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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - FCSSSLP (First Chemoselective Synthesis and Structural Studies of Lasso Peptides)

Teaser

Summary

Resistance to antibiotics is a response of pathogen microorganisms to the use of these drugs. Unfortunately, abuse implies their loss of efficacy. Bacteria stop being sensitive to their effects, being necessary novel generations of antibiotics to combat infections, with the concomitant more toxic to health.

The advantages in using peptides as drugs are their high specificity coupled to a large biological activity. They present a great diversity as therapeutic targets (antibiotics/antifungals, antivirals, cancer, etc). However, several difficulties have prevented them from being widely used as drugs. Peptides show low metabolic stability, they are rapidly degraded in vivo by proteases and the most common form of administration is intravenously, which highly prevent its degradation. In principle, these problems should be minimized with lasso-peptides. The rigidity and high packing (lasso-type) endow them with enormous stability against proteolytic degradation, physical/chemical denaturants. The first years of the project, at TSRI and under the guidance of Prof. Baran, we focused on exploring different synthetic methodologies with the final aim of preparing lasso peptides to study their recognition features and eventually their applications as putative drugs.

In the last year of the project, carried out at the Center for Cooperative Research in Biosciences (CIC bioGUNE) under the guidance of Professor Jimenez-Barbero, we focused on the study of protein-ligand molecular recognition processes of biomedical interest. In particular, I was trained in the applications of state-of-the-art Nuclear Magnetic Resonance (NMR) techniques and computational methods in this area.

Since the target Microcin J25 lasso peptide was elusive and could not be prepared either by organic or enzymatic synthesis, maintaining the essence of the DoA work-packages, we focused on the molecular recognition study of a biologically highly relevant system: the Heparan Sulfate/FGFR Ig2 system, of interest in cancer and inflammation.

Work performed

First, attempts to reproduce the enzymatic synthesis of Microcin J25 previously published were unsuccessful. At this point, maintaining the critical points of the different work packages, we decided to focus on the study of the molecular recognition of a biologically relevant system: the Heparan Sulfate / FGFR Ig2 system.

All the studies carried out during these last months have been done following the same strategy described in the DoA to keep my training objectives. Thus, I performed the overexpression of the FGFR-Ig2 growth factor following molecular biology methods, the structural elucidation of a biomacromolecule: Heparan Sulfate octasaccharide (13C/15N double labelled), and learnt molecular dynamics simulations protocols and applied them to this interacting system. MD simulations were carried out using AMBER, while docking methods used AutoDock. The NMR data were assisted by the computational protocols to obtain a detailed interaction 3D model, which is displayed below.

Final results

My contribution in the synthetic methodology provides an additional advance towards the synthesis of these molecules. However, the development of a robust organic synthesis method for Lasso peptides remains an open challenge. Indeed, a total synthesis has not yet been developed. In parallel, during the last 3 years, new lasso peptides such as subterisin , cattlecin , specialicin and squaraine have been isolated. Therefore, important advances are expected in this field and I would like in the future to continue working on these challenging molecules. Additionally, my contribution to the elucidation of an biologically important molecular complex, such as that formed by HS and FGFR provides a clear example of how state-of-the-art NMR methodology, as that employed herein synergically using receptor- and ligand-based NMR methods coupled to 13C/15N double labelled ligands and receptors, opens new avenues to the elucidation of the 3D structure of extremely important interacting systems related to disease. Further development of this methodology will enormously impact science in different manners, from both the basic and applied research perspectives.