RESUMEN
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 to 42.5 mM, 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 (45a), 5-OH (45c) and 5-propyl ether (45d) moieties. Among them, the OH 45c and propyl ether 45d retained PPI inhibition, with propyl ether 45d being the most active with a PPI inhibition IC50 = 7.3 mM. This is 2x more potent than Pitstop® 2 and 3x more potent than Pitstop 1.
RESUMEN
The minor phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is crucial for neurotransmission and has been implicated in Parkinson's disease. Here, we present a staining protocol for the analysis of activity-dependent changes of PI(4,5)P2 at synapses. We describe steps for stimulating and fixing murine hippocampal neurons, staining with probes for PI(4,5)P2 and a synaptic marker, and analysis by high-resolution microscopy. Our approach gives insights into local PI(4,5)P2 synthesis and turnover at synapses and can be extended to phosphoinositide lipids other than PI(4,5)P2. For complete details on the use and execution of this protocol, please refer to Bolz et al.1.