Development of new AI-2 prodrugs and chemical probes to study the microbiota of the mammalian gastro-intestinal tract

Project Information

AI-2 Prodrugs

Grant agreement ID: 101090282

DOI

10.3030/101090282

Project closed

EC signature date 3 June 2022

Start date 1 June 2022

End date 31 May 2024

Funded under

• Widening participation and spreading excellence

Total cost

No data

EU contribution

€ 164 698,56

Coordinated by

UNIVERSIDADE NOVA DE LISBOA
Portugal

Periodic Reporting for period 1 - AI-2 Prodrugs (Development of new AI-2 prodrugs and chemical probes to study the microbiota of the mammalian gastro-intestinal tract)

Reporting period: 2022-06-01 to 2024-05-31

Summary of the context and overall objectives of the project

Bacteria, as simple organisms, do not inhabit their environment as isolated individuals. Instead, they form single or multi-species communities. These communities have macroscopic manifestations and are often observable to the naked eye. Well-known examples include biofilms, the production of antibiotics on an industrial scale, the generation of virulent factors that contribute to bacterial infections, and the enchanting bioluminescence seen in sea waves in certain parts of the world. These phenomena are a result of coordinated behaviour among bacteria, driven by communication at the molecular level, known as quorum sensing. A significant molecule in this process is autoinducer-2 (AI-2), which plays a crucial role in interspecies communication. AI-2 originates from (4S)-4,5-dihydroxypentanedione (DPD) which can exist in various chemical forms in equilibrium.
The human gut microbiota, essential for health, is an extraordinary niche hosting diverse bacterial population. These bacteria are vital for nutrient production, immune system maturation, and pathogen defence. Recent research has highlighted the connection between gut microbiota and brain degenerative diseases like Alzheimer’s and Parkinson’s, suggesting that imbalances in the gut microbiome may contribute to these conditions.
AI-2 is particularly important in regulating the colonization and stability of the gut microbiota. Manipulating AI-2 signal has potential therapeutic benefits, as demonstrated by experiments using engineered Escherichia coli strains to alter AI-2 levels in the mouse gut. These manipulations affected the balance between Firmicutes and Bacteroidetes, two key bacterial phyla crucial for host health.
This project aimed to develop methods for maintaining healthy gut microbiota and protecting against pathogenic bacteria by understanding the AI-2 sensing and manipulating AI-2 signal. This involved synthesis and validation of AI-2 prodrugs for specific AI-2 delivery to the mice gut and development of a method for detecting AI-2 in biological. Second objective of this project was the development of new chemical tools for the detection of unknown AI-2 receptors. As part of this project, we also focused on the synthesis of completely new AI-2 analogs.
While the project was rooted in basic research, its findings have significant implications for the medical field, particularly concerning gut health.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project yielded significant conclusions. First, we engineered mutant gut commensal Klebsiella strains that accumulate AI-2 and successfully colonised mice intestines. We also developed a reproducible and accurate GC-MS method for measuring AI-2 in biological samples, involving the first synthesis of [1-D3]-DPD as an internal standard. This method, combined with double derivatization and a simple extraction protocol, precisely measured AI-2 in the ceca of mice inoculated with engineered Klebsiella ARO112 strains, showing excellent specificity and linearity for microbiota research.
Second, we synthesized AI-2 prodrugs designed for safe intestinal delivery, with AI-2 attached to D-glucose or D-galactose via glycosidic bonds. This specific approach is called colon specific drug delivery system. AI-2, thus bound to a sugar molecule, passes intact through the upper gastrointestinal tract and, after being transported to the intestine, is subsequently released by bacterial glycosyl hydrolases.
Hydrolysability of AI-2 prodrugs was tested in vitro using commercial glycosidases and AI-2 release was demonstrated using the Vibrio harveyi bioluminescence assay. Testing with real mouse intestinal content samples confirmed the presence of necessary enzymes, indicating that the intestinal microflora can break down these AI-2 prodrugs.

Additionally, a review article on AI-2 analogs was published, summarizing the synthesis and biological activities of known AI-2 derivatives. This theoretical work provided insights into structure-activity relationships, leading to the synthesis of a new class of AI-2 analogs, which were tested in the Vibrio harveyi bioluminescence assay during this project.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Quorum sensing (QS) therapies are gaining attention, particularly as alternatives to traditional antibiotics for controlling bacterial infections. While anti-QS therapies are being explored, we propose QS therapies that leverage AI-2's ability to regulate colonisation and species interactions in multi-species communities. Our aim is to develop strategies that maintain healthy gut microbiota and protect against pathogenic bacteria.
New AI-2 prodrugs and stable AI-2 analogues have potential pharmacological applications. These innovations have significant potential for societal impact and further research, as manipulating native signals and interactions using AI-2 prodrugs could prevent infections, obesity, diabetes, inflammatory bowel diseases, and gastrointestinal cancers. Moreover, these molecules could offer therapeutic solutions without promoting resistance, a major issue with traditional antibiotics.
The synthesis of AI-2 analogues, a key part of this project, showed promising activity and stability, potentially leading to patentable discoveries. In collaboration with the ITQB Technology Transfer Office, we will ensure proper intellectual property protection and knowledge transfer.
The new [1-D3]-DPD synthesis provides the scientific community with an important analytical standard for studying microbiome-related processes in vivo. Accurate AI-2 quantification enables researchers to investigate its concentration changes in the gut and its role in microbiota dynamics. This method is applicable to other complex biological samples, offering valuable data for QS studies across various scientific disciplines.

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