Puig Mora

Nanostructure Formation Enhances the Activity of LPS‐Neutralizing Peptides

Peptides that interact with lipopolysaccharide (LPS) can provide the basis for the development of new antisepsis agents. In this work, several LPS‐neutralizing acyl peptides derived from LALF, BPI, and SAP were prepared, structurally characterized, and biologically evaluated. In all cases, peptides with long acyl chains showed greater LPS‐neutralizing activities than the original acetylated peptides. Structural analysis of these peptides revealed that N‐acylation with long acyl chains promotes the formation of micellar or fibril‐like nanostructures, thus proving a correlation between anti‐LPS activity and nanostructure formation. The results of this study provide useful structural insight for the future design of new acyl peptides that strongly bind LPS and therefore act as antisepsis drugs. Furthermore, this nanostructure–biological activity correlation can be translated into other therapeutic areas.

Design of bioactive and structurally well‐defined peptides from conformationally restricted libraries

Libraries of peptides and proteins can be categorized according to the function of their origin in gene‐ and synthetic‐based libraries. Both kinds of libraries have the potential to generate the same grade of molecular diversity, although the limits imposed by the synthetic methods have been lately a matter of discussion. However, the use of synthetic strategies allows incorporation of non‐natural amino acids. The development of conformationally restricted synthetic peptide libraries can be considered as a point of convergence of the two methodologies. In these libraries the diversity is grafted into scaffolds that are defined by stable secondary structural motifs, and the deconvolution protocols can be directed towards the identification of biologically active molecules and the analysis of determinants of folding of protein domains.

Solvent-Exposed Residues Located in the Beta-Sheet Modulate the Stability of the Tetramerization Domain of P53--A Structural and Combinatorial Approach.

The role of hydrophobic amino acids in the formation of hydrophobic cores as one of the major driving forces in protein folding has been extensively studied. However, the implication of neutral solvent‐exposed amino acids is less clear and available information is scarce. We have used a combinatorial approach to study the structural relevance of three solvent‐exposed residues (Tyr327, Thr329, and Gln331) located in theβ‐sheet of the tetramerization domain of the tumor suppressor p53 (p53TD). A conformationally defined peptide library was designed where these three positions were randomized. The library was screened for tetramer stability. A set of p53TD mutants containing putative stabilizing or destabilizing residue combinations was synthesized for a thermodynamic characterization. Unfolding experiments showed a wide range of stabilities, with Tm values between 27 and 83°C. Wild type p53TD and some highly destabilized and stabilized mutants were further characterized. Thermodynamic and biophysical data indicated that these proteins were folded tetramers, with the same overall structure, in equilibrium with unfolded monomers. An NMR study confirmed that the main structural features of p53TD are conserved in all the mutants analyzed. The thermodynamic stability of the different p53TD mutants showed a strong correlation with parameters that favor formation and stabilization of the β‐sheet. We propose that stabilization through hydrophobic interactions of key secondary structure elements might be the underlying mechanism for the strong influence of solvent‐exposed residues in the stability of p53TD. Proteins 2008.

Solid-phase Chemistry: A Useful Tool to Discover Modulators of Protein Interactions

The Solid phase synthesis (SPS) concept, first developed for biopolymers, has spread in every field where organic synthesis is involved. While the potential of the solid-phase method was obvious in 1959 to its discoverer, Prof. R. B. Merrifield, it was unpredictable its dominance in peptide synthesis and especially in combinatorial chemistry, an area not yet conceived. SPS paved the way for solid-phase combinatorial approaches (extensively reviewed in (Choong, I. C. and Ellman, J. A.: 1996, Annu. Rep. Med. Chem. 31, 309–318; Obrecht, D. and Villalgordo, J. M.: 1998, Solid-supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries. Pergamon Press Ltd., Oxford, UK; Chabala, J. C.: 1995, Curr. Opin. Biotechnol. 6, 632–639; Kamal, A., Reddy, K. L., Devaiah, V., Shankaraiah, N., Reddy, D. R.: 2006, Mini Rev. Med. Chem. 6, 53–69; Whitehead, D. M., McKeown, S. C., Routledge, A.: 2005, Comb. Chem. HTS 8, 361–371; Nefzi, A., Ostresh, J. M., Houghten, R. A.: 1997, Chem. Rev. 97, 449–472; Gordon, E. M., Gallop, M. A., Patel, D. V.: 1996, Acc. Chem. Res. 29, 144–154)) as many laboratories and companies focused on the development of technologies and chemistry suitable to this new methodology. This resulted in the spectacular outburst of combinatorial chemistry, which profoundly changed the approach for new drug discovery. Combinatorial chemistry is currently considered a valid approach for a wide range of biomedical applications, such as, target validation and drug discovery.

Bioactive peptides derived from the Limulus anti-lipopolysaccharide factor: structure-activity relationships and formation of mixed peptide/lipid complexes.

The design of peptides that would interact and neutralise bacterial endotoxins or LPS could have benefited from the analysis of comparative structure–activity relationships among close‐related analogues. Here, we present a comparative structural characterisation of selected peptides derived from the LALF obtained by single‐amino‐acid replacement, which differ in biological activity. The peptides were characterised in solution using nuclear magnetic resonance, circular dichroism and fluorescence spectroscopies. Membrane mimetic peptide interactions were studied using fluorescence resonance energy transfer with the aid of extrinsic fluorescent probes that allowed the identification of mixed peptide/lipid complexes. Copyright

Design and facile solid-phase synthesis of peptide-based LPS-inhibitors containing PEG-like functionalities.

LPS release from Gram‐negative bacteria can result in sepsis, a serious systemic inflammatory response to infection that can lead to septic shock and multiple organ failure. Thus, easy‐to‐synthesize, effective, and safe LPS‐inhibitors are required to develop new agents for the treatment of sepsis. On the basis of the chemical features of the toxic part of LPS, lipid A, here we present peptide‐based LPS‐neutralizers that can be readily obtained using solid‐phase methodologies. The presence of PEG‐like moieties yielded the most active compounds, thereby indicating that these functionalities may be of great value in the design of new inhibitors. In this regard, the substitution of several amino acids by PEG‐like chains in a previously reported cyclic anti‐LPS peptide (the peptide RLKWc) rendered a new derivative that retained the activity of the original peptide. We foresee that this strategy could be successfully applied to other LPS‐neutralizing peptides.

Tiratricol neutralizes bacterial endotoxins and reduces lipopolysaccharide-induced TNF-alpha production in the cell.

The screening of a commercially available library of compounds has proved a successful strategy for the identification of a lead compound in a drug discovery programme. Here, we analysed 880 off‐patent drugs, which initially comprised the Prestwick Chemical library, as sources of bacterial endotoxin neutralizers. We identified 3,3′,5‐triiodo‐thyroacetic acid (tiratricol) as a non‐antibacterial compound that neutralizes the toxic lipopolysaccharide.