Inhibition Clathrin Mediated Endocytosis: Pitstop 1 and Pitstop 2 Chimeras

Twenty-five chimera compounds of Pitstop 1 and 2 were synthesised and screened for their ability to block the clathrin terminal domain-amphiphysin protein-protein interaction (NTD-PPI using an ELISA) and clathrin mediated endocytosis (CME) in cells. Library 1 was based on Pitstop 2, but no notable clathrin PPI or in-cell activity was observed. With the Pitstop 1, 16 analogues were produced with 1,8-naphthalic imide core as a foundation. Analogues with methylene spaced linkers and simple amides showed a modest to good range of PPI inhibition (7.6-42.5 μM, naphthyl 39 and 4-nitrophenyl 40 respectively) activity. These data reveal the importance of the naphthalene sulfonate moiety, with no des-SO₃ analogue displaying PPI inhibition. This was consistent with the observed analogue docked poses within the clathrin terminal domain Site 1 binding pocket. Further modifications targeted the naphthalene imide moiety, with the installation of 5-Br (45 a), 5-OH (45 c) and 5-propyl ether (45 d) moieties. Among them, the OH 45 c and propyl ether 45 d retained PPI inhibition, with propyl ether 45 d being the most active with a PPI inhibition IC₅₀=7.3 μM. This is 2x more potent than Pitstop 2 and 3x more potent than Pitstop 1.