Different strategies to target the epigenome

Despite the overriding importance of the role covers by the epigenetic modifications in the control of several cellular processes such as differentiation and development, dysfunctional gene regulation and relative expression is responsible for the onset of many human diseases, first of all cancer. On these bases, the modulation of epigenetic processes is presently and strongly considered an innovative and challenging therapeutic strategy. SIRT4 is one of the three mitochondrial sirtuins and exhibits mainly ADP-ribosyltransferase activity but also deacylase, lipoamidase properties with which affects different targets. Given these recent evidences the emerging role of such human mitochondrial sirtuin generated considerable interest, because could offer new therapeutic opportunities in various disorders such as obesity, diabetes, metabolic syndrome, cardiac hypertrophy and, although the role of SIRT4 is entirely in discussion in this disease, in cancer. No specific and potent SIRT4 inhibitors (SIRT4i) have been reported so far. Starting from a docking screen and a homology model of SIRT4 , the research group of our collaborator prof. Sippl (Martin Luther Universitat of Halle Wittenberg, Germany), identified two inhibitor scaffolds and relative hit candidates: UBCS191 (IC50(SIRT4)= 66.7 µM) and UBCS178 (IC50 (SIRT4)= 45.6 µM). Prof. Steegborn and coworkers (University of Bayreuth, Germany), instead, highlighted the SIRT4 capability to recognize and remove the 3-hydroxy-3-methylglutaric (HMG) residue if linked to the -amino group of a lysine (in addition to the other already accepted enzymatic activities). Since SIRT4 shows little to no detectable deacetylase activity against acetylated histone in vitro, the synthesis of a substrate was needed in order to perform enzymatic assays to evaluate the potential inhibitory capability of the developed SIRT4i. Exploiting the new discovery, we designed an analog of the SIRT1-3 substrate Z-MAL (Z-Lys (Acetyl) AMC) which, instead of being characterized by an acetyl moiety linked to the ε-amino group of lysine, has the HMG residue (Z-Lys (HMG) AMC), toward which, indeed, such mitochondrial sirtuin shows a new catalytic activity. In addition, we were able to develop analogues of both the two main scaffolds identified by Prof. Sippl as well as a series of hybrid compounds between them as a first series of SIRT4i. Beyond the direct inhibition of a specific (epigenetic-)protein of interest ((e-)POI), another approach could be to reduce the levels of the latter by promoting its degradation. This can be reached through Protein-Targeting Chimeras knows as PROTACs. PROTACs molecules are composed of two portions connected with a linker that combine an E3 ligase recognition sequence with a moiety that targets the selected (e-)POI. The key aspect of the mechanism of action of PROTACs provides the selective induction of the degradation of its target protein at sub-stoichiometric concentrations through the recruitment of the ubiquitin-proteasome system (UPS), thus modulating the targeted protein's levels instead of its function To date, about 600 different types of E3 ligases have been identified, which differ in terms of their characteristics and specificity. In this framework, only degraders of bromodomains and BET family members, PCAF and GCN5, SIRT2 and HDAC are reported to literature so far. 34 novel Epi-PROTACs targeting different epigenetic targets in the category of “erasers” (such as LSD1 and JmjC KDMs) and “writers” (such as p300 and EZH2) characterized by different E3 ligase binders, pegylated and non-pegylated linkers and by already known epigenetic modulators to provide the degradation of LSD1, JmJC KDMs, p300 and EZH2 have been developed.