Development of Peptide-Based Sirtuin Defatty-Acylase Inhibitors Identified by the Fluorescence Probe SFP3 That Can Efficiently Measure Defatty-Acylase Activity of Sirtuin
Abstract
Sirtuins (SIRTs) are a family of nicotinamide adenine dinucleotide-dependent histone deacetylases that serve as epigenetic regulators of many physiological processes. Recent studies have shown that, in addition to their well-known deacetylase activity, sirtuins also exhibit deacylase activity, such as demyristoylase activity. Here, we show that our previously reported sirtuin fluorescence probe, SFP3, can measure the defatty-acylase activity of SIRT1-3, enabling selective assay of the deacylase activity. We further utilized this finding to develop the first inhibitors of SIRT2 defatty-acylase activity. Notably, most previously reported sirtuin inhibitors, including compound TM, AGK2, and SirReal2, showed almost no SIRT2 defatty-acylase inhibitory activity, but are essentially specific deacetylase inhibitors. These results suggest that the active sites catalyzing the deacetylase and defatty-acylase activities of sirtuins may be independent.
Introduction
Human sirtuins (SIRT1-7) are a family of NAD⁺-dependent histone deacetylases that function as epigenetic regulators, with their activities correlated to cellular energy levels via the NAD⁺/NADH ratio. Their catalytic activities toward histone and non-histone proteins, including transcription factors and metabolic enzymes, play important roles in metabolic regulation, genomic DNA stabilization, stress responses, and cancer.
For a long time, it was thought that sirtuins only catalyze deacetylation reactions of histones and ADP-ribosylation. However, recent findings show that some sirtuins (SIRT4-7) exhibit weak deacetylase activity but possess strong deacylase activities. For example, SIRT4 shows lipoamidase activity, SIRT5 has desuccinylase and demalonylase activities, SIRT6 is a demyristoylase, and SIRT7 exhibits desuccinylase activity. Additionally, SIRT1-3, which are strong deacetylases, can also efficiently remove long-chain fatty acid-derived acyl groups from the ε-amino group of lysine residues, demonstrating defatty-acylase activity. X-ray crystallographic data support the existence of a large hydrophobic pocket in SIRT2 and SIRT6 that can accommodate the myristoyl group.
Protein fatty acylation is important for anchoring proteins to the cell membrane and plays significant roles in cell signaling and protein-protein interactions. Early studies focused on N-terminal glycine myristoylation and cysteine palmitoylation, but the biological significance of lysine fatty acylation is less understood. Recent reports suggest that SIRT6 demyristoylase activity regulates TNF-α secretion, and SIRT2 defatty-acylase activity regulates the localization of the KRas4a oncoprotein and promotes cellular transformation. Notably, the SIRT6 G60A mutant, which has defatty-acylase activity but lacks deacetylase activity, demonstrates that these two activities regulate independent cellular functions.
Many SIRT2 “deacetylase” inhibitors have been reported, but their “defatty-acylase” inhibitory activities have rarely been examined. This is important because specific defatty-acylase inhibitors would be valuable for biological studies and as candidate therapeutic agents for inflammation and cancer.
Therefore, in this study, we first established an assay system for measuring SIRT defatty-acylase activity and then used the developed assay to discover SIRT2 defatty-acylase inhibitors based on the structure of the SIRT2 inhibitor RIK^TfaRY. We evaluated SIRT2 defatty-acylase activity using our recently reported fluorescence probe, SFP3, and a fluorescence probe, p53(Myr)-AMC, developed to monitor demyristoylase activity.
Chemistry
We prepared dabcyl derivatives whose carboxyl groups are linked to the ε-amino group of Fmoc-Lys-OH. The synthetic routes to S2DMi-1-14 and their intermediates are detailed in the supplementary schemes. A series of new peptide-based SIRT2 inhibitors bearing a Dabcyl moiety were synthesized and evaluated for SIRT2-inhibitory activity using the SFP3 assay.
Results
Our previously reported fluorescence resonance energy transfer-based probe, SFP3, consists of fluorescein, an H3K9 peptide, and a dabcyl propanoic acid (Dabcyl-PH) quencher attached to the ε-amino group of the lysine residue. In the presence of sirtuin and NAD⁺, Dabcyl-PH of SFP3 is cleaved by SIRT1-3 and 6, affording a strongly fluorescent product. We hypothesized that the hydrophobic Dabcyl quencher and long-chain fatty acyl groups are both recognized by the same selectivity pocket of SIRT2. Thus, SFP3 might efficiently measure the defatty-acylase activity of SIRT2.
A series of new peptide-based SIRT2 inhibitors (S2DMi-1-14) were synthesized. Among these, S2DMi-6, S2DMi-7, and S2DMi-9 showed potent defatty-acylase-inhibitory activity (IC₅₀ = 0.019–0.022 μM). In contrast, most previously reported SIRT2 inhibitors, including compound TM and SirReal2, showed only very weak SIRT2 defatty-acylase-inhibitory activity (IC₅₀ > 2.0 and >10 μM, respectively), indicating that they are specific deacetylase inhibitors.
The SFP3 assay results were strongly correlated with those obtained using the p53(Myr)-AMC probe, confirming that SFP3 can efficiently measure SIRT2 defatty-acylase activity. Notably, the SIRT2 defatty-acylase-inhibitory activities of the new compounds were not correlated with their SIRT2 deacetylase-inhibitory activities, suggesting that these two enzymatic activities are independent.
Selectivity studies showed that the new inhibitors also inhibited SIRT1 and SIRT3 defatty-acylase activities, but had weaker activity against SIRT6. Cell viability assays indicated that the new inhibitors did not inhibit cell growth, likely due to poor cell permeability or instability in cellular systems.
Kinetic analyses revealed that S2DMi-6 and S2DMi-9 exhibit mixed inhibition with respect to SFP3 and noncompetitive inhibition with respect to NAD⁺.
Discussion and Conclusions
This study demonstrates that SFP3 is a valuable probe for measuring the defatty-acylase activity of SIRT2 and for screening inhibitors of this activity. S2DMi-6, S2DMi-7, and S2DMi-9 are the first potent SIRT2 defatty-acylase inhibitors, but they also inhibit SIRT2 deacetylase activity. The lack of correlation between deacetylase and defatty-acylase inhibition suggests that these activities are catalyzed at independent active sites. The findings highlight the importance of peptide sequence and the Dabcyl-EH scaffold for potent inhibition. Future work will focus on developing SIRT defatty-acylase-specific inhibitors with improved selectivity and cell permeability.