Development of Second Generation Activity-Based Chemical Probes for Sirtuins

Abstract

NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin’s roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.

Multifunctional activity-based chemical probes for sirtuins

“…1). 22 These two series also carry the terminal alkyne to enable Cu( i )-catalyzed “click” conjugation to a reporter. Most recently, we reported the synthesis and characterization of the third generation ABPs featuring an azido group (Fig.…”

“…We took a "less is more" strategy to engineer a simple Ala-Ala-Lys tripeptide as the backbone. [21][22][23] The epsilon-amino of the lysine residue was functionalized with a thioacyl group. The rst generation ABPs harbor a benzophenone photoaffinity group for its synthetic easiness (Fig.…”

“…In the previous studies, the ABPs demonstrated isoformselective labeling of individual sirtuin isoform in puried proteins as well as cell lysates. [21][22][23] The labeled proteins were visualized on a SDS-PAGE gel or by cellular imaging upon conjugation to a uorophore. The versatility of the probes can be further illustrated in affinity enrichment of a particular sirtuin isotype.…”

“…[18][19][20] We took advantage of the distinct reactivity of thioacyllysines to develop several series of activity-based chemical probes (ABPs) for human sirtuins. [21][22][23] These probes are Ala-Ala-Lys tripeptide featuring a thioacyl "warhead". A benzophenone or diazirine photoaffinity group is included for the covalent tethering of the ABP with the enzyme target upon UV irradiation.…”

Plasmodium falciparum Nicotinamidase as A Novel Antimalarial Target

“…Percoll/sorbitol gradient separation allowed the recovery of 95% schizonts. NAD + contents in these cells were measured by adding 18 O-NAD + and processed for MS analysis as reported previously [ 31 , 49 , 50 ]. The intensity of the peak for 18 O-NAD + ( m / z = 666) was ratioed against peak of 16 O-NAD + ( m / z = 664) in MALDI-MS to determine the NAD + concentration in these cells to be 53 pmol/10 6 cells.…”