Potent mechanism-based sirtuin-2-selective inhibition by an in situ-generated occupant of the substrate-binding site, “selectivity pocket” and NAD+-binding site

SIRT2 is potently and selectively inhibited by in situ-generated KPM-2 (36)-ADP ribose conjugate.


Fig. S3
Deacetylation mechanism catalyzed by sirtuins and inhibition mode of thioacetyl-based inhibitors including 36.

Fig. S5
Evaluation of the interference of 26 and 36 with the Developer II reaction on the SIRTs assay. MALDI-TOF blank spectra carried out for the detection of the 36-ADP-ribose conjugate.

S11
Supplementary Table  Page  Table S1 Data collection and refinement for the 6-SIRT2 crystal structure.

Supplementary Methods
Page SIRT assays.

S35-S37
References 1 H and 13 Table   Table S1. Data  added to the wells and the fluorescence was measured for 0-20 min at 30 °C using a Victor X3 plate reader (λex = 355 nm; λem = 460 nm). The results were plotted using GraFit 7.0.3.
For the differentiation study, the medium was changed to DMEM supplemented with 2% FBS. After incubation with or without 36 for 72 h, the cell morphology was examined using a microscope (Olympus CKX41) and further analyzed with the Photomeasure software (Kenis Ltd.). The S16 differentiated cells were defined as those with at least one neurite that was longer than twice the diameter of the cell body. The results are expressed as the percentage of differentiated cells relative to the total number of counted cells. These experiments were carried out in triplicate. One-way ANOVA and Dunnett's post hoc tests were used to determine the significance among the groups.