Activation of Thoeris antiviral system via SIR2 effector filament assembly.

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems1-7. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components8-12. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD+ (refs. 12-14). Although the structure of ThsA has been solved15, the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD+ depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.

Synthesis of Terminal Ribose Analogues of Adenosine 5'-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2.

TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5'-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9 H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2'-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1″-ß- O-methyl-2″,3″- O-isopropylidene derivative was evaluated. Removal of either C1″ or C3″ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.

Structure of tRNA splicing enzyme Tpt1 illuminates the mechanism of RNA 2'-PO4 recognition and ADP-ribosylation.

Tpt1 is an essential agent of fungal tRNA splicing that removes the 2'-PO4 at the splice junction generated by fungal tRNA ligase. Tpt1 catalyzes a unique two-step reaction whereby the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-ADP-ribosyl intermediate that undergoes transesterification to yield 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. Because Tpt1 is inessential in exemplary bacterial and mammalian taxa, Tpt1 is seen as an attractive antifungal target. Here we report a 1.4 Šcrystal structure of Tpt1 in a product-mimetic complex with ADP-ribose-1″-phosphate in the NAD+ site and pAp in the RNA site. The structure reveals how Tpt1 recognizes a 2'-PO4 RNA splice junction and the mechanism of RNA phospho-ADP-ribosylation. This study also provides evidence that a bacterium has an endogenous phosphorylated substrate with which Tpt1 reacts.

Enhanced sensitivity to cholera toxin in female ADP-ribosylarginine hydrolase (ARH1)-deficient mice.

Cholera toxin, an 84-kDa multimeric protein and a major virulence factor of Vibrio cholerae, uses the ADP-ribosyltransferase activity of its A subunit to intoxicate host cells. ADP-ribosylation is a posttranslational modification of proteins, in which the ADP-ribose moiety of NAD+ is transferred to an acceptor. In mammalian cells, ADP-ribosylation of acceptors appears to be reversible. ADP-ribosyltransferases (ARTs) catalyze the modification of acceptor proteins, and ADP-ribose-acceptor hydrolases (ARHs) cleave the ADP-ribose-acceptor bond. ARH1 specifically cleaves the ADP-ribose-arginine bond. We previously demonstrated a role for endogenous ARH1 in regulating the extent of cholera toxin-mediated fluid and electrolyte abnormalities in a mouse model of intoxication. Murine ARH1-knockout (KO) cells and ARH1-KO mice exhibited increased sensitivity to cholera toxin compared to their wild-type (WT) counterparts. In the current report, we examined the sensitivity to cholera toxin of male and female ARH1-KO and WT mice. Intestinal loops derived from female ARH1-KO mice when injected with cholera toxin showed increased fluid accumulation compared to male ARH1-KO mice. WT mice did not show gender differences in fluid accumulation, ADP-ribosylarginine content, and ADP-ribosyl Gαs levels. Injection of 8-Bromo-cAMP into the intestinal loops also increased fluid accumulation, however, there was no significant difference between female and male mice or in WT and KO mice. Female ARH1-KO mice showed greater amounts of ADP-ribosylated Gαs protein and increased ADP-ribosylarginine content both in whole intestine and in epithelial cells than did male ARH1-KO mice. These results demonstrate that female ARH1-KO mice are more sensitive to cholera toxin than male mice. Loss of ARH1 confers gender sensitivity to the effects of cholera toxin but not of cyclic AMP. These observations may in part explain the finding noted in some clinical reports of enhanced symptoms of cholera and/or diarrhea in women than men.

Mono-ADP-Ribosylation Catalyzed by Arginine-Specific ADP-Ribosyltransferases.

Methods are described for determination of arginine-specific mono-ADP-ribosyltransferase activity of purified proteins and intact cells by monitoring the transfer of ADP-ribose from NAD+ to a model substrate, e.g., arginine, agmatine, and peptide (human neutrophil peptide-1 [HNP1]), and for the nonenzymatic hydrolysis of ADP-ribose-arginine to ornithine, a noncoded amino acid. In addition, preparation of purified ADP-ribosylarginine is included as a control substrate for ADP-ribosylation reactions.

Selective inhibition of TRPM2 channel by two novel synthesized ADPR analogues.

