PROJECT CODE PN-III-P1-1.1-PD-2021-0127

Grant number PD 65 / 2022

Title: Investigation of molecular mechanisms regarding the modulation of sirtuin activity by polyphenol metabolism/degradation products

Acronim: POLYSIRT

Funding authority – The National Authority for Scientific Research of Romania (UEFISCDI)

POSTDOCTERAL REASERCH PROJECTS

(https://uefiscdi.gov.ro/proiecte-de-cercetare-postdoctorala)

Budget: 250000 RON

Duration: 24 months

Team members

Project Leader: Ungurianu Anca

Mentor: Margină Denisa

Project Summary

The issue of polyphenol intake and supplementation/treatment, considering the myriad of protective effects reported, has been a long debated one. Although they have been the subject of numerous studies in the last decades, their most intimate mechanisms of action (i.e. nuclear regulation of gene expression) have yet to be fully comprehended. The key might be shifting the focus from parent compounds to their metabolites, their post-absorption kinetics and intracellular effects, as they might prove to be the ones behind the pluripotent status of some well-known polyphenols. Up to now, few main metabolites have been subjected to analysis, revealing antioxidant, anti-inflammatory and anticancer effects, but a closer look at their effects on gene expression, as well as their interaction with nucleic acids and epigenetic regulators is warranted. Sirtuins are a family of enzymes that regulate numerous physiological processes, which lately have become attractive therapeutic targets for ageing-associated diseases, such inflammatory or neurodegenerative maladies. In this project, we aim to explore the effects of some well-known polyphenols’ metabolites and sirtuin isoforms, also investigating the interaction of these molecules with plasma proteins and nucleic acids, in order to evaluate their possible pharmacokinetic interfering with chronic therapy.

The purpose of this post-doctoral project is to investigate key metabolites of some of the best-known polyphenols/related natural compounds – curcumin, quercetin (MPs), resveratrol acclaimed for their protective and disease preventing effects. The specific project objectives are to investigate the interaction with serum main plasma proteins employed for binding studies, due to their paramount role in the transport and distribution of a wide range of molecules; to evaluate the degree of DNA binding and interaction via detection of minor and major groove interactions and/or intercalation/displacement experiments; to carry out a molecular docking study as a novel, rapid and insightful method to obtain in-depth data about the possible molecular dynamics of the interaction between the selected MPs and the targeted biological structures (relevant serum proteins), nucleic acids – DNA or enzymes – sirtuins; to assess the stimulating/inhibitory action of MPs on the activity of sirtuins.

STAGE I (2022)

Budget: 71250 RON

The activities planned for this phase of the contract were:

• In-depth re-evaluation of the latest literature on MP is mandatory in order to ensure the appropriate selection of the most relevant molecules.
• Protein interaction studies: Spectroscopic and competitive binding experiments, synchronous fluorescence and 3D spectra assessments.
• DNA binding studies: UV-Vis spectroscopy and fluorescence measurements, intercalation/displacement experiments for the evaluation of the degree and types of DNA interaction.

Results 

All activities under the work plan were completed, with the following results:

• The literature survey was completed and led to the selection of the most appropriate MPs for further tests. Also, this activity led to the preparation of two manuscripts – one already published (Regulation of Gene Expression through Food—Curcumin as a Sirtuin Activity Modulator, Ungurianu A, Zanfirescu A, Margină D, Plants, 2022; 11(13):1741, eISSN 2223-7747, doi: 10.3390/plants11131741, WOS:000824517000001, 2021 IF 4.658, Q1 Plant Sciences), while the other is under review.
• Protein interaction studies were completed revealing the different binding affinities the main serum proteins (e.g. transferrin, albumin) and varying interactions of MPs compared to the parent compounds.
• DNA binding studies were carried out evaluating DNA-MP interaction via UV and fluorescence spectroscopy. Also, competitive binding studies with ethidium bromide were employed. This mainly highlighted groove interactions and to a lesser extents intercalation between base pairs.

STAGE II (2023)

Budget: 128750 RON

The activities planned for this phase were:

• To continue the molecular docking studies in order to obtain data about the possible molecular dynamics of the interaction between the MPs and the targeted biological molecules: proteins (HSA), nucleic acids (DNA) or enzymes (sirtuins). These will be followed by data analysis, writing, editing and/or publishing the results.
• Investigating the stimulatory or inhibitory effects of selected MPs on the activity of targeted sirtuin isoforms using sensitive molecular biology techniques. We will be able to report the stimulatory or inhibitory effects of MPs on the activity of sirtuins, determining the effective concentrations and/or selectivity of the chosen molecules.
• We estimate that we will publish one article in an ISI Web of Science (Clarivate Analytics)-indexed journal (IF>2) or at least submit a manuscript, meeting the overall project goal of 2 ISI-indexed (IF>2) articles.

Results 

All activities planned were completed, with the following results:

• We continued the literature survey initiated in the first stage of the project in order to better assess which of the SIRT isoforms are the most relevant regarding epigenetic regulation, metabolic and energetic processes, and are involved in the aging-associated phenomena and chronic diseases.
• We published an article in a scientific journal with an IF>2: Sirtuins, resveratrol and the intertwining cellular pathways connecting them, Ungurianu A., Zanfirescu A., Margină D., Aging Research Reviews, Volume 88, 2023, 101936, ISSN 1568-1637, doi: 10.1016/j.arr.2023.101936, WOS:000988700700001 (2022 IF: 13.1, Q1: Cell Biology, Geriatrics & Gerontology).
• We obtained and analyzed the parameters found in molecular docking studies (binding energy, ligand efficiency, etc.) regarding the interaction of MPs with serum proteins, especially targeting HSA – the main transporter protein of xenobiotics, and the interaction of MPs with sirtuins, focusing on SIRT1, SIRT2 and SIRT6.
• We evaluated the inhibitory/stimulatory effects of MP on two of the selected SIRT isoforms: SIRT1 and SIRT2.
• We presented partial results of the project at the 47th FEBS Congress, July 8-12, 2023, in Tours, France, as a poster presentation: The effects of quercetin, curcumin and their key metabolites on SIRT1 activity, Ungurianu A., Margină D ., the abstract being published in FEBS Open Bio, 2023, 13, Suppl. S2: 215, ISSN 2211-5463, DOI: 10.1002/2211-5463.13646, (FI 2022: 2,792).

STAGE III (2024)

Budget: 50000 RON

The activities planned for this phase were:

• Continuing the assessment of the stimulatory or inhibitory effects of MP on the activity of sirtuin isoforms followed by data analysis, writing, editing and/or publishing the results.
• Evaluation of the effect of MP on the activity of SIRT6, using fluorescence techniques, determining the effective concentrations and selectivity of the chosen molecules. We will be able to report the stimulatory or inhibitory effect on sirtuins activity.
• We estimate that we will publish one article in an ISI Web of Science (Clarivate Analytics)-indexed journal (IF>2) or at least submit a manuscript, meeting the overall project goal of 2 ISI-indexed (IF>2) articles.

Results 

All activities planned were completed, with the following results:

• The following manuscript – Exploring the Therapeutic Potential of Quercetin: A Focus on its Sirtuin-mediated Benefits, Ungurianu A., Zanfirescu A., Margină D., was accepted for publication in Phytotherapy Research (IF 2022: 7.2, Q1: Pharmacology & Pharmacy), doi: 10.1002/ptr.8168.
• We evaluated the inhibitory/stimulatory effects of MP on SIRT6 isoform.
• We submitted a manuscript including partial results of the experimental studies within the project, which is currently under review.