Computational and Structural Biotechnology Journal 13 (2015) 358–365

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Mini Review

Computer-aided Molecular Design of Compounds Targeting HistoneModifying Enzymes

Federico Andreoli a, Alberto Del Rio a,b,⁎a Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Alma Mater Studiorum, University of Bologna, Via S. Giacomo 14, 40126 Bologna, Italyb Institute of Organic Synthesis and Photoreactivity, National Research Council, Via P. Gobetti, 101 40129 Bologna, Italy

⁎ Corresponding author at: Institute of Organic SynthesResearch Council, Via P. Gobetti, 101 40129 Bologna,fax: +39 051 6398349.

E-mail addresses: alberto.delrio@gmail.com, alberto.de

http://dx.doi.org/10.1016/j.csbj.2015.04.0072001-0370/© 2015 Andreoli, Del Rio. Published by ElsevieCC BY license (http://creativecommons.org/licenses/by/4.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 October 2014Received in revised form 24 April 2015Accepted 30 April 2015Available online 7 May 2015

Keywords:EpigeneticsPost-translational modificationsHistoneComputer-aided molecular designDrug designDrug discovery

Growing evidences show that epigenetic mechanisms play crucial roles in the genesis and progression of manyphysiopathological processes. As a result, research in epigenetic grew at a fast pace in the last decade. In partic-ular, the study of histone post-translational modifications encountered an extraordinary progression and manymodifications have been characterized and associated to fundamental biological processes and pathological con-ditions. Histonemodifications are the catalytic result of a large set of enzyme families that operate covalent mod-ifications on specific residues at the histone tails. Taken together, these modifications elicit a complex andconcerted processing that greatly contribute to the chromatin remodeling and may drive different pathologicalconditions, especially cancer. For this reason, several epigenetic targets are currently under validation for drugdiscovery purposes and different academic and industrial programs have been already launched to producethe first pre-clinical and clinical outcomes. In this scenario, computer-aided molecular design techniques are of-fering important tools, mainly as a consequence of the increasing structural information available for these tar-gets. In this mini-review we will briefly discuss the most common types of known histone modifications andthe corresponding operating enzymes by emphasizing the computer-aided molecular design approaches thatcan be of use to speed-up the efforts to generate new pharmaceutically relevant compounds.© 2015 Andreoli, Del Rio. Published by Elsevier B.V. on behalf of the Research Network of Computational and

Structural Biotechnology. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3582. Type of Histone Modifications and Their Biological and Clinical Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3593. Chemical Mechanisms of Histone Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3604. Molecular Design Techniques Applied to Histone Modifying Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3615. Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

1. Introduction

In the last decades, major efforts have been done in genetics thoughit is evident that unraveling complex biological mechanisms like cancer

is and Photoreactivity, NationalItaly. Tel.: +39 051 6398308;

lrio@isof.cnr.it (A. Del Rio).

r B.V. on behalf of the Research Netw0/).

requires a larger framework of research that recently evolved towardthe better understanding of the immune system regulation [1], the re-discovery of metabolism [2] and, importantly, the role of epigenetics[3]. The term epigenetics refers to themechanisms of temporal and spa-tial control of gene activity that do not entail modification of the DNAsequence but influence the physiological and pathological developmentof an organism. Themolecularmechanisms bywhich epigenetic chang-es occur are complex and cover awide range of processes [4]. Epigeneticmechanisms can occur at biochemical level in three different ways:

ork of Computational and Structural Biotechnology. This is an open access article under the

359F. Andreoli, A. Del Rio / Computational and Structural Biotechnology Journal 13 (2015) 358–365

i) through histone post-translational modifications (PTMs), which willbe the object of this mini-review, as well as the molecular recognitionof non-catalytic readers of histones [5], ii) through the DNA methyla-tion, i.e. the methylation of cytosines to 5-methylcytosines, which isthe object of recent reviews [6–8], and iii) through regulation of geneexpression by non-coding RNA (ncRNA), which is also an emergingtopic of research, covered by recent reviews [9–11]. All these processescontribute to define the epigenetic mechanisms by which gene expres-sion is activated or silenced [12–16].

