SIRT1 Activation by Small Molecules: KINETIC AND BIOPHYSICAL EVIDENCE FOR DIRECT INTERACTION OF ENZYME AND ACTIVATOR

Han Dai,Lauren Kustigian,David Carney,April Case,Thomas Considine,Basil P Hubbard,Robert B Perni,Thomas V Riera,Bruce Szczepankiewicz,George P Vlasuk,Ross L Stein

doi:10.1074/jbc.m110.133892

Han Dai, Lauren Kustigian + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m110.133892

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Abstract

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 (Pacholec, M., Chrunyk, B., Cunningham, D., Flynn, D., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) J. Biol. Chem. 285, 8340–8351). Rather, the data suggest that STACs interact directly with SIRT1 and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.

Highlights

Where RVmax is the maximum relative velocity and EC50 is the activator concentration at which vx/vo ϭ (RVmax Ϫ1)/2
Structures and Steady-state Kinetic Parameters for SIRT1 Substrates—Table 1 summarizes steady-state kinetic parameters for the peptide substrates used in this study
Discovery and Initial Characterization of STACs—Our initial lead structures were identified in a high throughput screening campaign that used a fluorescence polarization assay, with

Summary

EXPERIMENTAL PROCEDURES

Materials—All peptides were prepared by BioPeptide (San Diego, CA) and shown to be at least 95% pure by analytical HPLC analysis. The other, used with peptides of general structure AcArg-His-Lys-LysAc-X, used a continuous, enzyme-coupled method reported previously [23], in which the SIRT1 product nicotinamide is first converted into nicotinic acid and ammonia by the action of PNC1, and the ammonia is used by glutamate dehydrogenase to convert ␣-ketoglutarate into glutamate This reaction occurs with oxidation of NADH to NADϩ, which is accompanied by a change in absorbance at 340 nm (⌬⑀340 ϭ Ϫ6,200 MϪ1 cmϪ1). Final concentrations of the coupling system components were: 1 ␮M PNC1, 0.23 mM NADH, 3.4 mM ␣-ketoglutarate, and 20 units/ml bovine glutamate dehydrogenase This solution contained peptide substrate, NADϩ, and activator, at concentrations indicated under “Results.” The final DMSO concentration was kept constant at 1%, and the buffer used in these experiments was 50 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.5. It can be seen that (EC1.5)Ϫ1 is analogous to Vmax/Km of standard steady-state enzyme kinetics

RESULTS

Relative velocity

DISCUSSION