Tuning a Polar Molecule for Selective Cytoplasmic Delivery by a pH (Low) Insertion Peptide

Drug molecules are typically hydrophobic and small in order to traverse membranes to reach cytoplasmic targets, but we have discovered that more polar molecules can be delivered across membranes using water-soluble, moderately hydrophobic membrane peptides of the pHLIP (pH low insertion peptide) family. Delivery of polar cargo molecules could expand the chemical landscape for pharmacological agents that have useful activity but are too polar by normal drug criteria. The spontaneous insertion and folding of the pHLIP peptide across a lipid bilayer seeks a free energy minimum, and insertion is accompanied by a release of energy that can be used to translocate cell-impermeable cargo molecules. In this study, we report our first attempt to tune the hydrophobicity of a polar cargo, phallacidin, in a systematic manner. We present the design, synthesis, and characterization of three phallacidin cargoes, where the hydrophobicity of the cargo was tuned by the attachment of diamines of various lengths of hydrophobic chains. The phallacidin cargoes were conjugated to pHLIP and shown to selectively inhibit the proliferation of cancer cells in a concentration-dependent manner at low pH

Protein dynamic properties: essential dynamics method vs. NMR backbone dynamics

Proteins are dynamic systems whose internal motions and resulting conformational changes are essential for their functional skills. While the rigidity is required to maintain the structure, flexibility is needed to perform the function. The study of protein flexibility can be handled by both experimental, such as NMR backbone dynamics in solutions, and computational methods, such as molecular dynamics simulations. The major problem with molecular dynamics simulations is due to the conformational sampling efficiency that requires long times of calculation. In recent decades a new computational approach, based on the essential dynamics sampling (EDS) has been applied to the study of protein flexibility, folding etc. In essential dynamics sampling, an usual molecular dynamics simulation is performed, but only those steps, not increasing the distance from a target structure, are accepted. This method offers the possibility of representing protein dynamics in the essential subspace only, so reducing the complex protein dynamics to its essential degrees of freedom. In this work we apply ED simulations to identify flexible regions in two protein systems previously studied by NMR backbone dynamics

A binding pocket for phenolic substrates in laccases

Fungal laccases, belonging to the multicopper oxidases, can catalyze the oxidation of a large number of aromatic (especially phenolic), inorganic and xenobiotic compounds. In multicopper oxidases, the T1 Cu site accepts electrons from the substrate and transfers them to a T2/T3 trinuclear Cu cluster, which binds and activates O2 for reduction to H2O. The first X-ray structure available for a laccase, from Coprinus Cinereus, reveals a molecular architecture consisting of three b-barrel cupredoxin-like domains.1 In order to assess the structural features helping the phenolic substrates recognition, we have performed a comparative structural study on three fungal laccases: the one from Coprinus Cinereus, and two (POXC and POXA1b) from Pleurotus Ostreatus. Homology models are employed for POXC and POXA1b. First, a channel providing access to the T1 copper site, about 7 Å deep, is located at the interface between the structural domains 2 and 3. The T1-copper coordinating His457, analogous to the coordinating histidines involved in the electron transfer to other copper-proteins, is solvent exposed at the channel bottom. Docking calculations are then performed in such a cleft for two phenolic compounds: 2,6-di-methoxyphenol (DMP) and 2,6-di-t-butylphenol (DTBP), respectively a good and a bad substrate2 for laccases. The hydroxyl reducing group of DMP is shown to be at catalytic distance from His457. A possible role in assisting the catalytic oxidation is also suggested for Asp205, which forms an H-bond with the phenolic hydroxyl group and is conserved among fungal laccases. On the contrary, DTBP is shown not to match the cleft. A structure based sequence alignment between fungal laccases and the homologous ascorbate oxidase (AO)3 is also presented, which allows to outline a significant correspondence between their predicted substrate pockets. References 1. Ducros V. et al. (1998) Nature Struct. Biol. 5, 310. 2. Xu F. (1996) Biochemistry 35, 323. 3. Messerschmidt A. et al. (1992) J. Mol. Biol. 224, 179

CD CONFORMATIONAL STUDIES ON SYNTHETIC PEPTIDES ENCOMPASSING THE PROCESSING DOMAIN OF THE OCYTOCIN-NEUROPHYSIN PRECURSOR

Synthetic peptides of different size, reproducing the proteolytic processing site of proocytocin, were studied by CD under several experimental conditions in order to ascertain the ability of different solvents to stabilize secondary structural motifs, such as alpha-helix tracts and beta-turns. A combination of deconvolution methods and empirical calculations subtracting the contributions due to unordered structures from the spectra suggests that in solution (a) mainly two distinct families of ordered conformers containing structurally different beta-turns are present, (b) the relative stability of the different conformers depends from the nature of the solvent, and (c) in the case of the larger peptides, a population containing an alpha-helical conformation is also present. From the biological point of view the presence of at least two families of ordered conformers could be in line with current theories assuming that the catalytic effect of the receptor microenvironment may be determinant in shifting the equilibrium toward the active conformation

Elucidation of the structure of constrained bicyclopeptides in solution by two-dimensional cross-relaxation spectroscopy: Amatoxin analogues

The evaluation of peptide structures in solution is made feasible by the combined use of two-dimensional NMR in the laboratory (NOESY) and rotating frames (ROESY), and by the use of molecular dynamics calculations. The present paper describes how both the NMR method and molecular dynamics calculations were applied to very rigid synthetic bicyclic peptides that are analogues of natural amatoxins. The NMR theory, which allows the estimate of interatomic distances between interacting nuclei, is briefly discussed. The experimental data were compared with those of known solid-state structures. Three amatoxin analogues have been examined. Of these, one is biologically active (S-deoxo γ[R] OH-Ile3-amaninamide) and its structure in the solid state has recently been worked out. The second and third analogues (S-deoxo-Ile3-Ala5-amaninamide and S-deoxo-D-Ile3-amaninamide, respectively) are inactive and their solid-state structures are unknown. The data presented confirm the authors' previous hypothesis that lack of biological activity of S-deoxo-Ile3-Ala5-amaninamide is due to the masking of the tryptophan ring by the methyl group of L-Ala and not to massive conformational changes of the analogue

Elucidation of the structure of constrained bicyclopeptides in solution by two-dimensional cross-relaxation spectroscopy: Amatoxin analogues

The evaluation of peptide structures in solution is made feasible by the combined use of two-dimensional NMR in the laboratory (NOESY) and rotating frames (ROESY), and by the use of molecular dynamics calculations. The present paper describes how both the NMR method and molecular dynamics calculations were applied to very rigid synthetic bicycle peptides that are analogues of natural amatoxins. The NMR theory, which allows the estimate of interatomic distances between interacting nuclei, is briefly discussed. The experimental data were compared with those of known solid-state structures. Three amatoxin analogues have been examined. Of these, one is biologically active (S-deoxo gamma[R] OH-Ile3-amaninamide) and its structure in the solid state has recently been worked out. The second and third analogues (S-dexo-Ile3-Ala5-amaninamide and S-deoxo-D-Ile3-amaninamide, respectively) are inactive and their solid-state structures are unknown. The data presented confirm the authors previous hypothesis that lack of biological activity of S-deoxo-Ile3-Ala5-amaninamide is due to the masking of the tryptophan ring by the methyl group of L-Ala and not to massive conformational changes of the analogue