Fig. 4: Characterization of small molecule-based CAST candidates in cells.

a Schematic of a small molecule-based CAST system. The ABM is initially constrained (gray) by the intramolecular association of NS3a and CP5. After the addition of small molecule inhibitors (Asunaprevir, Danoprevir, or Grazoprevir) that disrupt the NS3a:CP5 complex, the CAST transitions to an active state (blue) capable of F-actin binding (left). The primary sequence of small molecule-based CAST candidates tested (right). b Fluorescent micrographs of fixed HeLa cells expressing sm1-sm4 in the absence of small molecule inhibitors. smCAST (sm2) was selected for further study since its localization to F-actin was significantly reduced (yellow arrows). Scale bar 30 µm. c AlphaFold2 prediction of native ABS (blue, left) and smCAST (right). ABS alone is predicted to be an α-helix, the active form of the ABM, whereas within smCAST the ABS sequence is unstructured (right). d Quantification of F-actin binding inhibition for ABS and Lifeact sm1-sm4, where 0% is defined by ABS-only localization and 100% is defined by GFP-only localization. Bars represent mean ± SD. n = 40 biological replicates. e Fluorescent micrographs of smCAST in the presence or absence of Asu, Dano, or Grazo in live HeLa cells (left). Quantification of activation in the presence of drug for 30 min (right), where 0% is defined by inactive smCAST localization and 100% is defined by ABS-only localization. Scale bar 20 µm. Bars represent mean ± SD. n = 30 biological replicates. f Time-lapse imaging of the smCAST after the addition of Grazo shows increased localization of smCAST to F-actin (white arrows) over time. Scale bar 10 µm. g Kinetics of smCAST activation. The ratio of smCAST’s F-actin-localized fluorescence intensity to its cytoplasmic fluorescence intensity normalized to GFP-only localization is plotted at several timepoints, up to 90 min. t_1/2 = binding half-time. n = 3 biological replicates. Data were fit to a one-phase exponential association model (broken lines). P values were determined using a two-tailed unpaired t-test comparison with ABS-only control (d) or inactive smCAST (e). (ns not significant P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001). Source data are provided as a Source Data file.