Post-translational modifications of histones occur at the N-terminaltails of the protein chains and consist in covalent modifications thatare catalyzed by different classes of enzymes [17,18]. The ensemble ofthesemodifications is commonly referred as to be the histone code refer-ring to the idea that all histone PTMs determine the activity state of anunderlying gene [19]. One of the hallmarks of the histone code is thatit can be positively or negatively correlatedwith specific transcriptionalstates or organization of chromatin [20–23]. This is accomplishedthrough a fine regulation of histone PTMs controlled by an enzymaticmachinery, which existence and function have been elucidated partly,but with an extraordinary progression in the last years [23–29]. Impor-tantly, further understanding of epigenetic phenomena occurring onhistone proteins is critical to shed light on biological processes that areprogressively translating into the development of new medical options[29–31]. In this direction, different studies have highlighted how thehistone alterations contribute to the onset and growth of a variety ofcancers [7,23,24,27,32–41], amongother pathologies. Consequently, en-zymes operating PTMs on histones are constituting attractive therapeu-tic targets for the development of new therapies [13,31,42–44]. Itshould be noted that, while the resulting effects on chromatin collec-tively depend on the ensemble of histone PTMs, these are operated byprecise variations of physicochemical properties that we recentlyreviewed [17]. For these reasons, large efforts from both academic andindustrial settings have been dedicated in the last year to identify andevaluate new biologically active compounds against histone modifyingenzymes. Fuelled by the increasing availability of structural information,several endeavors have been initiated and helped by the usage ofcomputer-aided molecular design techniques. Thus, in this mini-review, we aim to highlight the aspects relating histone modificationsin the light of the future applications of computational techniques tothe research of new probe or lead-like epigenetic modulators.

2. Type of Histone Modifications and Their Biological andClinical Relevance

To understand the relevance of computational techniques inhistone-related epigenetic targets, it is important to highlightthat these post-translational modifications are functionalizations/defunctionalizations of specific residues, which are lysine, arginine, ser-ine, threonine, histidine, tyrosine, cysteine and glutamic acid, located atthe N-terminal tails of each chain. Fig. 1 summarizes all the most com-mon PTMs that can occur on histones. By far, lysine represents the res-idues with most chemical versatility, as it is capable to undergo severalkinds and grades of modifications. Consequently, histone methyltrans-ferases, demethylases, acetyltransferases and deacetylases have beenrecently ascribed an important role as new classes of biological targetsfor drug discovery [18,45–49]. Arginine represents also a residue thatis modified by enzymes recognized for drug development, in particularhistone methyltransferases. Differently to these previous cases, en-zymes thatmodify histone serines, threonines, histidines, tyrosines, cys-teines and glutamic acids have not been exploited yet for the discoveryof newmodulating compounds. Nevertheless, it is expected that furtherelucidation of their biological role and protein structure will spur suchendeavors. It is worth to note that other kinds of modifications likepropionylation, butyrylation, crotonylation, 2-hydroxyisobutyrylationhave been reported [50].

Different studies elucidate the impact that PTMs have on chromatinand their relevance in human physiology and pathology [16,18,25,26,31,51–57]. Interestingly, their biological role greatly varies, dependingon the kind of modification. Therefore, for instance, the acetylation ap-pears to be the most promiscuous histone modification and is alwaysassociated to transcriptional activity. Conversely, histone methylationhas a high degree of selectivity toward specific histone residues andcan be associated with both repression and transcription [58,59]. In ad-dition PTMs can “cross-talk”, meaning that modifications can occur in aconcerted or a subsequent manner [25,60–63].

