Inhibition of the clathrin terminal domain - amphiphysin protein-protein interaction. Probing the Pitstop® 2 aromatic moiety.

Abstract

Pitstop 2, (Z)-N-(5-(4-bromenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)naphthalene-1-sulfonamide (1), inhibits the clathrin terminal domain- amphiphysin interaction (NTD-PPI) and has been widely used to investigate endocytosis. In this work we report on the synthesis of 56 novel Pitstop 2 analogues via four discrete focused libraries. Specific modification to the 4-bromonenzylidene moiety were explored, and their ability to inhibit the NTD-PPI interaction examined by an ELISA. In cell endocytosis effects were measured for select analogues as was the inhibition of dynamin, another protein involved in the endocytosis process. The most NTD-PPI active analogues retained 2- and 4-disposed substituents on the aromatic head, with 2,3,4-trihydroxy (28) the most active (IC50 0.94 μM). Catechol free 2,3-dihydroxybenzo[b][1,4]dioxone (54) is a more promising lead with a NTD-PPI IC50 = 1.16 μM. The corresponding benzo[d][1,3]dioxole (53) was 3-fold less active suggesting ring size preference at this position. Nine analogues showed improved or comparable NTD-PPI activity to Pitstop 2 with IC50 values from 0.94 - 2.1 µM. Heterocyclic analogues were well tolerated and potent inhibitors of CME in U2OS cells, in particular, benzofuran 67 (NTD-PPI IC50 1.5 μM and CME IC50 6.8 ± 2.7 μM). This positions 67 as one of the most in cell active inhibitors of clathrin mediated endocytosis yet reported.