Turning the Actin Nucleating Compound Miuraenamide into Nucleation Inhibitors

Abstract

Natural compounds that either increase or decrease polymerization of actin into filaments have become indispensable tools for cell biology. However, to date, it was not possible to use them as therapeutics due to their overall cytotoxicity and their unfavorable pharmacokinetics. Furthermore, their synthesis is in general quite complicated. In an attempt to find simplified analogues of miuraenamide, an actin nucleating compound, we identified derivatives with a paradoxical inversion of the mode of action: instead of increased nucleation, they caused an inhibition. Using an extensive computational approach, we propose a binding mode and a mode of action for one of these derivatives. Based on our findings, it becomes feasible to tune actin-binding compounds to one or the other direction and to generate new synthetic actin binders with increased functional selectivity.

Figure 1

(A) Structures of the original natural compound miuraenamide A and the derivatives. The nomenclature from ref (7) is set in parentheses. (B) Miuraenamide A causes a clear perinuclear accumulation of F-actin and complete loss of the F-actin network compared to untreated controls. The derivatives LK701, LK703, LK717, and LK719 change morphology of F-actin in a completely different way with accumulations along the cell borders, indicating a different mode of action. Scale bar: 30 μm. (C) High-resolution images of single cells treated with different concentrations of Miuraenamide A or LK701. Scale bar: 10 μm. In (B) and (C), the respective concentrations of the compounds are indicated. In (B), the concentrations have been adapted to 5 times the EC₅₀ value in previous studies to guarantee comparability of the compounds. Blue: nuclear staining with Hoechst; red: actin fibers stained with rhodamine-phalloidine. (C) indicates untreated control.

Figure 2

(A) Top: representative images of in vitro formed actin filaments (TIRF microscopy). Scale bar: 5 μm. Bottom: Quantitative analysis of the number of filaments (normalized to control). All derivatives inhibit formation of filaments at a concentration of 500 nM. (B) Calculated elongation rate of actin filaments normalized to control. Mean +/– SD, *p < 0.05.

Figure 3

LK701 molecular docking and MD simulation results. The ligand is shown in stick, and the protein in cartoon and surface representation. Ligand atom color code: dark blue (nitrogen), red (oxygen), white (hydrogen), pink (iodine), orange (carbon). Nonpolar hydrogens are not shown for clarity. (A) Surface representation of the actin monomer highlighting the macrolide binding cleft (red), the subdomains D1–D4 are shown for orientation. (B) Surface representation of the actin trimer structure; the three subunits were color-coded and labeled n (cyan), n + 1 (gray), and n + 2 (light blue) from the barbed to the pointed end of the nucleus and the intersubunit binding cleft is highlighted by a red circle. (C) Macrolide binding cleft with equilibrated bound conformation of LK701 in the actin monomer LK701^mono. (D) Superposition of LK701^mono on subunit n + 2 of the stable actin apo nucleus showing a clash between LK701 and the D-loop of subunit n. (E) Most prominent structure of LK701 in the actin trimer LK701^tri during MD (orange). Docked position in black, position of the LK701^mono in green for reference. (F) Root-mean-square deviation (RMSD) of the position of LK701 relative to the bound position in the actin monomer LK701^mono as obtained during the MD simulation for the LK701–actin trimer complex.