In the last decade, epigenetic modifications of histones have beenmainly studied in the context of cancer, particularly histone deacetylasesof classes I, II and IV (HDACs) [64]. Indeed, abnormal activity of the en-zymes responsible for deacetylation of histones, modification that altersthe chromatin structure repressing transcription, has been shown to beimplicated in several diseases [18,65]. Because of these compelling evi-dences, HDACs have been recognized as consolidated drug target, in par-ticular for breast cancer, colorectal cancer, leukemia, lymphoma, ovarianand prostate cancer. In addition, another class of histone deacetylasesnamed sirtuins (or class III deacetylases), which uses NAD+ to catalyzethe removal of an acetyl group, also came into the light as new therapeutictargets [66,67]. This family of enzymes, in fact,was found to be involved inrelevant physiological and pathological processes, as well as in aging-related disorders, metabolic and inflammatory conditions and processesinvolving DNA regulation and integrity, including cancer [68–71]. Inter-estingly, also the correspondent families of enzymes that revert the cata-lytic activity of histone deacetylation, i.e. acetyltransferases (HAT), havealso met a great deal of interest. Two classes of HAT exist and consist ofenzymes able to acetylatemultiple sites in the histone tails and additionalsites on the globular histone core, for the first class, while the second classmostly consists of cytoplasmic enzymes able to acetylate newly synthe-sized histones prior to their deposition into chromatin. Abnormal regula-tion of HATs has been linked to leukemia and several studies connectingthem to prostate and gastric cancers have been realized [72,73].

The secondmost studied histone modification is methylation. As an-ticipated above, protein methyltransferases (PMT) emerged recently asnew important targets for cancer therapy, since they were found to beoverexpressed or repressed in several types of cancer, precisely in breastcancer, leukemia,myeloma, ovarian, prostate and kidney cancers. Sever-al recently published reviews describe mechanism and biological rolesof PMTs [6,41,45,46,74–81]. Indeed, due to their importance in differentpathological conditions, several drug discovery programs have beenlaunched in order to design specific compounds able to modulatethese targets [46]. Equally, the recent focus in understanding histonemethylation led to the characterization of histone demethylases(HDM). The first described protein has been the lysine specificdemethylase 1 (LSD1) [82]. Soon after, a new class of proteins havingdemethylase activity, the JMJC (Jumonji C) domain family, was discov-ered and characterized [83]. LSD and JMJC demethylases have been re-ported to be regulators of various cellular processes. Therefore, aspecial effort, as in the case of PMTs, is currently made, aimed to the dis-covery of small-molecule inhibitors with therapeutic potential [47,83].

Beside the above classes of enzymes, there is a mounting evidencethat also other type of modifications can constitute important paradigmin epigenetics and may underlie new biological target for therapeuticpurposes. For instance, some studies highlighted specific roles ofubiquitination [84–87], poly-ADP-ribosylation [88–96] and glycosyla-tion [61,97–101] to the epigenetic code. However, the therapeutic po-tential of these modifications still needs to be validated for thetherapeutic and drug discovery point of view. Equally, other modifica-tions like histone phosphorylation, citrullination (deamination) [102],biotinylation [103,104], tail clipping and proline isomerization [25],are still poorly understood and their role in human pathologies remainslargely unclear [25]. In the next paragraph,we aim to describe the state-of-the-art of these modifications, analyzing, from a chemical point ofview, their role on the dynamics of histone proteins.

Fig. 1.Most common type of post-translational modifications occurring on histone proteins.

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3. Chemical Mechanisms of Histone Modifications

The histone code collectively depends on the ensemble of post-translational modifications that is operated at the chromatin level, bysingle variations of physicochemical properties of the modified resi-dues. Consequently, the microenvironment and the biochemical differ-ences obtained by PTMs depend on the attachment or removal ofchemical functional groups, whose enzymes, mechanisms of actionand cofactors are overviewed in Table 1. We recently proposed that,from a chemical functionality point of view, histone PTMs can be divid-ed in two main groups [17]. The first group I encompassing PTMs lead-ing to the addition or the removal of monofunctional, generally small,organic substituents and a second group including polyfunctional, andin some case elaborate and large, organic molecules. Both groups arecomposed of writer or eraser enzymes, i.e. which add or remove fromhistone residues specific substituents.

