Figure 4.

A novel chemical series which modulates TFEB and TFE3 regulation and lysosome activity. (A, B) a series of novel analogs of NK140 was assessed in concentration response at 24 h treatment time for toxicity and activity across the panel of assays. (C) TFEB and TFE3 translocation was confirmed to be induced by the hit compound NK140 but not the negative compound NK164. TFEB and TFE3 translocation is also induced by the analogs NK176 and NK177. DQ Red BSA signal was seen to be slightly, but not-significantly, increased. NK140, NK176 and NK177 show a small reduction in nuclear count from 0.75 µM without increases in AK, suggesting a small anti-proliferative effect over 48 h treatment. n = 3 for all above assays, mean ± s.d. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001 Two-way ANOVA with Dunnett’s multiple comparisons test. (D) iPSC-derived i3Neurons treated with compounds as shown were stained with DAPI (blue), and immunostained for MAP2 (yellow) and TFE3 (red). Scale bars: 50 µm. (E) Nuclear translocation of TFE3 was quantified, showing a significant relocalization by the active compound NK177 (n = 3 for all above assays, mean ± s.d.; One-way ANOVA with Dunnett’s multiple comparisons test).