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Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

ABSTRACT: Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties.

SUBMITTER: Saccoccia F

PROVIDER: S-EPMC9486128 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

Saccoccia Fulvio F Pozzetti Luca L Gimmelli Roberto R Butini Stefania S Guidi Alessandra A Papoff Giuliana G Giannaccari Marialaura M Brogi Simone S Scognamiglio Viviana V Gemma Sandra S Ruberti Giovina G Campiani Giuseppe G

The Journal of biological chemistry 20220813 10

Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of ...[more]

PMID: 35970392

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Project description:BackgroundSchistosoma mansoni histone deacetylase 8 (SmHDAC8) has elicited considerable interest as a target for drug discovery. Invalidation of its transcripts by RNAi leads to impaired survival of the worms in infected mice and its inhibition causes cell apoptosis and death. To determine why it is a promising therapeutic target the study of the currently unknown cellular signaling pathways involving this enzyme is essential. Protein partners of SmHDAC8 were previously identified by yeast two-hybrid (Y2H) cDNA library screening and by mass spectrometry (MS) analysis. Among these partners we characterized SmRho1, the schistosome orthologue of human RhoA GTPase, which is involved in the regulation of the cytoskeleton. In this work, we validated the interaction between SmHDAC8 and SmRho1 and explored the role of the lysine deacetylase in cytoskeletal regulation.Methodology/principal findingsWe characterized two isoforms of SmRho1, SmRho1.1 and SmRho1.2. Co- immunoprecipitation (Co-IP)/Mass Spectrometry (MS) analysis identified SmRho1 partner proteins and we used two heterologous expression systems (Y2H assay and Xenopus laevis oocytes) to study interactions between SmHDAC8 and SmRho1 isoforms. To confirm SmHDAC8 and SmRho1 interaction in adult worms and schistosomula, we performed Co-IP experiments and additionally demonstrated SmRho1 acetylation using a Nano LC-MS/MS approach. A major impact of SmHDAC8 in cytoskeleton organization was documented by treating adult worms and schistosomula with a selective SmHDAC8 inhibitor or using RNAi followed by confocal microscopy.Conclusions/significanceOur results suggest that SmHDAC8 is involved in cytoskeleton organization via its interaction with the SmRho1.1 isoform. The SmRho1.2 isoform failed to interact with SmHDAC8, but did specifically interact with SmDia suggesting the existence of two distinct signaling pathways regulating S. mansoni cytoskeleton organization via the two SmRho1 isoforms. A specific interaction between SmHDAC8 and the C-terminal moiety of SmRho1.1 was demonstrated, and we showed that SmRho1 is acetylated on K136. SmHDAC8 inhibition or knockdown using RNAi caused extensive disruption of schistosomula actin cytoskeleton.

Project description:BackgroundSchistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known.Methodology/principal findingsTSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality.Conclusions/significanceGenome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression of dozens of histone reader genes involved in regulation of the epigenetic program in S. mansoni can be used as a starting point to look for possible novel schistosomicidal targets.

Project description:Hydroxamate, as a zinc-binding group (ZBG), prevails in the design of histone deacetylase 6(HDAC6) inhibitors due to its remarkable zinc-chelating capability. However, hydroxamate-associated genotoxicity and mutagenicity have limited the widespread application of corresponding HDAC6 inhibitors in the treatment of human diseases. To avoid such side effects, researchers are searching for novel ZBGs that may be used for the synthesis of HDAC6 inhibitors. In this study, a series of stereoisomeric compounds were designed and synthesized to discover non-hydroxamate HDAC6 inhibitors using α-amino amide as zinc-ion-chelating groups, along with a pair of enantiomeric isomers with inverted L-shaped vertical structure as cap structures. The anti-proliferative activities were determined against HL-60, Hela, and RPMI 8226 cells, and 7a and its stereoisomer 13a exhibited excellent activities against Hela cells with IC50 = 0.31 µM and IC50 = 5.19 µM, respectively. Interestingly, there is a significant difference between the two stereoisomers. Moreover, an evaluation of cytotoxicity toward human normal liver cells HL-7702 indicated its safety for normal cells. X-ray single crystal diffraction was employed to increase insights into molecule structure and activities. It was found that the carbonyl of the amide bond is on the different side from the amino and pyridine nitrogen atoms. To identify possible protein targets to clarify the mechanism of action and biological activity of 7a, a small-scale virtual screen using reverse docking for HDAC isoforms (1-10) was performed and the results showed that HDAC6 was the best receptor for 7a, suggesting that HDAC6 may be a potential target for 7a. The interaction pattern analysis showed that the α-amino amide moiety of 7a coordinated with the zinc ion of HDAC6 in a bidentate chelate manner, which is similar to the chelation pattern of hydroxamic acid. Finally, the molecular dynamics simulation approaches were used to assess the docked complex's conformational stability. In this work, we identified 7a as a potential HDAC6 inhibitor and provide some references for the discovery of non-hydroxamic acid HDAC6 inhibitors.

Dataset Information

Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

Publications

Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

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