Modifications of the first group are acetylation, methylation, phos-phorylation, deimination and palmitoylation (acylation). As seenabove, acetylation is the most common and studied PTM and consistsin the addition of an acetyl group on a lysine residue mediated by twomajor classes of histone acetyltransferases (HAT), Type-A (which

includes GNAT, p300/CBP, and MYST) and Type-B [18,65,72]. This func-tion can be removed by two categories of different catalytic activity en-zymes: classes I, II and IV histone deacetylases (HDAC) and class IIIdeacetylases, also known as sirtuins (SIRT) that work with a NAD+-dependent mechanism [105].

Methylation is the second most common PTM and lysine, as seenabove, is the residue that undergoes the widest number of methylationreactions. Indeed, lysine can bemono-, di-, or tri-methylated by histonemethyltransferases (HMT, also known as PKMT). HMTs can also mono-or di-methylate arginines and this class of enzymes is commonly re-ferred to as protein arginine methyltransferases (PRMT) [106]. Methylgroups can be erased only from lysines through the action of histonedemethylases (HDM or, in the case of lysines, KDM). Phosphorylationof histone has been observed on serine, threonine [107], tyrosine[107] and histidine [108] sites while phosphatases, which hydrolyzethe phosphoric monoesters or monoamides restoring the original resi-dues, represent the erasers of this histonemodification. Deimination oc-curs on arginines and methylarginines through the catalysis ofpeptidylarginine deiminases (PAD) [109].

Modifications of the second group are ubiquitylation, SUMOylation,biotinylation, glycosylation and ADP-ribosylation. While, in principle,

Table 1Mechanisms of histone modifications.

Histone modification Cofactor Leaving group Target residue Substituents Operating enzyme Type of enzyme

Acetylation Acetyl-CoA CoA Lys HAT WriterDeacetylation Zn2+, Fe2+, Co2+, Mn2+ CH3COO− Ac-Lys HDAC Eraser

NAD+ OAADPr Ac-Lys SIRT EraserPalmitoylation – – Cys – Writer

Methylation SAM SAH Arg, Lys PMT WriterDemethylation FAD HCHO Me-Lys LSD Eraser

Me2-LysFe2+, α-KG HCHO Me-Lys PHF8 Eraser

Me2-Lys JmjDMe3-Lys JHDM

Citrullination/deimination Ca2+ NH3 Arg PAD WriterMe-Arg

Phosphorylation ATP ADP Ser Kinase WriterThrTyrHis

Dephosphorylation – – Phos-Ser PhosphatasePhos-Thr

ADP-ribosylation NAD+ NAM Lys ADP-ribosyltransferase, SIRT4, SIRT6 WriterGlu

Glycosylation (O-GlcNAcylation) UDP-GlcNAc UDP Ser OGT WriterThr

Biotinylation – – Lys Biotin-protein ligase Writer

Ubiquitination ATP – Lys E1, E2, E3 proteins WriterDeubiquitination – – Ub-Lys Histone H2A deubiquitinase Eraser

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these modifications are catalyzed by writers and eraser enzymes, itis worth to note that decitrullination, depalmitoylation, deADP-ribosylation, debiotinylation and deglycosylation have not been de-scribed, yet. Mono- or poly-ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ART), clostridia-toxin-like (ARTC) or diphtheriatoxin-like (ARTD), which are most commonly known as PARP, andsome sirtuin isoforms (Table 1) [88,110,111]. Biotinylation and glyco-sylation are still poorly characterized and have been linked to the his-tone code recently [97,99,112,113]. Ubiquitylation takes place by theaction of E1, E2, and E3 enzymes,which catalyze the addition of anubiq-uitin (Ub) molecule to the target lysine via an isopeptide bond. The re-versal of this action is operated by deubiquitylating enzymes (DUB), sofar demonstrated to act only on histones H2A and H2B. SUMO (SmallUbiquitin-like MOdifier) proteins can be added on lysine residues in asimilar way.