Transient receptor potential melastatin-2 (TRPM2) channel critical for monitoring internal body temperature is implicated in the pathological processes such as neurodegeneration. However, lacking selective and potent TRPM2 inhibitors impedes investigation and validation of the channel as a drug target. To discover novel and selective TRPM2 inhibitors, a series of adenosine 5'-diphosphoribose analogues were synthesized, and their activities and selectivity were evaluated. Whole-cell patch-clamp recordings were employed for screen and evaluation of synthesized compounds. Two compounds, 7i and 8a, were identified as TRPM2 inhibitors with IC50 of 5.7 and 5.4 µm, respectively. Both 7i and 8a inhibited TRPM2 current without affecting TRPM7, TRPM8, TRPV1 and TRPV3. These two TRPM2 inhibitors can serve as new pharmacological tools for further investigation and validation of TRPM2 channel as a drug target, and the summarized structure-activity relationship (SAR) may also provide insights into further improving existing inhibitors as potential lead compounds.

Understanding the differences of the ligand binding/unbinding pathways between phosphorylated and non-phosphorylated ARH1 using molecular dynamics simulations.

ADP-ribosylhydrolases (ARH1, ARH2 and ARH3) are a family of enzymes to catalyze ADP-ribosylation, a reversible and covalent post-translational modification (PTM). There are four phosphorylated sites (Tyr-4, Tyr-19, Tyr-20, and Tyr-205) in ARH1. To explore the structural changes and functional impact induced by phosphorylation, molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed for the phosphorylated and non-phosphorylated ARH1 with the ligands. MD simulations results indicate that: (1) Glu-25 is more frequently in the α helix group in the phosphorylated state with the adenosine-5-diphosphate-ribosylarginine (ADP-RA) complex (51.56%) than that of the non-phosphorylated state(2.12%); (2) Ser-124 and Ser-264 become less flexible in the phosphorylated state with ADP-RA complex, which helps two residues form hydrogen bonds with ADP-RA; and (3) Tyr-211 is also less flexible in the phosphorylated state with ADP-RA complex, which helps stabilize the cation-π interaction of Y211-R119. All these changes facilitate ADP-RA to bind ARH1. In addition, according to the crystal structure of adenosine-5-diphosphate-ribose (ADP-ribose) in complex with non-phosphorylated and phosphorylated ARH1, the possible unbinding pathways of ADP-ribose from non-phosphorylated and phosphorylated ARH1 were explored respectively using SMD simulations. Our results show that phosphorylated ARH1 has more ordered structures than the non-phosphorylated type.

Principles of Chemical Biology: Iron and CSCs, Histone Acylation sans Enzymes, Eliciting Silent Gene Clusters, and 2'-deoxy-ADPR as a Second Messenger.

This month: Lysosomal iron linked to cell death in cancer stem cells, non-enzymatic catalyst SynCAc for histone acylation, cytotoxins ivermectin and etoposide bring new anti-fungals out of the crypt, and 2'-deoxy-ADPR as second messenger activating TRPM2.

2'-Deoxyadenosine 5'-diphosphoribose is an endogenous TRPM2 superagonist.

Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.

Synthesis of a Bioreversibly Masked Lipophilic Adenosine Diphosphate Ribose Derivative.

The design of a bioreversibly protected lipophilic sugar nucleotide as a potential membrane-permeable precursor of adenosine diphosphate ribose (ADPR) is described. ADPR is the most potent activator of the transient receptor potential melastatin 2 (TRPM2) ion channel. Membrane-permeable, lipophilic derivatives of ADPR are of great interest as tools for study of the mechanism of TRPM2. The approach described here was based on our recently disclosed "DiPPro" and "TriPPPro" prodrug approaches developed for the intracellular delivery of nucleotides. A lipophilic, bioreversibly masked ADPR analogue containing an enzymatically cleavable 4-pentanoyloxybenzyl (PB) mask at the phosphate moiety next to the 5'-position of adenosine, together with O-acetyl groups, was prepared in high yields. Chemical and enzymatic hydrolysis studies in phosphate buffer (pH 7.3) were performed to assess chemical stability and possible (selective) enzymatic demasking of the ADPR analogue. HPLC-MS revealed that the PB group was readily cleaved enzymatically. In addition, the formation of partially deacetylated ADPR compounds and also of fully unprotected ADPR was observed.

Designer small molecules to target calcium signalling.

