Double-site ligands for the inhibition of Cholera toxin

Project Information

DOUBLELICHT

Grant agreement ID: 251763

Closed project

Start date 1 January 2011

End date 31 December 2012

Funded under

• Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Total cost € 172 939,40

EU contribution € 172 939,40

Coordinated by

UNIVERSITA DEGLI STUDI DI MILANO
Italy

Final Report Summary - DOUBLELICHT (Double-site ligands for the inhibition of Cholera toxin)

Project context

Cholera is a life-threatening disease caused by the pathogen Vibrio cholerae. The toxicity of cholera is caused by the secreted protein Cholera toxin (CT), a protein formed by one toxic A-subunit (CTA) anchored in the middle of a ring formed by five B-subunits (CTB). The B-subunits are responsible for anchoring the toxin to human intestinal epithelial cells by binding to the ganglioside GM1 (Gal-beta3GalNAc-beta4[NeuAc-alfa3]Gal-beta4Glc-betaCer) present on their surface. The toxin is then internalised into the cells, where it exerts its effects leading to diarrhoea and dehydration. The two major biotypes of cholera are Classical and El Tor. They differ for the structure of two residues in the CTB portion of the toxin.

The severity of cholera symptoms caused by El Tor strains has been reported to depend on the patient's blood group. Blood group type is determined by ABH antigens, oligosaccharides that are expressed on cell surfaces, including those of the gastrointestinal tract. Blood group O individuals, carrying the H antigen, appear to be more at risk of developing severe cholera than those of other blood groups. This has been linked to the different structure of El Tor CTB which, in addition to the GM1 binding site, harbours a second binding region, able to interact with blood group oligosaccharides.

Project objectives

The goal of this project consisted in developing new molecules (ligands) able to bind to the cholera toxin so that it cannot bind to the intestinal cells. To increase the strength of these ligands, our specific proposal consisted in connecting two elements: one able to occupy the GM1 binding site, the second one able to engage the blood group binding region of El Tor toxin. Since the two sites are separated by a relatively large distance, the length of the linker was to be selected within a pool of possible structures by performing the connecting reaction dynamically, in the presence of (El Tor) CTB as a template (combinatorial target-guided dynamic synthesis).

The host laboratory at the University of Milano (Italy) had already developed appropriate ligands that efficiently target the GM1 binding site in CT. In order to design the blood group site binders, in phase 1 of this project Nuclear Magnetic Resonance ( NMR) studies were performed to analyse the interaction of blood group oligosaccharides with CT, determine the minimum binding epitope and select the appropriate blood group mimics.

Project results

This phase of the project resulted in the surprising outcome that the O antigen binds almost equally well to classical and El Tor CT. Following this important observation, the NMR work was extended relative to the initial proposal to include analysis of A and B antigens. The blood group antigen affinity for CT was estimated, and they all were found to interact measurably with the classical biotype toxin, in a non-competitive fashion relative to GM1. These results suggested that also classical CT must contain a second binding site similar to that of El Tor and different from the GM1 interaction region. Furthermore, the O-antigen has the strongest affinity for classical CT between all analysed blood group antigens.
%These studies shed a new light on the structural interpretation of the epidemiological data. They also allowed us to select the A-trisaccharide as the minimal blood group derivative for the design of double-site ligands for the inhibition of cholera toxin. Further efforts to reduce the size of this ligand using different alfa-fucosyl amides synthesised during the project were frustrated by the inability of these simplified ligands to engage in productive interaction with the toxin.

The second phase of the project was dedicated to the synthesis of ligands for cholera toxin inhibition that feature two pharmacophoric fragments, using the dynamic templated methodology described above. The first synthetic effort was dedicated to the preparation of the components of the dynamic library: two stereoisomeric GM1 mimics armed with the first reactive group (an aldehyde) and a series of blood group antigen derivatives (three alfa-N-fucosylamides and three compounds based on the blood group A trisaccharide) functionalised with ethylenglycol linkers of different length and armed with the second reactive group (an amine).

The final step of the project consisted in the synthesis of the adaptive combinatorial library, based on the imine exchange as linking reaction. Many different conditions were tested to shift the equilibrium of the reaction to the imine formation, but none of the conditions tested so far has yet given us the desired products. Optimisation of the reaction conditions is still necessary to generate the dynamic combinatorial library. Currently, the host laboratory and the fellow, in his new position at the CSIC in Sevilla (Spain) are working to find a synthetic alternative to complete the studies proposed in the project.

Project outcomes

In conclusion, many of the results planned were achieved: (a) NMR analysis of the interaction of blood group oligosaccharides and CT allowed the minimal binding epitope for the oligosaccharide to be identified. This phase of the work was particularly interesting because it established for the first time that wild-type CT binds to blood group oligosaccharides as efficiently as El Tor CT. Extension of this phase beyond the projected time frame reduced the time available for the conclusion of the project;
(b) The individual fragments required for generation of the library were synthesised;
(c) Analytical conditions were established for the analysis of the reaction mixtures using Hilic chromatography;
(d)The programme could not reach its final goal because the reductive amination planned to generate the library did not work under a set of different conditions;
(e) a new approach to dynamic library generation will be explored to complete the project in a follow-up collaboration between the host laboratory and the fellow.

The results obtained in the project may have an important socio-economic impact because they confirmed the existence of a secondary blood group binding site both in classical and 'El Tor' CT, two of the strains responsible of the current pandemic in Asia. These data will help the analysis of epidemiological data and open the possibility to design new kind of cholera toxin antagonists based on blood group antagonist for both major biotypes. Finally, it was demonstrated on a molecular basis that the blood group phenotype of individuals could have a relationship with the severity of the disease. In addition, the project has contributed to creating a novel inter-European collaboration between the host laboratory and the fellow, in his new position at CSIC in Sevilla.