4. Molecular Design Techniques Applied to HistoneModifying Enzymes

Computer-aided molecular techniques are widely used in academiaand industrial settings to assist the selection of new compounds that canmodulate biological targets. Several examples testify their successful ap-plications in the development of new chemical entities [114–116] and awide range of disciplines, including chemoinformatics, computationalchemistry, structural biology, biophysics, medicinal chemistry, organicchemistry and pharmacology, is at disposal of scientists working inthese fields. When applied to the discovery of compounds supposed tobecome future drugs, these techniques are commonly referred ascomputer-aided drug design (CADD) techniques. Certainly, amongthem, virtual screening acquired the greatest popularity due to its abil-ity to screen rapidly and cost-effectively large libraries of chemical com-pounds [117–119]. CADD techniques can be divided in two categories:ligand-based and structure-based drug design techniques (LBDD andSBDD), even if this classification is becoming nowadays loose, as severaltechniques offer technical advantages that are proper of both categories.

The first category usually takes advantage of the information of knownbioactive compounds (ligands), while the second usually exploit three-dimensional structure of the biological target (protein), in order to iden-tify new small-moleculemodulators of the protein activity. The growingavailability of protein structures resolved by X-ray crystallography orNMR technique has progressively raised the possibility to deploySBDD.However, ligand-based techniques still constitute useful tools, es-pecially when the structural information of a biological target is missingor when the molecular design effort is not directed toward a target-centric approach. Studies aimed at the modulation of cellular pathwaysor specific phenotypic traits without a precise knowledge of the mech-anism of action are representative of this case.

In the context of epigenetics, the research oriented toward the de-velopment of new therapeutically-relevant molecules has flourishedin the last years. Several works report rationales, targets, drugs, ap-proaches, compounds, tools and methodologies [32,120]. In addition,different reviews reporting the approaches for the discovery of newepidrugs or chemical probes for epigenetic targets have beenwritten re-cently [18,56,57,121,122]. An intriguing aspect in this field of research isthe fast pace aimed to extend the discovery of new compounds toepitargets that are currently not validated and that may offer new per-spective for the generation of new therapeutic agents. In this direction,particular aspect of epigenetics, for instance, the modulation of themicroRNA biogenesis pathway, have been recognized as new possibleway to achieve therapeutic target for human disease, in particular can-cer [9,123–125].

In all this framework of research, computational techniques areplaying a progressive role to help the identification of new molecularentities for epigenetic targets. Different techniques have been used toidentify new modulating compounds and to explain, mechanism of ac-tions, binding modes and protein dynamics [22,80,126–129]. Most ofthe research in this direction has beendone on classes I, II and IV histonedeacetylases. Indeed, a variety of quantitative structure–activity rela-tionship (QSAR) studies and computational works, elucidating retro-spectively protein dynamics, binding modes, binding affinities and

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selectivity among HDAC isoforms, have been published [130–139]. Forinstance, a recent and prospective work has been done to identify newinhibitors of SchistosomamansoniHDAC8bymeans of homologymodel-ing, molecular dynamics and molecular docking techniques [140]. It isinteresting to note that most of the prospective works have been ap-plied on other epigenetic targets than HDACs, reflecting the idea thatcomputational techniques are useful tool to help the identification ofnew biologically active compounds for investigational epigenetic tar-gets. In this direction two molecular docking studies have been appliedonHAT p300, resulting on the identification of two classes of new inhib-itors: benzothiazines and pyrazolone exomethylene vinyl compounds(1 and2, Fig. 2) [141,142]. Prospective high-throughput docking screen-ing and computational studies with molecular dynamic simulationswere performed on LSD1, leading to the identification of nanomolarN′-(1-phenylethylidene)-benzohydrazides (3, Fig. 2) and novel classesof short peptide inhibitors [143–146]. LSD1was also object of retrospec-tive studies based on virtual screening procedures, aiming at elucidatingligand selectivity with a closely related target, theMAO-B [147] and ex-tended molecular dynamic simulations were used to highlight new po-tential binding regions for drug-like molecules, peptides, proteinpartners and chromatin [144]. Docking studies were also applied toidentify new KDM4B inhibitors, which demonstrated an activity in thelowmicromolar range of concentration (4, Fig. 2), and Sirt1/Sirt2 inhib-itors, like thiobarbiturates (5, Fig. 2) [148,149].We also recently appliedthese techniques to the identification of new and selective inhibitors ofSirt6 (6, Fig. 2) [150]. Interestingly, pharmacophore techniques havebeen used less frequently than docking techniques on histone modify-ing enzymes. A successful applicative example of the first techniquehas been published by Sippl and colleagues for the identification ofnew PRMT1 inhibitors, which were found through a pharmacophore

