Periodic Reporting for period 4 - SIRFUNCT (Chemical Tools for Unravelling Sirtuin Function)
Okres sprawozdawczy: 2021-10-01 do 2022-03-31
The key ABP technology, at the pinnacle of project AIM-I, is now conceptually established, and we propose to develop targeted acyllysine-containing probes and utilize these to gain insight into the biological mechanisms involving post-translational lysine acylation, which is a rapidly growing research area. Together with the selective inhibitors developed in project AIM-II, we will be able to investigate the post-translational acylation events and sirtuin function at an unprecedented molecular level. Addressing these questions from two different directions, i.e. using both substrate-based probes and enzyme inhibitors, provides a unique opportunity to gain understanding of mechanisms involving sirtuin function in metabolic pathways, potential lifespan extension, and links to certain types of cancer. Thus, in addition to expanding the fundamental understanding of intracellular regulation, the knowledge generated through this project establishes a framework, which should underpin medicinal lead compound discovery efforts.
We currently have indications that several compounds developed in the Action have the potential to selectively kill cancer cells in culture and we shared selected compounds with a cancer biology laboratory in the US. Here, one of our compounds showed promising effect in a mouse model, providing hope that with further optimisation, we can develop these tool compounds into drug lead molecules. If successful, these efforts will have a great impact on society in generating therapeutics to help treat diseased people as well as generating jobs and revenue in the process.
We currently have indications that several compounds developed in the Action have the potential to selectively kill cancer cells in culture and we shared selected compounds with a cancer biology laboratory in the US. Here, one of our compounds showed promising effect in a mouse model, providing hope that with further optimisation, we can develop these tool compounds into drug lead molecules. If successful, these efforts will have a great impact on society in generating therapeutics to help treat diseased people as well as generating jobs and revenue in the process.
AIM-Ia
We have developed a new strategy for activity-based probes to address the members of the proteome that target N-acyllysine posttranslational modifications, which are highly prevalent and constitute a continuously expanding collection of chemical groups. Our results are highly encouraging, as they corroborate the hypothesis that we can install enzymatically stable amide bond analogues, while retaining the ability of native interaction partners in cell lysates to recognize the modification with high selectivity and specificity. In addition, we provide the first investigation of the importance of peptide sequence on performance of N-acyllysine-containing photo cross-linking probes (Bæk et al., Chem. Eur. J. 2019).
The developed methodology provides basis for detailed chemo proteomics studies by combining our protocol with tandem mass spectrometry-based methods.
AIM-Ib
With the enhanced structural integrity of the probes, we expect that potential reader domains can be identified as well. These efforts are initiated and are still ongoing beyond the timeframe of the Action, through a collaboration with the laboratory of Prof. Luca Laraia at the Technical University of Denmark.
Our work on erasers of epsilon-N-lactyllysine and epsilon-N-(beta-hydroxybutyryl)lysine, performed in collaboration with the laboratory of Prof. YingMing Zhao at the University of Chicago and published this year (Moreno-Yruela et al., Science Advances 2022), served as inspiration for the latest series of probes.
AIM-II
Development of potent and selective SIRT5 inhibitor:
Early in the Action we succeeded in publishing the most potent and selective inhibitors of SIRT5 enzyme activity (Rajabi et al. Angewandte Chemie 2017). This is a landmark achievement in inhibition of SIRT5, which could be of therapeutic interest. Furthermore, the developed compound has now been commercialized by a company specialized in assay materials and tool compounds, which makes the molecule available to the scientific community beyond the labs that have already requested samples from us.
We then embarked on two different strategies to improve cell uptake. Both our prodrug strategy and bioisostere strategy produced potent compounds that exhibited target engagement in living cells and were able kill SIRT5-dependent cancer cells (Rajabi, Hansen, et al., Angewandte Chemie 2022).
Finally, arylfluorosulfates have recently emerged as latent electrophiles (reviewed by us; Martin-Gago et al. Angewandte Chemie 2019). We have thus incorporated arylfluorosulfates into tour scaffold and demonstrated SIRT5 labeling in cells (Bolding et al.,Angewandte Chemie 2022).
