Selective bioconjugation of chemical probes to new modalities using acylases as biocatalysts

Abstract

The selective modification of peptides and proteins has been one of the cornerstones of the field of chemical biology for the past decade. From the production of biopharmaceuticals to the enhancement of pharmacokinetic properties, this field has thrived and seen the emergence of a myriad of bioconjugation methods. Nevertheless, many of the chemical methods that exist have shortcomings, yielding heterogeneous conjugates due to their low selectivity. In contrast, enzymatic methods have displayed high selectivities, allowing the modification of a certain residue over all the others present in the biomolecule. However, most enzymatic techniques require the introduction of tags, which necessitate protein engineering. Herein, we have sought (chemo)enzymatic techniques that allow for the selective modification of native proteins and peptides, without the need of introducing tags. First, the employment of lysine acetyltransferases (KATs) for bioconjugation was investigated. Up until now, it has been challenging to form the acyl-S-CoA substrate that many enzymes, including lysine acetyltransferases, employ to carry out acylation. To address this problem, the adenylation domain of carboxylic acid reductase (CARsr-A) was harnessed to generate the acyl-S-CoA substrate from the respective acid. This enzymatic system was harnessed to modify in one-pot the natural substrate of KATs, histone H4, with a range of bioorthogonal probes. The application system on non-natural and more complex substrates was also investigated, and KATs were able to modify hormone peptides as well as GFPuv. Moreover, CARsr-A was able to generate acyl-adenosine monophosphates which were observed to have high selectivity for the N-terminus, likely due to its lower pKa with respect to other nucleophilic residues present in the biomolecule. This enzymatic system, coined the Enzymatic Reagent Activation (ERA) method, was shown to work on a variety of peptides and proteins such as insulin, liraglutide or glucagon. Furthermore, it also accepted a variety of substrates, such as di-carboxylic acids, fatty acids, or bioorthogonal probes. The applicability of this system was studied for more complex biomolecules too, such as myoglobin, GFPuv or antibodies. It was demonstrated that the ERA method was compatible with these proteins too, although further optimisation is still required. In conclusion, we have expanded the toolbox of bioconjugation methods by developing two new (chemo)enzymatic methods. These methods do not require the introduction of tags and allow functionalisation of native peptides and proteins from the readily available acid substrates.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sabine Flitsch (Supervisor) & Perdita Barran (Supervisor)