Abstract

Part 1: Miuraenamide A, a novel actin stabilizing compound, selectively inhibits cofilin binding to F-actin Actin binding compounds such as phalloidin, jasplakinolide and latrunculin are widely used tools in cell biology regulating actin dynamics. Jasplakinolide is a prototypic actin stabilizer which binds to F-actin with no effect on ABPs (cofilin, gelsolin, profilin). In contrast miuraenamide A, as a new actin stabilizer, competes exclusively with cofilin for binding to F-actin. Notably, the molecular basis for this difference still remains to be determined. To investigate whether this difference is due to a specific binding site in miuraenamide A we performed molecular dynamics simulations. These simulations suggest that the bromophenol group of miurenamide A interacts with actin residues Tyr133, Tyr143, and Phe352. This interaction shifts the D-loop of the neighboring actin, creating tighter packing of the monomers, and blocks the binding site of cofilin. We found that miuraenamide A shows activity similar to jasplakinolide both in vitro with respect to polymerization, depolymerization, branching, nucleation and in vivo with respect to cell proliferation, migration. However, gene expression in HUVEC cells was differentially affected by both compounds, indicating functional differences. We found that relatively small changes in the molecular structure give rise to this selectivity, suggesting that actin binding compounds might serve as promising scaffolds for creating actin binders with specific functionality instead of just “stabilizers”. Part 2: Chivosazole A modulates protein-protein-interactions of actin Actin is a protein of central importance for many cellular key processes. Its function is regulated by local interactions with a large number of ABPs. To date, various compounds are known either increasing or decreasing polymerization dynamics of actin. However, no actin binding compound has been developed for clinical applications yet, due to selectivity issues. We provide a crystal structure of the natural product chivosazole A bound to actin, and show that – in addition to inhibiting nucleation, polymerization and severing of F-actin filaments – it selectively modulates binding of ABPs to G-actin: while unphysiological actin dimers are induced by chivosazole A, interaction with of gelsolin, profilin, cofilin and thymosin β4 is inhibited. Moreover, chivosazole A causes transcriptional effects differing from latrunculin B, an actin binder with a different binding site. Thus, our data show that chivosazole A and related compounds could serve as scaffolds for the development of actin binding molecules selectively targeting specific actin functions.

Abstract