Direct targeting of intracellular Gα subunits of G protein-coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359-based analogs from natural sources, synthetic cyclic peptides, as well as all so-far known G α inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of G in the specific FR/YM-binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on G and G proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of G and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than G .

Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide μ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.

Tridegin is a potent and specific 66mer peptide inhibitor of coagulation factor XIIIa with six cysteines involved in three disulfide bonds. Three of the fifteen possible 3-disulfide-bonded isomers have been identified, which share a bridge between cysteines 19 and 25. We synthesized the three possible 2-disulfide-bonded analogs using a targeted protecting-group strategy to investigate the impact of the C 19 -C 25 bond on tridegin's folding, stability, and function. The FXIIIa inhibitory activity of the analogs was retained, which was shown by in vitro fluorogenic activity and whole blood clotting assays. Molecular dynamics simulations of wild type tridegin and the analogs as well as molecular docking studies with FXIIIa were performed to elucidate the impact of the C 19 -C 25 bond on conformational stability and binding mode. The strategy of

The inhibition of the final step in blood coagulation, the factor XIIIa (FXIIIa) catalyzed cross-linking of fibrin monomers, is currently still a challenge in medicinal chemistry. We report synthesis, recombinant expression, disulfide connectivity, and biological activity of tridegin, the sole existing peptide representative displaying inhibitory activity on FXIIIa. Inhibition of the enzyme by this 66-mer cysteine-rich peptide is mediated by its C-terminal sequence, while the N-terminal part comprises structural information and contributes to inhibitor binding. Either of the production strategies examined leads to the formation of different disulfide-bridged isomers indicating the requirement of the correct fold for inhibitory activity. Molecular modeling and docking studies confirm disulfide bond isomer preference with respect to binding to FXIIIa, in turn, the knowledge of the enzyme−inhibitor interactions might bring about comprehensive ideas for the design of a suitable lead structure for addressing FXIIIa.

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