Inverting α-glucosidase inhibitors as potential drugs for SARS-CoV-2

Project Information

glyCoVdrugs

Grant agreement ID: 101063551

DOI

10.3030/101063551

Project closed

EC signature date 23 August 2022

Start date 1 September 2022

End date 31 August 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 203 464,32

Coordinated by

UNIVERSITEIT LEIDEN
Netherlands

Periodic Reporting for period 1 - glyCoVdrugs (Inverting α-glucosidase inhibitors as potential drugs for SARS-CoV-2)

Reporting period: 2022-09-01 to 2024-08-31

Summary of the context and overall objectives of the project

The project aimed to address an urgent global health need by developing new approaches to combat viral infections such as SARS-CoV-2, the virus responsible for COVID-19. The focus was on creating inhibitors that target α-glucosidase enzymes, which are essential for the replication of various viruses, including SARS-CoV-2. Inhibiting these enzymes causes protein misfolding, leading to the controlled death of infected cells. This research was part of a broader strategy to enhance global pandemic preparedness and response. It aligned with the EU's commitment to public health, innovation, and scientific excellence, directly supporting societal needs and global challenges.
The main objective was to develop a probe to screen existing libraries for new antiviral agents against SARS-CoV-2 and other viral pathogens. As powerful tool, Activity-Based Protein Profiling (ABPP) should be used as the screening method, which relies on Activity-Based Probes (ABPs, Figure 1). Since no effective covalent binding inhibitor was available, a key task of the project was the design and synthesis of suitable inhibitors. Such an inhibitor could then be converted into an ABP.
This was the first time targeting covalent inhibitors for inverting glycosidases, enhancing our understanding in inhibitor design. This laid the foundation for developing and commercializing these inhibitors, helping to prevent future pandemics and improve global public health. The project’s success will also strengthen the EU’s position in pharmaceutical innovation and pandemic preparedness, opening new pathways for treatments that address global health challenges.

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

The project aimed to develop a potential Activity-Based Probe (ABP) for ER α-glucosidase I (ER-I) by creating a covalent binding inhibitor, a type of molecule that had not been identified before this project. The design of potential inhibitors was guided by two principles: (1) mimicking the transition state of the substrate and (2) replacing a critical water molecule in the enzyme's binding pocket with a suitable electrophile (Figures 2A). This led to a series of promising inhibitors, which were further refined through in silico analysis to identify the best candidates for synthesis (Figure 2B). The project focused on creating different molecules, such as 1,2- and 1,5a-bridged cyclitols and inositol derivatives (Figure 2C). Synthesizing these molecules required new synthetic routes and repeated optimization to ensure their reliability and correct structure. This effort resulted in a library of several promising compounds.
In parallel, an assay was developed to test these compounds against the enzymes ER-I and ER-II. The assay utilized a reaction that produces fluorescence when the enzyme is active; successful inhibition by the compounds blocks this signal. The development of the assay involved creating a trisaccharide-fluorophore conjugate and optimizing the assay conditions. Finally, the assay led to the identification of a promising inhibitor candidate.
The project also advanced new synthetic methods, such as direct aziridination methods, useful for creating chemical groups often used in glycosidase inhibitors. Additionally, the work extended to other inhibitors, e.g. for α- and β-D-arabinofuranosidases. These inhibitors are currently under investigation as selective GBA-2 inhibitors or in binding studies towards different enzymes.

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)

The project made significant advancements, particularly in developing covalent inhibitors for inverting glycosidases. For the first time, a deliberate synthesis of inhibitors for ER-I was designed and executed. While the initial results are promising, further validation with techniques like crystallography and proteomics is needed to confirm their binding and activity. Once validated, these inhibitors will be optimized for effectiveness and specificity and can be converted into ABPs for inverting glycosidases, which are valuable for screening large compound libraries to discover novel antiviral drugs.
During the project an efficient assay for testing inhibition against ER-I and ER-II was developed, serving as a critical tool for future research and drug development. Additionally, the project contributed to synthetic chemistry with new compound for direct aziridinations, providing a tool for an efficient access to ABPs. Furthermore, the project provided new inhibitors for other enzymes, such as α- and β-D-arabinofuranosidases, currently under investigation as selective GBA-2 inhibitors with a potential use for therapeutic treatment.
Overall, these achievements mark a significant advancement in the field, offering new tools, methods, and compounds. The innovations derived from this work establish a solid foundation for further research and hold the potential to make meaningful contributions to drug development and analysis.

Figure 1: Concept of activity-based protein profiling (ABPP)

Figure 2: Inhibitor design.