Solid State and Solution Conformation of [Ala7]-Phalloidin: A Synthetic Phallotoxin Analogue

Abstract

Phallotoxins are toxic compounds produced by poisonous mushroom Amanita phalloides and belong to the class of bicyclic peptides with a transannular thioether bridge. Their intoxication mechanism in the liver involves a specific binding of the toxins to F-actin that, consequently, prevents the depolymerization equilibrium with G-actin. Even though the conformational features of phallotoxins have been worked out in solution, the exact mechanism of interaction with F-actin is still unknown. In this study a toxic phalloidin synthetic derivative, bicyclo(Ala1-D-Thr2-Cys3-cis-4-hydroxy-Pro4-Ala5-2-mercapto-Trp6-Ala7)(S-3-->6) has been synthesized. A substitution at position 7. with an Ala residue replaces the 4,5-dihydroxy-Leu present in the natural phalloidin. This analogue has formed crystals suitable for X-ray analysis, and represents the first case for such a class of compounds. The solid-state structure as well as the solution conformation have been evaluated. NMR techniques have been used to extract interproton distances as restraints in subsequent molecular dynamics calculations. Finally, a direct comparison between structures in solution and in the solid state is presented.

Phalloidin Synthetic Analogues: Structural Requirements in the Interaction with F-Actin

“…The CD spectrum of this analogue shows some similarities but also marked differences when compared to those of the bioactive phallopeptides. The three-dimensional structure of this molecule, as determined by twodimensional NMR and RMD analyses, shows significant structural modifications of the backbone, mainly due to isomerism about the Cys 3 ÀHyp 4 peptide bond (trans in natural PHD [11] and in the active analogues 1 e [9] and 1' f; cis in 1 h) and the different position of a type I b-turn (residues 3 ± 6 in PHD, [11] 1 e, [9] and 1' f; residues 6 ± 2 in 1 h). Therefore, the inversion of chirality of residue 3 leads to significant structural changes of the backbone with a concomitant loss of activity.…”

“…Specifically, 40 % and 60 % of the recovered bioactivity can be assigned to the b-hydroxyl group on the side chain of d-Thr 2 and to the branched side chain of Leu 7 , respectively. The replacement of d-Thr 2 in the [Ala 7 ]-phalloidin (1 e) [9] by an l-Thr 2 residue (analogues 1 f and 1' f) causes a pronounced loss of toxicity (Table 1). Analogues 1 f and 1' f correspond to two atropisomers obtained by two subsequent cyclization reactions of the appropriate linear heptapeptide precursor.…”

“…The thioether bridge in the X-ray structure of analogue 1 e, the only solid-state structure of a phallotoxin so far available and chosen as a starting model for structure calculations, is of the U-type. [9] Consequently, this orientation was initially assumed in the starting model for the three analogues. For analogues 1 h and 1' f, a very good agreement between the experimental data and the conformational parameters of the molecular model was observed for the U-type structures.…”

“…The 2-indolyl thioether moiety, the dissymmetric chromophoric system responsible for the UV spectrum with l max 292 nm and for the characteristic strongly positive Cotton effects in the CD spectrum at around 300 and 240 nm, is positively helical, as demonstrated by the X-ray structure of a related peptide model [8] and recently confirmed by an X-ray analysis of a synthetic bioactive phallotoxin analogue. [9] Any variation from the typical UV and CD spectra exhibited by bioactive phallotoxins is always associated with a notable or even total loss of toxicity.…”

“…[9] Retaining the necessary bicyclic structure, the influence of the side chain on the affinity towards F-actin has been analyzed by carrying out structure ± activity relationship (SAR) studies on both semisynthetic and totally synthetic analogues. [15±18] These studies have shown that the Cys 3 , 4-cishydroxy-Pro 4 (Hyp 4 ), Ala 5 , and Trp 6 residues are essential for toxicity.…”