Synthetic compounds open up new avenues to interrogate and manipulate intracellular Ca2+ signalling pathways. They may ultimately lead to drug-like analogues to intervene in disease. Recent advances in chemical biology tools available to probe Ca2+ signalling are described, with a particular focus on those synthetic analogues from our group that have enhanced biological understanding or represent a step towards more drug-like molecules. Adenophostin (AdA) is the most potent known agonist at the inositol 1,4,5-trisphosphate receptor (IP3R) and synthetic analogues provide a binding model for receptor activation and channel opening. 2-O-Modified inositol 1,4,5-trisphosphate (IP3) derivatives that are partial agonists at the IP3R reveal key conformational changes of the receptor upon ligand binding. Biphenyl polyphosphates illustrate that simple non-inositol surrogates can be engineered to give prototype IP3R agonists or antagonists and act as templates for protein co-crystallization. Cyclic adenosine 5'-diphosphoribose (cADPR) can be selectively modified using total synthesis, generating chemically and biologically stable tools to investigate Ca2+ release via the ryanodine receptor (RyR) and to interfere with cADPR synthesis and degradation. The first neutral analogues with a synthetic pyrophosphate bioisostere surprisingly retain the ability to release Ca2+, suggesting a new route to membrane-permeant tools. Adenosine 5'-diphosphoribose (ADPR) activates the Ca2+-, Na+- and K+-permeable transient receptor potential melastatin 2 (TRPM2) cation channel. Synthetic ADPR analogues provide the first structure-activity relationship (SAR) for this emerging messenger and the first functional antagonists. An analogue based on the nicotinic acid motif of nicotinic acid adenine dinucleotide phosphate (NAADP) antagonizes NAADP-mediated Ca2+ release in vitro and is effective in vivo against induced heart arrhythmia and autoimmune disease, illustrating the therapeutic potential of targeted small molecules.

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue ß2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site.

Tuning IL-2 signaling by ADP-ribosylation of CD25.

Control of immunologic tolerance and homeostasis rely on Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5, and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells.

ADP-Ribosylargininyl reaction of cholix toxin is mediated through diffusible intermediates.

BACKGROUND: Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase. RESULTS: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates. CONCLUSIONS: We discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.

Stereoselective ribosylation of amino acids.

The glycosylation properties of ribofuranosyl N-phenyltrifluoroacetimidates toward carboxamide side chains of asparagine and glutamine were investigated. Conditions were found that promote nearly exclusive formation of the α-anomerically configured N-glycosides. The strategy allows for the synthesis of Fmoc-amino acids suitably modified for the preparation of ADP-ribosylated peptides. Furthermore, ribosylation of serine with these donors proved to be completely α-selective, and for the first time, α-ribosylated glutamic and aspartic acid, the naturally occurring sites for poly-ADP-ribosylation, were synthesized.

ADP-ribosylarginine hydrolase regulates cell proliferation and tumorigenesis.

Protein ADP-ribosylation is a reversible posttranslational modification of uncertain significance in cancer. In this study, we evaluated the consequences for cancer susceptibility in the mouse of a genetic deletion of the enzyme responsible for removing mono-ADP-ribose moieties from arginines in cellular proteins. Specifically, we analyzed cancer susceptibility in animals lacking the ADP-ribosylarginine hydrolase (ARH1) that cleaves the ADP ribose-protein bond. ARH1(-/-) cells or ARH1(-/-) cells overexpressing an inactive mutant ARH1 protein (ARH1(-/-)+dm) had higher proliferation rates than either wild-type ARH1(+/+) cells or ARH1(-/-) cells engineered to express the wild-type ARH1 enzyme. More significantly, ARH1(-/-) and ARH1(+/-) mice spontaneously developed lymphomas, adenocarcinomas, and metastases more frequently than wild-type ARH1(+/+) mice. In ARH1(+/-) mice, we documented in all arising tumors mutation of the remaining wild-type allele (or loss of heterozygosity), illustrating the strict correlation that existed between tumor formation and absence of ARH1 gene function. Our findings show that proper control of protein ADP-ribosylation levels affected by ARH1 is essential for cancer suppression.

ADP ribose is an endogenous ligand for the purinergic P2Y1 receptor.

The mechanism by which extracellular ADP ribose (ADPr) increases intracellular free Ca(2+) concentration ([Ca(2+)](i)) remains unknown. We measured [Ca(2+)](i) changes in fura-2 loaded rat insulinoma INS-1E cells, and in primary ß-cells from rat and human. A phosphonate analogue of ADPr (PADPr) and 8-Bromo-ADPr (8Br-ADPr) were synthesized. ADPr increased [Ca(2+)](i) in the form of a peak followed by a plateau dependent on extracellular Ca(2+). NAD(+), cADPr, PADPr, 8Br-ADPr or breakdown products of ADPr did not increase [Ca(2+)](i). The ADPr-induced [Ca(2+)](i) increase was not affected by inhibitors of TRPM2, but was abolished by thapsigargin and inhibited when phospholipase C and IP(3) receptors were inhibited. MRS 2179 and MRS 2279, specific inhibitors of the purinergic receptor P2Y1, completely blocked the ADPr-induced [Ca(2+)](i) increase. ADPr increased [Ca(2+)](i) in transfected human astrocytoma cells (1321N1) that express human P2Y1 receptors, but not in untransfected astrocytoma cells. We conclude that ADPr is a specific agonist of P2Y1 receptors.

