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Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin's lymphoma characterized by poor prognosis. The complexity of MCL pathogenesis arises from aberrant activities of diverse signaling pathways, including BTK, PI3K-AKT-mTOR and MYC-BRD4. Here, we report that MCL-related signaling pathways can be altered by a single small molecule inhibitor, SRX3305. Binding and kinase activities along with resonance changes in NMR experiments reveal that SRX3305 targets both bromodomains of BRD4 and is highly potent in inhibition of the PI3K isoforms α, γ and δ, as well as BTK and the drug-resistant BTK mutant. Preclinical investigations herein reveal that SRX3305 perturbs the cell cycle, promotes apoptosis in MCL cell lines and shows dose dependent anti-proliferative activity in both MCL and drug-resistant MCL cells. Our findings underscore the effectiveness of novel multi-action small molecule inhibitors for potential treatment of MCL., (© 2022. The Author(s).)

The past two decades have witnessed rapid advances in the identification and characterization of epigenetic readers, capable of recognizing or reading post-translational modifications in histones. More recently, a new set of readers with the ability to interact with the nucleosome through concomitant binding to histones and DNA has emerged. In this review, we discuss mechanistic insights underlying bivalent histone and DNA recognition by newly characterized readers and highlight the importance of binding to DNA for their association with chromatin., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Methylation of lysine residues plays crucial roles in a wide variety of cell signaling processes. While the biological importance of recognition of methylated histones by reader domains in the cell nucleus is well established, the processes associated with methylation of non-histone proteins, particularly in the cytoplasm of the cell, are not well understood. Here, we describe a search for potential methyllysine readers using a rapid structural motif-mining algorithm Erebus, the PDB database, and knowledge of the methyllysine binding mechanisms., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma and one of the most challenging blood cancers to combat due to frequent relapse after treatment. Here, we developed the first-in-class BTK/PI3K/BRD4 axis inhibitor SRX3262, which simultaneously blocks three interrelated MCL driver pathways - BTK, PI3K-AKT-mTOR and MYC. SRX3262 concomitantly binds to BTK, PI3K, and BRD4, exhibits potent in vitro and in vivo activity against MCL, and overcomes the Ibrutinib resistance resulting from the BTK-C481S mutation. Our results reveal that SRX3262 inhibits IgM-induced BTK and AKT phosphorylation and abrogates binding of BRD4 to MYC loci. SRX3262 promotes c-MYC destabilization, induces cell cycle arrest and apoptosis, and shows antitumor activity in in vivo xenograft models. Together, our study provides mechanistic insights and rationale for the use of the triple BTK/PI3K/BRD4 activity inhibitors as a new approach to treat MCL., Competing Interests: D.L.D. and G.A.M. are consultants for and/or have equity interests in SignalRx Pharmaceuticals, Inc., have financial conflicts of interest regarding the SRX3239, SRX3240 and SRX3262 compounds and have a patent related to this work, patent number WO2020023340A1. T.G.K. is a member of the iScience Editorial Board. Other authors declare no competing interests., (© 2021.)

The extensive length, compaction, and interwound nature of DNA, together with its controlled and restricted movement in eukaryotic cells, create a number of topological issues that profoundly affect all of the functions of the genetic material. Topoisomerases are essential enzymes that modulate the topological structure of the double helix, including the regulation of DNA under- and overwinding and the removal of tangles and knots from the genome. Type II topoisomerases alter DNA topology by generating a transient double-stranded break in one DNA segment and allowing another segment to pass through the DNA gate. These enzymes are involved in a number of critical nuclear processes in eukaryotic cells, such as DNA replication, transcription, and recombination, and are required for proper chromosome structure and segregation. However, because type II topoisomerases generate double-stranded breaks in the genetic material, they also are intrinsically dangerous enzymes that have the capacity to fragment the genome. As a result of this dualistic nature, type II topoisomerases are the targets for a number of widely prescribed anticancer drugs. This article will describe the structure and catalytic mechanism of eukaryotic type II topoisomerases and will go on to discuss the actions of topoisomerase II poisons, which are compounds that stabilize DNA breaks generated by the type II enzyme and convert these essential enzymes into "molecular scissors." Topoisomerase II poisons represent a broad range of structural classes and include anticancer drugs, dietary components, and environmental chemicals.

Human ubiquitin ligase HERC2, a component of the DNA repair machinery, has been linked to neurological diseases and cancer. Here, we show that the ZZ domain of HERC2 (HERC2 ZZ ) binds to histone H3 tail and tolerates posttranslational modifications commonly present in H3. The crystal structure of the HERC2 ZZ :H3 complex provides the molecular basis for this interaction and highlights a critical role of the negatively charged site of HERC2 ZZ in capturing of A1 of H3. NMR, mutagenesis, and fluorescence data reveal that HERC2 ZZ binds to H3 and the N-terminal tail of SUMO1, a previously reported ligand of HERC2 ZZ , with comparable affinities. Like H3, the N-terminal tail of SUMO1 occupies the same negatively charged site of HERC2 ZZ in the crystal structure of the complex, although in contrast to H3 it adopts an α-helical conformation. Our data suggest that HERC2 ZZ may play a role in mediating the association of HERC2 with chromatin., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Since its discovery, several ligands of the ZZ domain have been identified; however, molecular and structural information underlying binding of these ligands remains limited. Here, we describe a protocol for biochemical and structural analysis of the ZZ domain of human E3 ubiquitin ligase HERC2 (HERC2 ZZ ) and its interaction with its ligands: the N-terminal tails of histone H3 and SUMO1. This methodology could be applied for characterization of binding activities of other histone readers. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Competing Interests: Conflict of interestD.L.D. and G.A.M. acknowledge financial interest in SignalRx Pharmaceuticals, Inc.

