Luigia Auriemma

Structure-activity relationship, conformational and biological studies of temporin l analogues

Temporins are naturally occurring peptides with promising features, which could lead to the development of new drugs. Temporin-1Tl (TL) is the strongest antimicrobial peptide, but it is toxic on human erythrocytes and this fact makes the design of synthetic analogues with a higher therapeutic index vital.We studied the structure-activity relationships of a library of TL derivatives focusing on the correlation between the α-helix content of the peptides, the nature of their cationic residues, and their antibacterial/antiyeast/hemolytic activities. We found that the percentage of helicity of TL analogues is directly correlated to their hemolytic activity but not to their antimicrobial activity. In addition, we found that the nature of positively charged residues can affect the biological properties of TL without changing the peptides helicity. It is noteworthy that a single amino acid substitution can prevent the antimicrobial activity of TL, making it a lytic peptide presumably due to its self-association. Last, we identified a novel analogue with properties that make it an attractive topic for future research.

The effect of d-amino acid substitution on the selectivity of temporin L towards target cells: Identification of a potent anti-Candida peptide ☆ ☆☆

The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.

Novel α-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity

Previous investigations indicate that α-melanocyte-stimulating hormone (α-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broad-spectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(2′)-7, Phe-12]-MSH(6–13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8–13 is a key factor in antimicrobial activity. Synthetic analogues of α-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity.

Alanine scanning analysis and structure–function relationships of the frog‐skin antimicrobial peptide temporin‐1Ta

The increasing resistance of bacteria and fungi to the available antibiotic/antimycotic drugs urges for a search for new anti‐infective compounds with new modes of action. In line of this, natural CAMPs represent promising and attractive candidates. Special attention has been devoted to frog‐skin temporins, because of their short size (10–14 residues long) and their unique features. In particular, temporin‐1Ta has the following properties: (i) it is mainly active on Gram‐positive bacteria; (ii) it can synergize, when combined with temporin‐1Tl, in inhibiting both gram‐negative bacterial growth and the toxic effect of LPS; (iii) it preserves biological activity in the presence of serum; and (iv) it is practically not hemolytic. Rational design of CAMPs represents a straightforward approach to obtain a peptide with a better therapeutic index. Here, we used alanine scanning analogs to elucidate the contribution of the side chains of each amino acid residue to the peptides antimicrobial and hemolytic activity. Beside providing insight into the biophysical attributes and the critical positions within the peptide sequence, which govern the antimicrobial/hemolytic activity of this temporin isoform, our studies assist in optimizing the design of temporin‐based lead structures for the production of new anti‐infective agents. Copyright

New insight into the binding mode of peptide ligands at Urotensin-II receptor: structure-activity relationships study on P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized several analogues of P5U and urantide in which the Asp(4) residue in N-terminus position was replaced with coded and noncoded amino acids. The replacement of the Asp(4) residue by Tic led to an analogue, compound 14, more potent as antagonist (pK(B) = 8.94) compared to urantide. Furthermore, a different SAR was observed for the P5U compared to the urantide analogues. NMR and docking studies revealed a different binding mode for the agonist and antagonist ligands which could explain the observed SAR.

Structure–function Relationships and Conformational Properties of α-MSH(6–13) Analogues with Candidacidal Activity

α‐Melanocyte‐stimulating hormone (α‐MSH) is an endogenous linear tridecapeptide with potent anti‐inflammatory effects. We firstly demonstrated that α‐MSH and its C‐terminal sequence Lys‐Pro‐Val [α‐MSH(11–13)] have antimicrobial effects against two major and representative pathogens: Staphylococcus aureus and Candida albicans. Successively, in an attempt to improve the candidacidal activity of α‐MSH and to better understand the peptide structure–antifungal activity relations, we have recently designed and synthesized novel peptide analogues. We focused on the sequence α‐MSH(6–13), which contains the invariant melanocortin core sequence His‐Phe‐Arg‐Trp (6–9) and also contains the sequence Lys‐Pro‐Val (11–13) important for antimicrobial activity. In that structure–activity study, we discovered several compounds that have greater candidacidal activity than α‐MSH, among which the peptide [d‐Nal‐7,Phe‐12]‐α‐MSH(6–13) was the most potent. Here, we report a detailed conformational analysis by spectroscopic and computational methods of three peptides, α‐MSH(6–13) (1), [d‐Nal‐7,Phe‐12]‐α‐MSH(6–13) (2) and [d‐Nal‐7,Asp‐12]‐α‐MSH(6–13) (3). Peptides were chosen on the basis of their candidacidal activities and were studied in membrane mimetic environment (SDS micelles). Different turn structures were observed for the three peptides and a conformation–activity model was developed based on these results. This study offers a structural basis for the design of novel peptide and non‐peptide analogues to be used as new antimicrobial agents.

Lead Optimization of P5U and Urantide: Discovery of Novel Potent Ligands at the Urotensin-II Receptor

We have optimized 1 (P5U) and urantide, two important ligands at the h-UT receptor, designing several analogues by the exchange of the Tyr9 residue with different unnatural aromatic amino acids. This study allowed us to discover novel ligands with improved activity. In particular, the replacement of the Tyr9 residue by (pCN)Phe or (pNO2)Phe within the urantide sequence led to compounds 13 (UPG-83) and 15 (UPG-95), respectively, which showed pure antagonist activity toward UT receptor in a rat aorta bioassay. More interestingly, the replacement of the Tyr9 in 1 sequence with the Btz or the (3,4-Cl)Phe residues led to superagonists 6 (UPG-100) and 10 (UPG-92) with pEC50 values at least 1.4 log higher than that of 1, being the most potent UT agonists discovered to date. Compounds 10 and 13 showed also a good stability in a serum proteolytic assay. These ligands represent new useful tools to further characterize the urotensinergic system in human physiopathology.

New insight into the binding mode of peptides at urotensin‐II receptor by Trp‐constrained analogues of P5U and urantide

Urotensin II (U‐II) is a disulfide bridged peptide hormone identified as the ligand of a G‐protein‐coupled receptor. Human U‐II (H‐Glu‐Thr‐Pro‐Asp‐c[Cys‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) has been described as the most potent vasoconstrictor compound identified to date.

Efficient Synthesis in Solid-Phase of Freidinger-like Lactams by Microwave Irradiation

Introduction Among the numerous strategies toward the conformational restriction of peptides, incorporating the backbone into a “Freidinger” lactam structure has proven useful in the design of a variety of medicinally relevant targets, especially peptidase/protease inhibitors [1]. Such cyclization of the peptide backbone fixes the amide bond in the trans rotameric form, places severe limitations on ψ1 rotation, and would be expected to bias neighboring φ1 and φ2 torsional angles. Several different synthetic strategies have been developed toward Freidinger lactams, including some stereoselective methods that allow control over the C-3 center (amino substituent) or the glycyl side chain (R1 in Fig. 1) [2]. However, no one method has proved completely facile for the stereoselective synthesis of Freidinger lactams of various ring sizes containing a spectrum of C-terminal amino acid residues. Here we report a new synthetic methodology to perform Freidinger lactams, developed under microwave irradiation. The structures were synthesized starting from iodine derivative of Asp opportunely protected that was reacted with an amino acid linked on Wang resin (Fig. 2). The iodine derivative of Asp was prepared as previously reported in literature. In this preliminary study, Gly, Phe, Lys, and Asp were used as amino acids loaded on Wang resin.