Development of Activity-Based Chemical Probes for Human Sirtuins

Abstract

Sirtuins consume stoichiometric amounts of nicotinamide adenine dinucleotide (NAD+) to remove an acetyl group from lysine residues. These enzymes have been implicated in regulating various cellular events and have also been suggested to mediate the beneficial effects of calorie restriction (CR). However, controversies on sirtuin biology also peaked during the past few years because of conflicting results from different research groups. This is partly because these enzymes have been discovered recently and the intricate interaction loops between sirtuins and other proteins make the characterization of them extremely difficult. Current molecular biology and proteomics techniques report protein abundance rather than active sirtuin content. Innovative chemical tools that can directly probe the functional state of sirtuins are desperately needed. We have obtained a set of powerful activity-based chemical probes that are capable of assessing the active content of sirtuins in model systems. These probes consist of a chemical “warhead” that binds to the active site of active enzyme and a handle that can be used for the visualization of these enzymes by fluorescence. In complex native proteome, the probes can selectively “highlight” the active sirtuin components. Furthermore, these probes were also able to probe the dynamic change of sirtuin activity in response to cellular stimuli. These chemical probes and the labeling strategies will provide transformative technology to allow the direct linking of sirtuin activity to distinct physiological processes. They will create new opportunities to investigate how sirtuins provide health benefits in adapting cells to environmental cues and provide critical information to dissect sirtuin regulatory networks.

Photo Cross‐Linking Probes Containing ϵ‐N‐Thioacyllysine and ϵ‐N‐Acyl‐(δ‐aza)lysine Residues

“…Here, we employed coupling of biotin to the protein‐ABP conjugate instead of the fluorophore, followed by enrichment on streptavidin‐coated beads, and protein identification using western blot analysis. Most ABPs developed for investigation of sirtuins have only been evaluated using recombinant enzymes or with overexpressing cell lines thus far . A notable exception to this is work from Li and co‐workers, who have demonstrated enrichment of endogenous SIRT3 and SIRT5 from HeLa whole cell lysate using probes based on Kac and Kmal, respectively .…”

“…Both were selected due to their ability to form so‐called stalled intermediates with the NAD + co‐substrate in the sirtuin active site, resulting in decreased rates of hydrolysis . Encouragingly, Cen and co‐workers recently reported on an ABP, containing a ϵ‐ N ‐thioacetyllysine that could successfully label overexpressed SIRT2 in HEK293 cells, providing further impetus for this concept …”

Novel and efficient bridged bis(N‐heterocyclic carbene)palladium(II) catalysts for selective carbonylative Suzuki–Miyaura coupling reactions to biaryl ketones and biaryl diketones

“…[56] ORTEP3 was used for molecular graphics. [57] All hydrogen atoms were included at calculated positions using a riding model with C-H distances of 0.93 Å for sp 2 carbons and 0.96 Å for sp 3 carbons. The isotropic displacement parameters were U iso (H) = 1.2U eq (C) and U iso (H) = 1.5U eq (C).…”

“…Biaryl ketones or butterfly‐shaped ketones are very valuable organic compounds that are recognized by their thermal, optical, and electrochemical properties 1 . They are attractive compounds in biological and medical applications, such as following medication dosing, monitoring DNA activities, and internal radiography dosing, sun blockers 2–4 . These compounds are used to achieve full efficiency of delayed color brilliance due to a slight difference in energy between their single and trigeminal states 5 .…”

“…[1] They are attractive compounds in biological and medical applications, such as following medication dosing, monitoring DNA activities, and internal radiography dosing, sun blockers. [2][3][4] These compounds are used to achieve full efficiency of delayed color brilliance due to a slight difference in energy between their single and trigeminal states. [5] In addition, benzophenone derivatives are widely used in the preparation of industrial organic compounds, pharmaceuticals, cosmetics, and agrochemicals.…”