Figure 1.
Inhibition of SIRT3/SIRT1 by nicotinamide and its analog.
(A) Nicotinamide/isonicotinamide inhibition assays showing percent change in deacetylation activity as a function of NAM/isoNAM concentration. Data for the SIRT1 enzyme are indicated with blue squares (filled as NAM, and no filled as isoNAM); data for the SIRT3 enzyme are indicated with red circles (filled as NAM, and no filled as isoNAM). (B) The inset table lists the IC50s of the two inhibitors for these enzymes.
Figure 2.
Inhibition of SIRT3/SIRT1 against NAD+ by Nicotinamide.
(A) Recombinant human SIRT1 was incubated for 0, 10, 20, 30, 60, 120, 180, and 240 min at 37°C in the presence of 50, 125, 750, 1500 µM NAD+ and 0, 50, and 100 µM NAM. (B) Recombinant human SIRT3 was incubated for 0, 10, 20, 30, 60, 120, 180, and 240 min at 37°C in the presence of 100, 375, 750, 1500, 3000 µM NAD+ and 0, 25, 100, and 200 µM NAM. Reactions were terminated by the addition of developer and samples were analyzed by fluorometry (excitation set at 355 nm and emission at 460 nm). Data are globally nonlinear fitted to base exchange inhibition model (Eq. 1) and shown as a double-reciprocal plot of 1/v versus 1/[NAD+]. In (C) and (D), the intersection points of the double reciprocal plots are enlarged for SIRT1 and SIRT3, respectively.
Table 1.
Model parameter estimates from global nonlinear fitting of mixed inhibition models for SIRT3 and SIRT1 inhibition by isoNAM (Eq. 2) and NAM (Eq. 1).
Figure 3.
Deacetylation rate as a function of nicotinamide concentration.
Dixon plot (1/v vs [NAM]) of the deacetylation rates for (A) SIRT1 and (B) SIRT3 enzymes. Experimental data were fit to a linear equation.
Figure 4.
Inhibition of SIRT3 against NAD+ by Isonicotinamide.
Recombinant human SIRT3 was incubated for 0, 10, 20, 30, 60, 120, 180, and 240 min at 37°C in the presence of 100, 375, 750, 1500, 3000 µM NAD+ and 0, 2.5, 10, and 40 mM isoNAM. Reactions were terminated by the addition of developer and samples were analyzed by fluorometry (excitation set at 355 nm and emission at 460 nm). Data are globally nonlinear fitted to mixed inhibition model (Eq. 2) and shown as a double-reciprocal plot of 1/v versus 1/[NAD+].
Figure 5.
Inhibition of SIRT3 by nicotinamide in the presence of IsoNAM.
Recombinant human SIRT3 was incubated with 50, 500, 700 and 900 µM of isoNAM for 40 min at 37°C in the presence of 500 µM NAD+, and 100 µM NAM. Reactions were terminated by the addition of developer and samples were analyzed by fluorometry (excitation set at 355 nm and emission at 460 nm).
Figure 6.
Alignment of ternary complex (Sir2Tm:Ac-p53:NAD+) from MD averaged structure with respect to crystal structure.
Ribbon representation of crystal structure (PDB ID: 2H4F) is colored by secondary structure (sheets in cyan, helixes in red and coils in gray), MD averaged structure (10 frames over last 10 ps) in yellow green color. Carbon atoms in crystal structure are in white, and are in yellow green for MD averaged structure. Alignment was made using residues 15–27, 182–242, which forms the stable A binding pocket in Rossmann fold domain.
Figure 7.
A B, and C pockets of the MD averaged structure of Sir2Tm complex with NAM (Sir2Tm:Ac-p53:NAD+:NAM).
Carbon is colored in yellow for MD averaged structure of 10 frames over last 10 ps, in yellow green for reference NAD+ structure from MD averaged structure over last 10 ps of Sir2Tm ternary complex.
Table 2.
MD/MM-PB(GB)SA binding affinity estimates for NAD+ in SIRT3:Ac-CS2:NAD+and Sir2Tm:Ac-p53:NAD+ in catalytically productive (without NAM) binding mode.
Table 3.
MD/MM-PB(GB)SA binding affinity estimates for NAD+ in SIRT3:Ac-CS2:NAD+ and Sir2Tm:Ac-p53:NAD+ in catalytically unproductive (with NAM) binding modes.
Figure 8.
Snapshots taken from MD simulations of SIRT3:NAD+ binary complex.
Averaged structures of 10 frames over 10 ps at 22, 24, 26, 28, 30 and 32 ns from MD simulation of binary complex (SIRT3:NAD+). Alignment was made using residues 139–151, 313–378 in Rossmann fold domain that forms A binding pocket with respect to SIRT3 ternary complex crystal structure 4FVT.
Figure 9.
A, B and C binding pockets identified in MD averaged structure of different SIRT3 complexes.
(A) A and C pockets from the MD averaged structure of 10 frames over last 10 ps of ternary complex (SIRT3:Ac-CS2:NAD+, carbon in yellow green), reference NAD+ structure (carbon in white) from crystal structure 4FVT; (B) A, B and C pockets from the MD averaged structure of 10 frames over last 10 ps of SIRT3 complex with NAM (SIRT3:Ac-ACS2:NAD+:NAM, carbon in yellow), reference NAD+ structure (carbon in yellow green) from MD averaged structure over last 10 ps of SIRT3 ternary complex; (C) A, B and C pockets from the MD averaged structure of 10 frames over last 10 ps of SIRT3 complex with isoNAM (SIRT3:Ac-ACS2:NAD+:isoNAM, carbon in plum), reference NAD+ structure (carbon in yellow) from MD averaged structure over last 10 ps of SIRT3 complex with NAM.
Table 4.
MD/MM-PB(GB)SA binding affinity estimates for NAM and isoNAM in SIRT3:Ac-CS2:NAD+ and Sir2Tm:Ac-p53:NAD+.
Figure 10.
General model for the inhibition of sirtuins by NAM.
(A) Reaction scheme for the sirtuin deacetylation reaction and base exchange inhibition. For simplicity, deacetylation and AADPR+Pr dissociation are assumed to occur together (kcat denotes the rate constant for deacetylation and dissociation of AADPR+Pr from E). Binding of NAM to E.Ac-Pr is not depicted in this scheme since base exchange is responsible for NAM inhibition of sirtuins and direct competition between NAM and NAD+ can be neglected, as discussed in the text. (B) Simplified reaction network for base exchange inhibition. In the presence of saturating Ac-Pr, E is rapidly converted into E.Ac-Pr and NAM binding to E can be neglected, resulting in a simplified reaction network with 5 species. ADPR, adenosine diphosphate ribose; AADPR, O-acetyl-adenosine-diphosphate-ribose.
Figure 11.
Mixed noncompetitive base exchange inhibition kinetics of sirtuin enzymes: mechanistic interpretation.
Figure 12.
NAM/isoNAM interaction diagrams of MD averaged structures.
(10 frames from last 10 ps) (A) SIRT3 complex with NAM; (B) SIRT3 complex with isoNAM.
Figure 13.
(A) Sir2Tm-NAD+ interaction diagrams of MD averaged structures (10 frames from last 10 ps) - ternary complex (Sir2Tm:Ac-p53:NAD+); (B) SIRT3-NAD+ interaction diagrams of MD averaged structures (10 frames from last 10 ps) - ternary complex (SIRT3:Ac-CS2:NAD+).