Probing pattern and dynamics of disulfide bridges using synthesis and NMR of an ion channel blocker peptide toxin with multiple diselenide bonds

Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin containing four disulfide bridges, is a high affinity blocker of the voltage-gated potassium channel Kv1.3, but also blocks Kv1.2. To improve potential therapeutic use of the toxin, we have designed a double substituted analog, [N17A/F32T]-AnTx, which showed comparable Kv1.3 affinity to the wild-type peptide, but also a 2500-fold increase in the selectivity for Kv1.3 over Kv1.2. In the present study we have achieved the chemical synthesis of a Sec-analog in which all cysteine (Cys) residues have been replaced by selenocysteine (Sec) forming four diselenide bonds. To the best of our knowledge this is the first time to replace, by chemical synthesis, all disulfide bonds with isosteric diselenides in a peptide/protein. Gratifyingly, the key pharmacological properties of the Sec-[N17A/F32T]-AnTx are retained since the peptide is functionally active. We also propose here a combined experimental and theoretical approach including NOE- and Se-77-based NMR supplemented by MD simulations for conformational and dynamic characterization of the Sec-[N17A/F32T]-AnTx. Using this combined approach allowed us to attain unequivocal assignment of all four diselenide bonds and supplemental MD simulations allowed characterization of the conformational dynamics around each disulfide/diselenide bridge

Probing pattern and dynamics of disulfide bridges using synthesis and NMR of an ion channel blocker peptide toxin with multiple diselenide bonds

Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin containing four disulfide bridges, is a high affinity blocker of the voltage-gated potassium channel Kv1.3, but also blocks Kv1.2. To improve potential therapeutic use of the toxin, we have designed a double substituted analog, N17A/F32T-AnTx{,} which showed comparable Kv1.3 affinity to the wild-type peptide{,} but also a 2500-fold increase in the selectivity for Kv1.3 over Kv1.2. In the present study we have achieved the chemical synthesis of a Sec-analog in which all cysteine (Cys) residues have been replaced by selenocysteine (Sec) forming four diselenide bonds. To the best of our knowledge this is the first time to replace{,} by chemical synthesis{,} all disulfide bonds with isosteric diselenides in a peptide/protein. Gratifyingly{,} the key pharmacological properties of the Sec-N17A/F32T-AnTx are retained since the peptide is functionally active. We also propose here a combined experimental and theoretical approach including NOE- and 77Se-based NMR supplemented by MD simulations for conformational and dynamic characterization of the Sec-N17A/F32T-AnTx. Using this combined approach allowed us to attain unequivocal assignment of all four diselenide bonds and supplemental MD simulations allowed characterization of the conformational dynamics around each disulfide/diselenide bridge

Gliko és foszfopeptidek szintézisének lehetőségei = Synthesis of glyco and phosphopeptides

Módszereket dolgoztunk ki oligo-szachariddal glikozilált N-glikopeptidek szintéziséremind konvergens, mind építőkő módszerrel. Védőcsoportkombinációkat optimalizáltunk O-glikopeptidek szintézisére, valamint optimalizáltuk a kapcsolási reakciót a glikán és a védett aminosav között. Számos foszfopeptidet és sejtpenetráló peptidet szintetizáltunk, részben fluoreszcensen jelzett formában is. Új foldamer molekulát (N-amino prolin) állítottunk elő és építettünk be béta-aminosav oligomerekbe. Megvizsgáltuk a kapott oligomerek térszerkezeti és stabilitásviszonyait. | We improved the applicable methods for the synthesis of N-glycopeptides including convergent and building block approach. The protecting group combinations for the synthesis of O-glycopeptides was optimized and we improved the coupling conditions for the reaction of the glycosyl donor and acceptor. Numerous phospho and cell-penetrating peptide were synthesized and a part of them was fluorescently labelled. A new foldamer molecule (N-amino proline) was prepared and incorporated into beta-amino acid oligomers. The conformation and the stability of the new oligomers was investigated

An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility

The voltage-gated Kv1.3 K(+) channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels

Probing pattern and dynamics of disulfide bridges using synthesis and NMR of an ion channel blocker peptide toxin with multiple diselenide bonds

Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin contg. four disulfide bridges, is a high affinity blocker of the voltage-gated potassium channel Kv1.3, but also blocks Kv1.2. To improve the potential therapeutic use of the toxin, we have designed a double substituted analog, [N17A/F32T]-AnTx, which showed comparable Kv1.3 affinity to the wild-type peptide, but 2500-fold increase in the selectivity for Kv1.3 over Kv1.2. In the present study we have achieved the chem. synthesis of a Sec-analog in which all cysteine (Cys) residues have been replaced by selenocysteine (Sec) forming four diselenide bonds. To the best of our knowledge this is the first time to replace, by chem. synthesis, all disulfide bonds with isosteric diselenides in a peptide/protein. Gratifyingly, the key pharmacol. properties of the Sec-[N17A/F32T]-AnTx are retained since the peptide is functionally active. We also propose here a combined exptl. and theor. approach including NOE- and 77Se-based NMR supplemented by MD simulations for conformational and dynamic characterization of the Sec-[N17A/F32T]-AnTx. The use of such combined approach allowed us to attain unequivocal assignment of all four diselenide bonds and supplemental MD simulations allowed to characterize the conformational dynamics around each disulfide/diselenide bridge. [on SciFinder(R)

Antiamnesic properties of analogs and mimetics of the tripeptide human urocortin 3

Amnesia is a deficit in memory caused by brain damage, disease, or trauma. Until now, there are no successful medications on the drug market available to treat amnesia. Short analogs and mimetics of human urocortin 3 (Ucn 3) tripeptide were synthesized and tested for their action against amnesia induced by eletroconvulsion in mice. Among the 16 investigated derivs. of Ucn 3 tripeptide, eight compds. displayed antiamnesic effect. Our results proved that the configuration of chiral center of glutamine does not affect the antiamnesic properties. Alkyl amide or isoleucyl amide at the C-terminus may lead to antiamnesic compds. As concerned the N-terminus, acetyl, Boc, and alkyl ureido moieties were found among the active analogs, but the free amino function at the N-terminus usually led to an inactive derivs. These observations may lead to the design and synthesis of small peptidomimetics and amino acid derivs. as antiamnesic drug candidates, although the elucidation of the mechanism of the action requires further investigations. [on SciFinder(R)

Short analogs and mimetics of human urocortin 3 display antidepressant effects in vivo

Peptide analogs of urocortin 3[36-38] (Ucn 3[36-38]), obtained with deletion or replacement of amino acids of the original human urocortin 3 sequence, were designed, synthesized, and tested in vivo for treatment of depression. Based on the results of the biol. tests of the peptide analogs, several new peptidomimetics of the above short analogs of urocortin 3, including urea- and azapeptides, were also designed and synthesized and found to preserve the antidepressant-like effect of the 38 amino acid long original neuropeptide. The mol. modifications of urocortin 3[36-38] led to an improved understanding of the relationship between mol. structure and biol. activity of this peptide, and the novel peptidomimetics could be further tested for possible clin. treatment of depression. [on SciFinder(R)