Sirtuins catalyze NAD؉ -dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophorecontaining peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

Sequence-specific resonance assignments are a prerequisite for structural and dynamical interpretation of protein NMR spectra. For proteins smaller than 10 kDa assignment strategies have relied upon through-bond correlations in homonuclear COSY and TOCSY spectra to identify resonances associated with particular spin systems. Conformation-dependent nuclear Overhauser effects are then employed to sequentially connect these spin systems (1-5). In larger proteins, however, extensive resonance overlap and decreased sensitivity of experiments utilizing 'H-'H scalar couplings have hindered this approach.'H-I%-15N triple-resonance experiments provide a conformation-independent approach for the assignment of backbone resonances in I%-15N-labeled large proteins (6-13). In addition these experiments allow accurate measurement of coupling constants in proteins with large linewidths (14,15). These experiments exploit large heteronuclear one-bond couplings to transfer magnetization with the sensitivity of indirect detection. As demonstrated in calmodulin ( 16, 17) backbone assignments utilize four triple-resonance experiments [ HNCa, HNCO, HCaCO, and HCA(CO)N] and the 3D

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Proton NMR spectroscopy of the low-spin cyanide complex of horseradish peroxidase, HRPCN, in H20 solution was used to examine exchangeable resonances of functionally important amino acids in the heme pocket and their role in hydrogen-bonding networks, which have been proposed to facilitate peroxidase catalysis. Spectra were analyzed by use of nuclear Overhauser effect and saturation-transfer spectroscopies, along with consideration of paramagnetic shift and relaxation.Definitive assignments could be made in spite of the size of the proteins (42 kDa), its inherent paramagnetism (iron(III), 5 = */2), and the relatively few resolved resonances, suggesting that these NMR methods may be applicable to even larger heme proteins. The resonance identification was made for labile protons of the proximal His-170, distal His-42, and a heme peripheral contact, Tyr-185, which confirm the close similarity of the heme pocket stereochemistry of horseradish and cytochrome c peroxidases. The resonance assignments enabled determination of several key facets of the structure of the heme cavity of HRPCN. The proton taken up in concert with anionic ligand binding is shown to be present on the His-42 imidazole ring, forming a imidazolium side chain that hydrogen bonds sufficiently strongly to the nitrogen of the bound cyanide to induce a detectable isotope effect on the electronic structure of the heme. The strength of the hydrogen bond is confirmed by the stability of this network over the pH range 4-11. The labile ring proton of the proximal His-170 in HRPCN was located in a position that is consistent with an imidazolate description of the axial ligand but with the proton transferred to a nearby amino acid acceptor site less than 1 Á removed. The structural changes in the heme and proximal and distal amino acid residues upon ligand binding is discussed as a model for HRP compound II formation.

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