Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

Abstract

Sirtuins are NAD + -dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC 50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.

Galectin-3, histone deacetylases, and Hedgehog signaling: Possible convergent targets in schistosomiasis-induced liver fibrosis

“…During its life cycle, S . mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications, including the effects of hSirt2 inhibitors, recently considered a new antiparasite target [ 63 ]. On this basis, a Schistosome Epigenetics Consortium (targets, regulation, and new drugs) was launched to study the potential utility of HDACs in the treatment of adult schistosomiasis and to develop alternate treatments for prazinquantel-resistant Schistosoma.…”

The Current State of NAD+‐Dependent Histone Deacetylases (Sirtuins) as Novel Therapeutic Targets

“…) was reported as a weakly selective Sirt1 inhibitor with an IC 50 value in the low micromolar range . In a focused library screening MS3 was also identified as an inhibitor of the schistosomal lysine deacylase smSirt2 in vitro . Moreover, Maurer et al.…”

Epigenetics in Schistosomes: What We Know and What We Need Know

“…This result suggests that inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development and that HATs can be used as a drug target against schistosomiasis. Regarding HDAC, the NAD + -dependent HDAC, smSirt2 in S. mansoni was identified using fluorescence-based screening assays (Schiedel et al, 2015). The class I HDAC s, SmHDAC1, 3, and 8, were identified in S. mansoni (Oger et al, 2008; Marek et al, 2013; Singh and Pandey, 2015).…”

“…All S. mansoni sirtuins have been expressed throughout the parasitic life cycle and characterized (Lancelot et al, 2013). Initial experiments in adult worms and schistosomula have demonstrated strong effects of hSirt2 inhibitors on both life span and reproduction (Lancelot et al, 2013), and Schiedel et al found that the smSirt2 IC50 was less than 50 μM (Schiedel et al, 2015). These results suggest that schistosome sirtuins could be potential therapeutic targets and validate screening for selective sirtuin inhibitors as a strategy for the development of new drugs against schistosomiasis.…”