CE07 - Chimie moléculaire et procédés associés pour une chimie durable

Ugi Reaction for the Site-Specific Bioconjugation of Native Proteins – BioconjUgi


Ugi Reaction for the Selective Bioconjugation of Native Proteins.

Origin and ambition of the project

The chemical modification of proteins - bioconjugation - is of major interest, particularly in the therapeutic field, thanks to the additional properties it can confer: better stability, use in targeted therapies, fluorescence, etc. <br />The conjugation methods currently available focus on the use of artificial proteins - mutant proteins, created by biochemical engineering, allowing precise and efficient modifications. However, the development of such proteins is costly and time-consuming, two limitations that could be overcome by using natural (or native) proteins, which are cheap and easily accessible. In practice, however, the use of natural proteins comes up against a major obstacle: the very great heterogeneity of the products obtained. <br />Unlike artificial proteins, natural proteins possess hundreds of potential modification sites, and the precise conjugation of just one of them is extremely delicate. The development of a method for modifying natural proteins as precisely and efficiently as that offered by artificial proteins would therefore have a decisive advantage.

To address the issues detailed above, we propose to use a classical organic chemistry reaction - the Ugi reaction. This reaction uses four different components, two of which - let's call them components A and B - would be provided by the natural protein to be modified. The other two - C and D - would be small molecules that are either commercial or synthesised in the laboratory.
While most protein modification methods focus on the conjugation of a single component - either A or B - the simultaneous modification of these two components - A and B - would greatly restrict the number of sites that can be conjugated and thus allow the development of a precise - selective - natural protein conjugation method.

After carefully varying several parameters - C and D structures, temperature, pH, reaction time, etc. - we were able to identify conditions that led to the modification of only one site (among more than a hundred possible) on the surface of a specific family of natural proteins: antibodies. These selectively modified antibodies could be produced in large quantities and showed excellent stability and activity, both proof that the modification did not alter their properties.

We now wish to take advantage of our new conjugation method by producing new types of proteins: antibody dimers. This type of compounds is of growing interest for their potential therapeutic properties and is currently produced almost exclusively by biochemical engineering, again with prohibitive costs and certain technical limitations. Access to a simple and efficient chemical method for their production would therefore open the way to numerous studies of major interest.

A state-of-the-art review of chemical protein conjugation methods has just been accepted for publication in the Royal Society Open Science journal.

Protein bioconjugation can be described as the chemical or enzymatic coupling between a protein and any other molecule (e.g. a small molecule, another biomolecule) to generate a complex possessing the combined properties of its individual components. One of the major applications of protein bioconjugation is in the therapeutic field, where immunoconjugates (antibodies to which cytotoxic compounds such as drugs, toxins or radioisotopes have been conjugated) have become one of the main tools in the fight against cancer.
Optimal use of these conjugates requires a strict control of the amount of cytotoxic agent grafted as well as the precise site of conjugation in order to have a repeatable method allowing the production of adducts with well-defined structures and pharmacological properties. However, very few bioconjugation methods allow access to site-selectively modified native proteins and the bioconjugates classically generated are thus often heterogeneous complexes with different physico-chemical properties.
This is due to the very structure of proteins, which have multiple nucleophilic functions with similar reactivity, borne by the side chains of their amino acids components. While methods exist to target only certain families of residues (lysine, cysteine), higher levels of complexity (targeting one lysine among more than 80 for example) are poorly accessible, putting a curb on the development of conjugates with enhanced therapeutic properties.
While all bioconjugation methods are limited to targeting only one residue so far, this project aims to develop a strategy conjugating two residues at once, via a multicomponent Ugi reaction. Such an approach drastically reduces the number of potential conjugation sites, thus increasing the chances of accessing a site-specific method that could be applied to a wide range of native and natural proteins.
By developing three major work packages and collaborations with mass spectrometry specialists and computational chemists, this project aims to explore and exploit the full potential of this technique and develop new approaches for the generation of original protein complexes.

Project coordinator

Monsieur Guilhem Chaubet (Laboratoire de Conception et Application de Molécules Bioactives (UMR 7199))

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


CAMB _ UNISTRA Laboratoire de Conception et Application de Molécules Bioactives (UMR 7199)

Help of the ANR 244,188 euros
Beginning and duration of the scientific project: May 2020 - 42 Months

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