating that it also blocks clathrin-independent endocytosis [105,106]. One study [105] based much of this conclusion on the inability of Pitstop 2-treated cells to internalize major histocompatibility complex I, which was later shown to be a clathrin-dependent process [107].…”

“…Moreover, further development to make isoform-specific dynamin inhibitors will pave the way for more selective inhibitors. Currently, a number of dynamin and clathrin inhibitors have been suggested to have some off-target effects, which should be kept in mind while investigating their suitability [101,105,106], as it is still unclear how this will affect their therapeutic feasibility and ability to prevent intoxication.…”