A minimal Cys-cyclobutene non-canonical amino acid for bioorthogonal imaging of proteins in live cells

Informations projet

MinBioTag

N° de convention de subvention: 701473

DOI

10.3030/701473

Projet clôturé

Date de signature de la CE 24 Février 2016

Date de début 1 Avril 2016

Date de fin 31 Mars 2018

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 183 454,80

Contribution de l’UE

€ 183 454,80

Coordonné par

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 1 - MinBioTag (A minimal Cys-cyclobutene non-canonical amino acid for bioorthogonal imaging of proteins in live cells)

Période du rapport: 2016-04-01 au 2018-03-31

Résumé du contexte et des objectifs généraux du projet

Nature has produced an intricate machinery to diversify the structure of proteins after their synthesis in the ribosome. Recently and in an attempt to mimic nature, reactions that allow for post-expression modification of proteins at selected sites had been developed. These reactions are used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they can be used to study and monitor disease-associated proteins or to conjugate cytotoxic molecules to antibodies to improve efficacy and reduce side effects of cancer treatments. The aim of my research is to develop a new non-natural small amino acid, tagged with a cyclobutene moiety, that can be introduced in proteins followed a very fast and selective chemical reaction. Establishment of this new chemical site-selective bioorthogonal protein labelling strategy will allow us, as a proof of principle, to modify the C2A Domain of Synaptotagmin-I in a chemoselective fashion with no toxicity for living cells in order to image apoptotic cells without interfere with the protein’s innate structure, function, activity and localisation as well as cellular functions.. We expect this project find wide application as the strategy here outline will allow to label and monitor proteins with a minimal interference on their function and localisation, creating a valuable future tool for chemists and biologists.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

The non-natural cysteine aminoacid tagged with a cyclobutene moiety was synthesized and characterized. The IEDDA reaction between the cyclobutene moiety and different tetrazines was characterised by NMR and kinetics were measured by UV absorbance. However, the tag could not be introduced in the protein C2Am, possibly due to the fact that the cyclobutene ring is not stable in the alkylation reactions. Different proteins and alkylation conditions were tested with no positive results.
At this point, a change in my approach was needed, so I envised a small-molecule cyclobutene conjugate to interrogate key biological aspects of cancer. As a small molecule I selected azetolamide, a ligand of carbonic anhydrase IX. Carbonic anhydrase IX is a membrane protein overexpressed in several cancers, including lung, stomach, pancreas or brain. Besides the ubiquitous presence of this protein in cancer, there is still some controversy about whether this molecule is internalised or not upon ligand binding. In order to answer this question, an azetolamide-cyclobutene conjugate was designed and synthesized. As an IEDDA partner for this conjugate a tetrazine-bodipy was synthesized. The bodipy selected had been previously used for super-resolution microscopy and we expect that the reaction between the azelotamide-cyclobutene conjugate and the tetrazine-bodipy in cells will help us to deepen our knowledge of carbonic anhydrase IX.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

We were able to confirm that the cyclobutene tag is reactive in IEDDA reactions with suitable tetrazines with good kinetic rates, making it a suitable handle for the study of biological processess. As a proof of concept, a azetolamide-cyclobutene conjugate was synthesized and its reaction with a fluorogenic tetrazine-bodipy conjugate was studied. The reaction between these two handles in cells overexpressing carbonic anhydrase IX will help us to understand better the role of this protein in certain types of cancer.

We can expect two main types of results from this project. First, the development of a new dienophile for IEDDA reactions that may find wide application as a tool for both chemists and biologist to interrogate biology. Second, a deeper insight into the biological processes carbonic anhydrase IX is involved. This will, hopefully, have wide impact in the development of antibody-drug conjugates directed against this target, overexpressed in tumor hypoxia and certain types of cancer as lung, pancreas or breast.

Objectives of the project