Date of Presentation
5-5-2022 12:00 AM
College
School of Osteopathic Medicine
Poster Abstract
Sirtuins are a class of proteins belonging to the Sir2 (Silencing information regulator 2) family of NAD+ dependent protein lysine deacylases. Different Isoforms (SIRT1-SIRT7) differ in their specific deacylase activity and cellular location. They have roles in DNA repair, glucose metabolism, and cellular proliferation which make them highly desirable targets for carcinoma therapeutics. We previously used 1-aminoanthracene’s (AMA) fluorescent properties when bound with SIRT2 (Kd of 37 μM) to develop a high-throughput screen to identify novel ligands that inhibit SIRT2’s enzymatic activities. We hope to reveal other potential probes for future high-throughput screening with all the sirtuin isotopes. 1-AMA’s fluorescence along with fluorescent labeled peptides “Cy3-PEG4-H4K16(myr)” and “FAM-PEG4-H4K16(myr)” were used in binding assays to determine their affinities with SIRT2, SIRT3, and SIRT6. Further, we determined 1-AMA’s ability to bind sirtuin isoforms when they were equilibrated with 100 μM of various acyl-peptides. 1-AMA displays weak binding to SIRT3 and SIRT6 when compared to SIRT2. FAM-PEG4-H4K16(myr) binds SIRT2 with a Kd of 7nM which is much higher than its interaction with SIRT3 and SIRT6 (Kd of 6 μM and 2 μM, respectively). Cy3-PEG4-H4K16(myr) binds as expected SIRT2,3,6 although its affinity for SIRT6 changes minimally with the addition of ADP-ribose, which suggests Cy3 may facilitate binding in the absence of SIRT6’s co-factor. Future work will test additional probes with the other sirtuin proteins and establish their competency to be utilized for high-throughput screening.
Keywords
Sirtuins, Carcinoma, Therapeutics, 1-aminoanthracene, High-Throughput Screening Assays
Disciplines
Investigative Techniques | Laboratory and Basic Science Research | Medical Molecular Biology | Medicine and Health Sciences | Neoplasms | Therapeutics
Document Type
Poster
Included in
Investigative Techniques Commons, Laboratory and Basic Science Research Commons, Medical Molecular Biology Commons, Neoplasms Commons, Therapeutics Commons
Interaction of Fluorescent Probes with Sirtuin Proteins
Sirtuins are a class of proteins belonging to the Sir2 (Silencing information regulator 2) family of NAD+ dependent protein lysine deacylases. Different Isoforms (SIRT1-SIRT7) differ in their specific deacylase activity and cellular location. They have roles in DNA repair, glucose metabolism, and cellular proliferation which make them highly desirable targets for carcinoma therapeutics. We previously used 1-aminoanthracene’s (AMA) fluorescent properties when bound with SIRT2 (Kd of 37 μM) to develop a high-throughput screen to identify novel ligands that inhibit SIRT2’s enzymatic activities. We hope to reveal other potential probes for future high-throughput screening with all the sirtuin isotopes. 1-AMA’s fluorescence along with fluorescent labeled peptides “Cy3-PEG4-H4K16(myr)” and “FAM-PEG4-H4K16(myr)” were used in binding assays to determine their affinities with SIRT2, SIRT3, and SIRT6. Further, we determined 1-AMA’s ability to bind sirtuin isoforms when they were equilibrated with 100 μM of various acyl-peptides. 1-AMA displays weak binding to SIRT3 and SIRT6 when compared to SIRT2. FAM-PEG4-H4K16(myr) binds SIRT2 with a Kd of 7nM which is much higher than its interaction with SIRT3 and SIRT6 (Kd of 6 μM and 2 μM, respectively). Cy3-PEG4-H4K16(myr) binds as expected SIRT2,3,6 although its affinity for SIRT6 changes minimally with the addition of ADP-ribose, which suggests Cy3 may facilitate binding in the absence of SIRT6’s co-factor. Future work will test additional probes with the other sirtuin proteins and establish their competency to be utilized for high-throughput screening.