Fig. 2. Representative molecular entities able to interfere with histone modifying enzymes. M(http://www.chemaxon.com).

hypothesis built on the basis of the PRMT1–allantodapsone interactionmodel (7, Fig. 2) [151].

Computational chemistry and protein modeling is also activelysupporting the basic research on epigenetic targets. A valuable exampleis the set of tools that are currently used to for detect protein plasticity,dynamics of catalytic sites, analysis of allosteric pockets and interactionswith other proteins that may constitute a useful way to study new pro-tein–protein inhibitors (PPI). Some recent reviews discussing these as-pects have been written recently on HDACs and other epigeneticplayers [152–154]. Interestingly, also quantum mechanical methodslike QM/MM or DFT have found utility in this field, especially to explorethe role metal-containing enzymes and their influence on catalytic ac-tivities and the inhibition mechanism by small-molecule modulators[155–157]. Combined computational techniques have been also suc-cessfully exploited for the de novo design of inhibitory peptides for his-tone methyltransferase [158] and chemoinformatic data mining toolslike self-organizing maps (SOM) have been exploited for the computa-tional prediction of common non-epigenetic drugs as epigenetic modu-lators [159]. It is worth to note also that emerging works driven bycomputational-based techniques are also starting to focus on food andnatural components, known to be able to influence the epigeneticcode [37,160–164]. Indeed, dietary components like complementaryand/or alternative medicines from green tea, genistein from soybean,isothiocyanates from plant foods, curcumin from turmeric, resveratrolfrom grapes, and sulforaphane from cruciferous vegetables, have beenstudied for their ability to target the epigenome, especially in differentcancer pathologies [18]. Nevertheless, the mechanisms of action ofthese compounds are still poorly understood and computer-aided tech-niques are expected to help the comprehension of thesemechanisms. Inparticular, we believe that the availability of compound databases of

arvin was used for drawing and displaying chemical structures, Marvin 6.0.0, ChemAxon

363F. Andreoli, A. Del Rio / Computational and Structural Biotechnology Journal 13 (2015) 358–365

natural and dietary sources could constitute an effective step toward theidentification, development and pharmacological definition of naturaland dietary-derived components, affecting epigenetic mechanism, thathold the advantage of pharmaceutical formulation based on natural-occurring scaffold.

5. Summary and Outlook

Major research efforts are currently directed toward the discovery ofnew small-molecules able tomodulate epigenetic writers or erases thatare involved in chromatin remodeling. Recent successful stories docu-ment the possibility to interfere with the epigenetic codewith small or-ganic molecules. Indeed, first pre-clinical and clinical results, especiallyfor HDACs, testify thatmany other epidrugsmight be effective as combi-nation therapies to control the process of genesis and progression ofseveral forms of cancer, among other pathologies. It is unquestionablethat epigenetics framework will play a major role in the near future todevelop new therapies against these diseases and molecular designtechniques offer the chance to tackle these challenges.

Acknowledgments

This work was supported by the Italian Association for CancerResearch (Start Up grant N.6266).

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