Inhibitors of TRP channels reveal stimulus-dependent differential activation of Ca2+ influx pathways in human neutrophil granulocytes.

A pharmacological characterization of Ca(2+) influx pathways in neutrophil granulocytes is problematic because of the lack of specific inhibitors. The activation of transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channels by intracellular adenosine diphosphate ribose (ADPR), well characterized in neutrophils, is reportedly inhibited by 8-bromo-ADPR (8Br-ADPR). TRPM2 is blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA) interfering with the pore, but ACA is as well effective on other transient receptor potential channels, especially transient receptor potential canonical (TRPC) channels. We wished to analyze whether ACA and 8Br-ADPR were suitable probes to demonstrate that different Ca(2+) entry pathways are activated in human neutrophil granulocytes by the receptor-dependent stimuli N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and platelet-activating factor (PAF) and the receptor-independent thapsigargin. Ca(2+)-influx-related increases in [Ca(2+)](i) were calculated by comparing aliquots of fluo-3-loaded neutrophils in the presence and absence of extracellular Ca(2+). Moreover, Mn(2+) quenching was used in fura-2-loaded cells. We compared 8Br-ADPR with ACA. 8Br-ADPR was exclusively effective when Ca(2+) influx (or Mn(2+) quenching) was induced by fMLP; it did not affect influx when PAF or thapsigargin was the stimulus. ACA inhibited Ca(2+) influx significantly more strongly when this was induced by PAF than by fMLP. Moreover, it reduced thapsigargin-induced Ca(2+) influx. The contribution of TRPM2 to Ca(2+) influx in neutrophils strongly depends on the stimulus; it is sizeable in the case of fMLP and minimal in the case of PAF. PAF induces Ca(2+) entry pathways different from TRPM2; the inhibition by ACA suggests the contribution of channels of the TRPC family.

Precursor ion scanning and sequencing of arginine-ADP-ribosylated peptide by mass spectrometry.

Arginine (Arg)-specific ADP-ribosylation is one of the posttranslational modifications of proteins and is thought to play an important role in reversibly regulating functions of the target proteins in eukaryotes. However, the physiological target protein has not been established. We examined the fragmentation pattern of both ADP-ribosyl-Arg (ADP-R-Arg) and Arg-ADP-ribosylated peptides by quadrupole tandem mass spectrometry and found a specific cleavage of ADP-R-Arg into N-(ADP-ribosyl)-carbodiimide (ADP-R-carbodiimide) and ornithine. Based on this specific fragmentation pattern, we successfully identified the modification site and sequence of Arg-ADP-ribosylated peptide using a two-step collision and showed that ADP-R-carbodiimide is an excellent marker ion for precursor ion scanning of Arg-ADP-ribosylated peptide. We propose that a combination of the precursor ion scanning with ADP-R-carbodiimide as a marker ion and two-step collision is useful in searching for physiological target proteins of Arg-ADP-ribosylation.

Inhibition of human sirtuins by in situ generation of an acetylated lysine-ADP-ribose conjugate.

A new type of small-molecular sirtuin inhibitor was designed on the basis of the proposed catalytic mechanism for deacetylation of acetylated lysine substrates by sirtuins. Among the compounds thus designed and synthesized, we found that 2k, which contains an ethoxycarbonyl group at the alpha position to the acetamide of acetylated lysine substrate analogue 1, showed potent inhibitory activity in an in vitro assay using recombinant SIRT1, with high selectivity over SIRT2 and SIRT3. Mechanistic study by means of kinetic analysis, mass spectroscopy, and computation indicated that the enol form of compound 2k nucleophilically attacks NAD(+) in the active site of SIRTs to afford the stable compound 2k-ADP-ribose conjugate 5, leading to inhibition of the enzyme activity. Compound 2k also caused a dose-dependent increase of p53 acetylation in human colon cancer HCT116 cells, indicating inhibition of SIRT1 in the cells. These results have implications for the development of selective sirtuin inhibitors by means of mechanism-based drug design.