Insights into mechanism-based inhibitors of SIRT1–3:
We have evaluated a series of mechanism-based inhibitors of SIRT1–3. Notably, we have shown that short-chain thioamide-based inhibitors can be efficiently deacylated by SIRT1–3 to an unprecedented degree (Rajabi, Nielsen, et al., ACS Med. Chem. Lett. 2020).
The collective insight gained in this study is essential for future sirtuin inhibitor design and underscores the crucial need for comprehensive compound assessment.
Development of potent and selective SIRT2 inhibitors:
We have built on previous knowledge to develop the most potent SIRT2 inhibitors reported to date, which exhibit inhibition of cell migration and invasion in breast cancer cells in culture (Nielsen et al., RSC Chem. Biol. 2021).
Development of potent and selective SIRT3 inhibitor:
To achieve selectivity for SIRT3 over SIRT1 and SIRT2, a mitochondrial targeting sequence was incorporated into the inhibitors. Thus, achieving selectivity by sub-cellular localization rather than selective affinity for the target enzyme alone (Troelsen et al., RSC Chem. Biol. 2021).
We have developed a new strategy for activity-based probes to address the members of the proteome that target N-acyllysine posttranslational modifications, which are highly prevalent and constitute a continuously expanding collection of chemical groups. Our results are highly encouraging, as they corroborate the hypothesis that we can install enzymatically stable amide bond analogues, while retaining the ability of native interaction partners in cell lysates to recognize the modification with high selectivity and specificity. In addition, we provide the first investigation of the importance of peptide sequence on performance of N-acyllysine-containing photo cross-linking probes (Bæk et al., Chem. Eur. J. 2019).
The developed methodology provides basis for detailed chemo proteomics studies by combining our protocol with tandem mass spectrometry-based methods.
AIM-Ib
With the enhanced structural integrity of the probes, we expect that potential reader domains can be identified as well. These efforts are initiated and are still ongoing beyond the timeframe of the Action, through a collaboration with the laboratory of Prof. Luca Laraia at the Technical University of Denmark.
Our work on erasers of epsilon-N-lactyllysine and epsilon-N-(beta-hydroxybutyryl)lysine, performed in collaboration with the laboratory of Prof. YingMing Zhao at the University of Chicago and published this year (Moreno-Yruela et al., Science Advances 2022), served as inspiration for the latest series of probes.
AIM-II
Development of potent and selective SIRT5 inhibitor:
Early in the Action we succeeded in publishing the most potent and selective inhibitors of SIRT5 enzyme activity (Rajabi et al. Angewandte Chemie 2017). This is a landmark achievement in inhibition of SIRT5, which could be of therapeutic interest. Furthermore, the developed compound has now been commercialized by a company specialized in assay materials and tool compounds, which makes the molecule available to the scientific community beyond the labs that have already requested samples from us.
We then embarked on two different strategies to improve cell uptake. Both our prodrug strategy and bioisostere strategy produced potent compounds that exhibited target engagement in living cells and were able kill SIRT5-dependent cancer cells (Rajabi, Hansen, et al., Angewandte Chemie 2022).
Finally, arylfluorosulfates have recently emerged as latent electrophiles (reviewed by us; Martin-Gago et al. Angewandte Chemie 2019). We have thus incorporated arylfluorosulfates into tour scaffold and demonstrated SIRT5 labeling in cells (Bolding et al.,Angewandte Chemie 2022).
Insights into mechanism-based inhibitors of SIRT1–3:
We have evaluated a series of mechanism-based inhibitors of SIRT1–3. Notably, we have shown that short-chain thioamide-based inhibitors can be efficiently deacylated by SIRT1–3 to an unprecedented degree (Rajabi, Nielsen, et al., ACS Med. Chem. Lett. 2020).
The collective insight gained in this study is essential for future sirtuin inhibitor design and underscores the crucial need for comprehensive compound assessment.