Development of small molecule compounds that target several cancer drivers has shown great therapeutic potential. Here, we developed a new generation of highly potent thienopyranone (TP)-based inhibitors for the BET bromodomains (BDs) of the transcriptional regulator BRD4 that have the ability to simultaneously bind to phosphatidylinositol-3 kinase (PI3K) and/or cyclin-dependent kinases 4/6 (CDK4/6). Analysis of the crystal structures of the complexes, NMR titration experiments and IC 50 measurements reveal the molecular basis underlying the inhibitory effects and selectivity of these compounds toward BDs of BRD4. The inhibitors show robust cytotoxic effects in multiple cancer cell lines and induce cell-cycle arrest and apoptosis. We further demonstrate that concurrent disruption of the acetyllysine binding function of BRD4 and the kinase activities of PI3K and CDK4/6 by the TP inhibitor improves efficacy in several cancer models. Together, these findings provide further compelling evidence that these multi-action inhibitors are efficacious and more potent than single inhibitory chemotypes.

Chromosomal NUP98-PHF23 translocation is associated with an aggressive form of acute myeloid leukemia (AML) and poor survival rate. Here, we report the molecular mechanisms by which NUP98-PHF23 recognizes the histone mark H3K4me3 and is inhibited by small molecule compounds, including disulfiram that directly targets the PHD finger of PHF23 (PHF23PHD). Our data support a critical role for the PHD fingers of NUP98-PHF23, and related NUP98-KDM5A and NUP98-BPTF fusions in driving leukemogenesis, and demonstrate that blocking this interaction in NUP98-PHF23 expressing AML cells leads to cell death through necrotic and late apoptosis pathways. An overlap of NUP98-KDM5A oncoprotein binding sites and H3K4me3-positive loci at the Hoxa/b gene clusters and Meis1 in ChIP-seq, together with NMR analysis of the H3K4me3-binding sites of the PHD fingers from PHF23, KDM5A and BPTF, suggests a common PHD finger-dependent mechanism that promotes leukemogenesis by this type of NUP98 fusions. Our findings highlight the direct correlation between the abilities of NUP98-PHD finger fusion chimeras to associate with H3K4me3-enriched chromatin and leukemic transformation.

Microrchidia 3 (MORC3), a human ATPase linked to several autoimmune disorders, has been characterized both as a negative and positive regulator of influenza A virus. Here, we report that the CW domain of MORC3 (MORC3-CW) is targeted by the C-terminal tail of the influenza H3N2 protein NS1. The crystal structure of the MORC3-CW:NS1 complex shows that NS1 occupies the same binding site in CW that is normally occupied by histone H3, a physiological ligand of MORC3-CW. Comparable binding affinities of MORC3-CW to H3 and NS1 peptides and to the adjacent catalytic ATPase domain suggest that the viral protein can compete with the host histone for the association with CW, releasing MORC3 autoinhibition and activating the catalytic function of MORC3. Our structural, biochemical, and cellular analyses suggest that MORC3 might affect the infectivity of influenza virus and therefore has a role in cell immune response., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The YEATS domain has been identified as a reader of histone acylation and more recently emerged as a promising anti-cancer therapeutic target. Here, we detail the structural mechanisms for π-π-π stacking involving the YEATS domains of yeast Taf14 and human AF9 and acylated histone H3 peptides and explore DNA-binding activities of these domains. Taf14-YEATS selects for crotonyllysine, forming π stacking with both the crotonyl amide and the alkene moiety, whereas AF9-YEATS exhibits comparable affinities to saturated and unsaturated acyllysines, engaging them through π stacking with the acyl amide. Importantly, AF9-YEATS is capable of binding to DNA, whereas Taf14-YEATS is not. Using a structure-guided approach, we engineered a mutant of Taf14-YEATS that engages crotonyllysine through the aromatic-aliphatic-aromatic π stacking and shows high selectivity for the crotonyl H3K9 modification. Our findings shed light on the molecular principles underlying recognition of acyllysine marks and reveal a previously unidentified DNA-binding activity of AF9-YEATS.

