The dRYBP-SCF complex regulates the intrinsic apoptotic pathway

The validation and characterization of the activators that were identified by the high throughput screen of the ChemBioNet library was performed by Louisa Hill during her master thesis (Hill 2012).

Figure 3.32: Chemical structures of potential SirT1 activators

The validation of the activators with the MAL deacetylation assay showed that of 12 activators, seven (Figure 3.32) induced a potent activation of SirT1 activity (Figure 3.33).

The activators 1, 2, as well as 3 showed the strongest activation of SirT1, whereas the compounds 4 to 7 showed a moderate activation of SirT1.

Figure 3.33: Identification of SirT1 activators

The influence of potential SirT1 activators (1-7) was determined by MAL deacetylation assay. The control reaction containing DMSO was set to 100 %. IC₅₀ values and curve fitting were performed using GraphPad.

Values correspond to average enzyme activity of a 3-fold measurement ± standard deviation. The MAL deacetylation assays were performed by Louisa Hill (Hill 2012).

Furthermore, the effect of these potential activators was measured by the HPLC-based p53 deacetylation assay, and this showed that none of them was able to enhance the activity of SirT1 (Hill 2012). As opposed to this, surprisingly, compound 1 (Figure 3.32) strongly inhibited SirT1 activity in the HPLC-based p53 deacetylation assay. This suggested that the influence of a small molecule on the SirT1 activity depends on the environment of the deacetylation substrate.

Furthermore, the effect of these potential activators was measured with the SirT1 truncations (see 3.2.1) and with ySir2 in the MAL deacetylation assay.

Figure 3.34: Influence of compound 1 on SirT1 truncations

The influence of compound 1 on SirT1 truncations was determined by MAL deacetylation assay. The control reaction containing DMSO was set to 100 %. IC₅₀ values and curve fitting were performed using GraphPad.

Values correspond to average enzyme activity of a 3-fold determination ± standard deviation. The MAL deacetylation assays were performed by Louisa Hill (Hill 2012).

Surprisingly, the shorter constructs SirT1_D, SirT1_E as well as SirT1_F were inhibited by compound 1 (Figure 3.34). These results indicated that the N-terminal region of SirT1 had an

influence of the enhancement of the SirT1 activity. Furthermore, compound 1 may be a more potent inhibitor when SirT enzymes with a shortening N-terminal region were used.

Based on these results from Louisa Hill, we measured the effect of compound 1 on the activity of ySir2 as well as of SirT2 in the MAL deacetylation assay, because both have a relatively short N-terminal region in comparison to SirT1. Notably, compound 1 inhibited both enzymes in the MAL-deacetylation assay (Figure 3.35A) with an IC50 value of 58.2 µM for ySir2 and 88.2 µM for SirT2. Additionally, SirT2 was also inhibited in the HPLC-based p53 deacetylation assay (Figure 3.35B), where a lower concentration was required to inhibit 50 % of SirT2 activity than the IC50 value of the MAL deacetylation assay.

Figure 3.35: Inhibitory effect of compound 1 on SirT2 and yeast Sir2

(A) The inhibitory effect of compound 1 on SirT2 and yeast Sir2 activity was measured by fluorescence-based assay. IC₅₀ curve fitting was performed using GraphPad. (B) The inhibitory effect of 50 µM, 100 µM and 200 µM of compound 1 on SirT2 was analyzed by HPLC-based assay and displayed as % of SirT2 activity. In both assays (A and B) the control reaction containing DMSO was set to 100 %. Values correspond to the average enzyme activity of three independent experiments ± standard deviation.

These results suggested that compound 1 might be a more potent inhibitor of SirT enzymes with a short N-terminal region independently of the deacetylation substrate.

The IC50 value of compound 1 (58 µM) for ySir2 was comparable with the IC50 value of splitomicin (60 µM; (Bedalov et al. 2001)). For this reason we performed HM silencing assay as described in 3.3.5. However no inhibition of ySir2-dependent HM silencing was observed.

This indicated that compound 1 was not able to inhibit ySir2 in vivo, or its in vivo influence was insufficient to observe an effect.

A B

4. Discussion

Sirtuin family members, homologs of the Silent Information Regulator Two (Sir2) protein from yeast, are best known as NAD⁺-dependent deacetylases, and some family members possess ribosyltransferase, demalonylase and desuccinylase activities (Frye 1999; Landry et al. 2000; Du et al. 2011). They are conserved from bacteria to humans and target both histones and a broad range of non-histone proteins. The best and most extensively studied sirtuin is SirT1 that deacetylates a number of important transcription factors such as p53, FOXO family members, PGC1-α and many more. Therefore, SirT1 is involved in a variety of biological processes including cell survival, apoptosis, cancer development and stress resistance (reviewed in (McGuinness et al. 2011)). Based on these roles of sirtuins, small molecules that influence the activity of sirtuins are deemed promising candidates for therapies of a variety of different diseases. So far, a variety of modulators exist that inhibit or activate sirtuins, but most of them have a low bioavailability, interact with multiple targets or have a high IC50 value. Furthermore, many details of the sirtuin reaction mechanism and regulation are still unclear. The SirT5/suramin complex has provided important insights into the inhibitory mechanism of suramin (Schuetz et al. 2007), but this to date is the only sirtuin structure bound to an inhibitor, which limits our knowledge about sirtuins, especially considering that many other sirtuin inhibitors with greater selectivity have been identified (Yuan and Marmorstein 2012). Additionally, the effect of putative activators of sirtuins (Howitz et al. 2003; Milne et al. 2007) have been shown to be substrate and assay dependent (Borra et al. 2004; Kaeberlein et al. 2005; Beher et al. 2009; Pacholec et al. 2010). In this study, we have identified inhibitors of SirT1 as well as SirT2 that can be developed further for anticancer therapy.