Development of potent and selective SIRT2 inhibitors:
We have built on previous knowledge to develop the most potent SIRT2 inhibitors reported to date, which exhibit inhibition of cell migration and invasion in breast cancer cells in culture (Nielsen et al., RSC Chem. Biol. 2021).
Development of potent and selective SIRT3 inhibitor:
To achieve selectivity for SIRT3 over SIRT1 and SIRT2, a mitochondrial targeting sequence was incorporated into the inhibitors. Thus, achieving selectivity by sub-cellular localization rather than selective affinity for the target enzyme alone (Troelsen et al., RSC Chem. Biol. 2021).
AIM-I
Beyond state-of-the-art.
The development of ABPs was pioneered by Professors Cravatt, Bogyo, and Powers, and has been used in a wide variety of biological systems.
In preliminary studies, we synthesized peptide probes containing an N-crotonyllysine residue, a benzophenone, and an alkyne and showed that these could be used to pull down HDAC3 and sirtuin 1. However, we tested the stability of these probes by incubation with recombinant sirtuins and HDACs, which revealed rapid hydrolysis of the modifications of interest. We therefore improved the molecular design by introducing non-cleavable N-acyllysine modifications. I strongly believe that this development will prove to be a significant step beyond the current state-of-the-art, which will be broadly applicable to investigation of novel lysine PTMs. Follow-up generations of probes have been prepared and successfully applied in various pulldown experiments in our laboratory, showing capture of for example the YEATS domain. These studies are still ongoing and will be published upon finalization of the collaborative MS/MS efforts.
AIM-II
Beyond state-of-the-art.
Since the first discovery of human sirtuin enzymes in 1999, the class has received widespread attention, not least due to the reports suggesting activation of SIRT1 to play a role in lifespan extension. More recently, it was questioned whether up or downregulation of individual sirtuins are beneficial. Combined with the discovery of novel lysine PTMs targeted by sirtuins, this made the time just right for our program devoted to efficient and selective sirtuin inhibitors, which will be powerful tools to probe their biology and may provide novel lead compounds to address sirtuin malfunction. We have thus pushed beyond the previous state of the art related to inhibition of SIRT 2, 3, and 5, and are dedicatedly pursuing yet novel chemotypes targeting SIRT5 as well as efforts to inhibit SIRT6 and SIRT7.
The developed probes enable cell culture investigations but will be further optimized to enable in vivo studies in animal models in the future.
Beyond state-of-the-art.
The development of ABPs was pioneered by Professors Cravatt, Bogyo, and Powers, and has been used in a wide variety of biological systems.
In preliminary studies, we synthesized peptide probes containing an N-crotonyllysine residue, a benzophenone, and an alkyne and showed that these could be used to pull down HDAC3 and sirtuin 1. However, we tested the stability of these probes by incubation with recombinant sirtuins and HDACs, which revealed rapid hydrolysis of the modifications of interest. We therefore improved the molecular design by introducing non-cleavable N-acyllysine modifications. I strongly believe that this development will prove to be a significant step beyond the current state-of-the-art, which will be broadly applicable to investigation of novel lysine PTMs. Follow-up generations of probes have been prepared and successfully applied in various pulldown experiments in our laboratory, showing capture of for example the YEATS domain. These studies are still ongoing and will be published upon finalization of the collaborative MS/MS efforts.
AIM-II
Beyond state-of-the-art.
Since the first discovery of human sirtuin enzymes in 1999, the class has received widespread attention, not least due to the reports suggesting activation of SIRT1 to play a role in lifespan extension. More recently, it was questioned whether up or downregulation of individual sirtuins are beneficial. Combined with the discovery of novel lysine PTMs targeted by sirtuins, this made the time just right for our program devoted to efficient and selective sirtuin inhibitors, which will be powerful tools to probe their biology and may provide novel lead compounds to address sirtuin malfunction. We have thus pushed beyond the previous state of the art related to inhibition of SIRT 2, 3, and 5, and are dedicatedly pursuing yet novel chemotypes targeting SIRT5 as well as efforts to inhibit SIRT6 and SIRT7.
The developed probes enable cell culture investigations but will be further optimized to enable in vivo studies in animal models in the future.