Etoposide is an anticancer drug that acts by inducing topoisomerase II-mediated DNA cleavage. Despite its wide use, etoposide is associated with some very serious side-effects including the development of treatment-related acute myelogenous leukemias. Etoposide targets both human topoisomerase IIα and IIβ. However, the contributions of the two enzyme isoforms to the therapeutic vs. leukemogenic properties of the drug are unclear. In order to develop an etoposide-based drug with specificity for cancer cells that express an active polyamine transport system, the sugar moiety of the drug has been replaced with a polyamine tail. To analyze the effects of this substitution on the specificity of hybrid molecules toward the two enzyme isoforms, we analyzed the activity of a series of etoposide-polyamine hybrids toward human topoisomerase IIα and IIβ. All of the compounds displayed an ability to induce enzyme-mediated DNA cleavage that was comparable to or higher than that of etoposide. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward topoisomerase IIβ than IIα. Modeling studies suggest that the enhanced specificity may result from interactions with Gln778 in topoisomerase IIβ. The corresponding residue in the α isoform is a methionine., (Published by Elsevier Ltd.)

Polycomb repressive complex 2 (PRC2) is a chief epigenetic regulator. In a new article, Chen et al. describe the crystal structure of the heterotetrameric PRC2 holo complex, which provides important mechanistic insights into the organization of its subunits and the association of PRC2 with chromatin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Yaf9 is an integral part of the NuA4 acetyltransferase and the SWR1 chromatin remodeling complexes. Here, we show that Yaf9 associates with acetylated histone H3 with high preference for H3K27ac. The crystal structure of the Yaf9 YEATS domain bound to the H3K27ac peptide reveals that the sequence C-terminal to K27ac stabilizes the complex. The side chain of K27ac inserts between two aromatic residues, mutation of which abrogates the interaction in vitro and leads in vivo to phenotypes similar to YAF9 deletion, including loss of SWR1-dependent incorporation of variant histone H2A.Z. Our findings reveal the molecular basis for the recognition of H3K27ac by a YEATS reader and underscore the importance of this interaction in mediating Yaf9 function within the NuA4 and SWR1 complexes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Epigenetic marks, including DNA methylation and posttranslational modifications (PTMs) in histones, are important factors in determining the fate of replicating cells. In this issue of Molecular Cell, Ishiyama et al. (2017) reveal yet another layer in a remarkably complex mechanism of maintenance DNA methylation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mitosis in metazoans is characterized by abundant phosphorylation of histone H3 and involves the recruitment of condensin complexes to chromatin. The relationship between the 2 phenomena and their respective contributions to chromosome condensation in vivo remain poorly understood. Recent studies have shown that H3T3 phosphorylation decreases binding of histone readers to methylated H3K4 in vitro and is essential to displace the corresponding proteins from mitotic chromatin in vivo. Together with previous observations, these data provide further evidence for a role of mitotic histone H3 phosphorylation in blocking transcriptional programs or preserving the 'memory' PTMs. Mitotic protein exclusion can also have a role in depopulating the chromatin template for subsequent condensin loading. H3 phosphorylation thus serves as an integral step in the condensation of chromosome arms.

Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is an antineoplastic agent that intercalates into DNA and alters topoisomerase II activity. Unfortunately, this compound displays a number of adverse properties. Therefore, to investigate new ellipticine-based compounds for their potential as topoisomerase II-targeted drugs, we synthesized two novel derivatives, N-methyl-5-demethyl ellipticine (ET-1) and 2-methyl-N-methyl-5-demethyl ellipticinium iodide (ET-2). As determined by DNA decatenation and cleavage assays, ET-1 and ET-2 act as catalytic inhibitors of human topoisomerase IIα and are both more potent than the parent compound. Neither compound impairs the ability of the type II enzyme to bind its DNA substrate. Finally, the potency of ET-1 and ET-2 as catalytic inhibitors of topoisomerase IIα appears to be related to their ability to intercalate into the double helix., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Two metabolites from the ascomycete fungus Septofusidium berolinense were recently identified as having antineoplastic activity [Ekiz et al. (2015) J. Antibiot. , DOI: 10.1038/ja.2015.84]. However, the basis for this activity is not known. One of the compounds [3,6-dihydroxy-2-propylbenzaldehyde (GE-1)] is a hydroquinone, and the other [2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2)] is a quinone. Because some hydroquinones and quinones act as topoisomerase II poisons, the effects of GE-1 and GE-2 on DNA cleavage mediated by human topoisomerase IIα were assessed. GE-2 enhanced DNA cleavage ∼4-fold and induced scission with a site specificity similar to that of the anticancer drug etoposide. Similar to other quinone-based topoisomerase II poisons, GE-2 displayed several hallmark characteristics of covalent topoisomerase II poisons, including (1) the inability to poison a topoisomerase IIα construct that lacks the N-terminal domain, (2) the inhibition of DNA cleavage when the compound was incubated with the enzyme prior to the addition of plasmid, and (3) the loss of poisoning activity in the presence of a reducing agent. In contrast to GE-2, GE-1 did not enhance DNA cleavage mediated by topoisomerase IIα except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. These results suggest that topoisomerase IIα may play a role in mediating the cytotoxic effects of these fungal metabolites.

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10-100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption.