《药物化学》课程文献资料（Medicinal Chemistry）Epigenetic protein families - a new frontier for drug discovery.pdf

Although all cells in an organism inherit the same genetic material, the ability of cells to maintain the unique physical characteristics and biological functions of specific tissues and organs is due to heritable differences in the packaging of DNA and chromatin. These differences dictate distinct cellular gene expression programmes but do not involve changes in the underlying DNA sequence of the organism. Thus, epigenetics (which literally means ‘above genetics’) underpins the fundamental basis of human physiology. Importantly, the epigenetic state of a cell is malleable; it evolves in an ordered manner during the cellular differentiation and development of an organism, and epigenetic changes are responsible for cellular plasticity that enables cellular reprogramming and response to the environment. Because epigenetic mechanisms are responsible for the integration of environmental cues at the cellular level, they have an important role in diseases related to diet, lifestyle, early life experience and environmental exposure to toxins1 . Thus, epigenetics is of therapeutic relevance in multiple diseases such as cancer, inflammation, metabolic disease and neuropsychiatric disorders, as well as in regenerative medicine2–4. The dynamic nature of epigenetics means that it may be possible to alter disease-associated epigenetic states through direct manipulation of the molecular factors involved in this process. Several interrelated molecular mechanisms contribute to epigenetic gene regulation, including chromatin remodelling via ATP-dependent processes and exchange of histone variants, regulation by non-coding RNAs, methylation and related modifications of cytosines on DNA, as well as covalent modification of histones5 (FIG. 1). Inhibitors of DNA methylation and histone deacetylase (HDAC) inhibitors are approved for clinical use in haematological malignancies, thus providing proof of concept for epigenetic therapies6 . Over the past decade, knowledge of the proteins involved in the post-translational modification of histones has grown tremendously. These proteins comprise several families of related enzymes and chromatin-interacting proteins, and are a rich source of potential therapeutic targets. Here, we review the proteins involved in depositing, removing or binding to acetyl and methyl groups — the two most abundant histone post-translational modifications (which are commonly referred to as histone marks). We focus on the mediators of acetyl and methyl histone marks because of their prominent role in several diseases, as well as the emerging realization that many of these proteins are susceptible to inhibition by small molecules. Defining the druggable epigenome Acetylation and methylation networks define a large component of the human epigenome. Although several histone post-translational modifications — including phosphorylation and ubiquitylation — are important components of the epigenome, acetyl and methyl marks are the most abundant and among the most widely 1Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada. 2Ontario Cancer Institute, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada. 3Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada. Correspondence to C.A. e-mail: carrow@uhnresearch.ca doi:10.1038/nrd3674 Published online 13 April 2012 Epigenetic protein families: a new frontier for drug discovery Cheryl H. Arrowsmith1,2,3, Chas Bountra4, Paul V. Fish5, Kevin Lee6* and Matthieu Schapira1,7 Abstract | Epigenetic regulation of gene expression is a dynamic and reversible process that establishes normal cellular phenotypes but also contributes to human diseases. At the molecular level, epigenetic regulation involves hierarchical covalent modification of DNA and the proteins that package DNA, such as histones. Here, we review the key protein families that mediate epigenetic signalling through the acetylation and methylation of histones, including histone deacetylases, protein methyltransferases, lysine demethylases, bromodomain-containing proteins and proteins that bind to methylated histones. These protein families are emerging as druggable classes of enzymes and druggable classes of protein–protein interaction domains. In this article, we discuss the known links with disease, basic molecular mechanisms of action and recent progress in the pharmacological modulation of each class of proteins. Chromatin The fibres in which DNA and genes are packaged in the nucleus of a cell. Chromatin consists of the DNA double helix wrapped around a complex of histone proteins — together called the nucleosome. REVIEWS 384 | MAY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved

Epigenetics Heritable changes in gene expression or phenotype that are stable between cell divisions, and sometimes between generations, but do not involve changes in the underlying DNA sequence of the organism. Differentiation The process by which a stem cell, or other precursor cell, commits towards a more specialized cell type with a specific function, and represents an exit from self-renewal. Differentiation is controlled by cell signalling pathways and maintained through epigenetic mechanisms. Post-translational modification A chemical modification of proteins that acts as a signal to other proteins that recognize the modification. In the context of epigenetic signalling, post-translational modifications are often called ‘marks’. Epigenome The combination of histone and DNA post-translational modifications and related interacting proteins that together package the genome and help define the transcriptional programme in a given cell. Heterochromatin A tightly packed form of DNA associated with transcriptionally silent or repressed genes. It is highly correlated with di- and trimethlyated H3K9 (Lys9 of histone 3) marks. Euchromatin A more loosely packed form of DNA that is associated with transcriptionally active genes. studied, and have a large number of druggable proteins that mediate their dynamic activity. A feature of epigenetic regulation that is mediated by histone marks is the collaboration among combinations of marks to affect specific cellular outcomes — often referred to as the histone code hypothesis7–10 (FIG. 1). For example, the recent mapping of nine acetyl and methyl histone marks across the genomes of nine different cell types showed that combinations of marks defined 15 chromatin states related to the transcriptional activity of surrounding genes11. Individual marks and combinations of marks are recognized by several classes of conserved protein domains, usually within the context of larger multiprotein complexes. Thus, histone marks and the multiprotein complexes that bind to them contribute to the physical make-up of chromatin and to the recruitment of specific proteins to genomic loci that contain specific histone marks. For example, most of the enzymes that are ‘writers’ of methyl or acetyl histone marks are large proteins that, in addition to their catalytic domain, contain other domains or regions that ‘read’ histone marks and/or interact with DNA or other proteins. Together, these proteins form complexes that integrate upstream cellular and environmental signals to establish and maintain cellular identity and contribute to the genesis and/ or maintenance of disease states10. Owing to remarkable progress over the past decade, we now know the basic complement of regulatory proteins that ‘read’, ‘write’ and ‘erase’ the major histone marks. These are summarized in TABLE 1, and further delineated in FIG. 2 as phylogenetic trees of structurally and evolutionarily related families of proteins. Histone acetylation. Since the first description of histone acetylation in 1964 (REF. 12), it has been established that this is a highly dynamic process that is regulated by two families of enzymes — histone acetyltransferases (HATs) and HDACs — that operate in an opposing manner. HATs use acetyl-CoA as a cofactor and catalyse the transfer of an acetyl group to the ε-amino group of lysine side chains on the histone protein. This neutralizes the positive charge on lysine, thus reducing the affinity of the histone tail that protrudes from the nucleosome core of DNA. As a result, chromatin adopts a more relaxed structure, enabling the recruitment of the transcriptional machinery. HDACs oppose the effects of HATs and reverse the acetylation of lysine residues to restore their positive charge and stabilize the local chromatin architecture. Among the various sites of histone lysine acetylation, Lys16 of histone 4 (H4K16) appears to be crucial in the regulation of chromatin folding and in the switch from heterochromatin to euchromatin13. In addition to the acetylation of histone tails, there are several lysine substrates within the globular core of the histone proteins (such as H3K56), which suggests that acetylation can also directly affect the interaction between histones and DNA14. There is evidence that histone acetylation, particularly of H4K5 and H4K12, is important for the recognition of chaperones during histone assembly and deposition into DNA. Histone acetylation also promotes transcription by providing binding sites for proteins that are involved in gene activation. In particular, the bromodomain-containing family of proteins recognize (that is, ‘read’) modified lysine residues within histone proteins. Bromodomains are a common feature in a diverse set of proteins united by their importance in transcriptional co-activation, and the ability of bromodomains to identify and bind to acetylated lysine residues within histone proteins is key to their activity15,16. Histone methylation The significance of and the associated mechanisms of histone methylation have been gradually elucidated over the past decade. Lysine residues on histones can be monomethylated, dimethylated or trimethylated. Arginine residues are also subject to monomethylation and dimethylation. Dimethylation of arginine residues can occur in a symmetric manner (via monomethylation of both terminal guanidino nitrogens) or in an asymmetric manner (via dimethylation of one of the terminal guanidino nitrogens). As with acetylation, methylation is dynamic. Methyl marks are written by S-adenosylmethionine (SAM)-dependent methyltransferases and erased by either the Jumonji family of 2-oxoglutarate-dependent demethylases17 or the flavindependent enzymes lysine-specific histone demethylase 1 (LSD1; also known as KDM1A) and LSD2 (also known as KDM1B)18. Because methylation does not change the charged state of a lysine or arginine residue, it does not appear to effect chromatin structure directly. Instead, the various methyl marks act as binding sites for other proteins that compact nucleosomes together19,20 or bring additional regulatory proteins to chromatin sites marked by methylation21,22. Each type of mark constitutes a specific signal that is recognized by highly evolved methyl-lysinebinding domains that recognize the level of methylation and, in many cases, the surrounding amino acid sequence (TABLE 1). Thus, trimethylated Lys4 of histone 3 (H3K4me3), H3K9me3 and H4K20me2 each interact with a distinct set of reader domains. Histone lysine methylation can be associated with either transcriptional activation or repression. For example, H3K4me3 is a hallmark of transcriptionally active genes, whereas H3K9me3 and H3K27me3 (REFS 23,24) are associated with silenced genes. Although protein arginine methylation is abundant and has been known for a long time, histone arginine methylation has only recently become recognized as an important transcriptional Author addresses 4 Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7LD, UK. 5 Worldwide Medicinal Chemistry, Pfizer, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK. 6 Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK. 7 Department of Pharmacology & Toxicology, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada. *Present address: Pfizer Biotherapeutics, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140, USA. REVIEWS NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | MAY 2012 | 385 © 2012 Macmillan Publishers Limited. All rights reserved

Nucleus Writers Erasers Readers Nature Reviews | Drug Discovery Histone acetyltransferases, Histone methyltransferases Histone deacetylases, Lysine demethylases Bromodomains, chromodomains, PHD fingers, malignant brain tumour domains, Tudor domains, PWWP domains DNA methylation Me Me Me Me Chromatin fibre Chromosome Nucleosome Bromodomain An evolutionarily conserved, ~110-amino-acid motif composed of four left-handed, antiparallel α-helices. regulatory mechanism25. Arginine methylation of histones can promote or antagonize the interaction of nuclear factors with other nearby histone marks, thereby increasing the complexity of the histone code26,27. Disease association The readers, writers and erasers of epigenetic marks can contribute to or drive disease via two primary mechanisms. First, aberrant activity due to mutation or altered expression of epigenetic factors can alter subsequent cellular gene expression patterns that lead to or even drive and maintain disease states. Second, because the readers, writers and erasers are general factors that work in concert with many other cellular proteins, especially tissue-specific and environmentally responsive DNAbinding transcription factors, they can mediate altered gene expression patterns driven by upstream signals10. Importantly, the latter case offers the opportunity to target disease pathways whose primary drivers (for example, certain transcription factors or external stimuli) may not be druggable. Cancer. Epigenetic mechanisms have long been known to be involved in cancer, beginning with the observation that levels of DNA methylation were dramatically altered in most cancers. Although cancer is fundamentally a genetic disease that is driven by irreversible genomic mutations that subsequently activate oncogenes or inactivate tumour suppressor genes, there is increasing evidence that many epigenetic regulatory proteins are among those dysregulated in cancer, and that histone marks are globally and locally altered within cancer epigenomes28. This knowledge stimulated the development of inhibitors of DNA methyltransferases and HDACs that are clinically effective in several cancers, attesting to the value of epigenetic therapies in oncology28. However, these Figure 1 | Covalent modification of histones and DNA are key mechanisms involved in epigenetic regulation of gene expression. DNA is packaged into chromatin by wrapping around histone proteins (two copies each of histones H2A, H2B, H3 and H4) to form a nucleosome. Nucleosomes are further compacted by additional protein factors to form chromatin, with the degree of compactness dependent on the types of post-translational modification present on the histones, especially on their terminal residues, which protrude from the nucleosome particle. Acetylated histones tend to be less compact and more accessible to RNA polymerase and the transcriptional machinery, thereby enabling transcription of nearby genes. Methylated histones can be either repressive or activating, depending on the site and degree of methylation. The combination of modifications on each histone and/or nucleosome establishes a code that relates to the transcriptional properties of the nearby genes. The primary protein families that mediate histone post-translational modifications are illustrated in the inset. Proteins that covalently attach acetyl or methyl groups produce (or ‘write’) the code (these include histone acetyltransferases and histone methyltransferases) and are termed ‘writers’. Proteins that recognize and bind to histone modifications are termed ‘readers’ of the code (these include bromodomains, plant homeodomains (PHDs) and members of the royal family of methyl-lysine-binding domains). Enzymes that remove histone marks are termed ‘erasers’ (these include histone deacetylases and lysine demethylases). REVIEWS 386 | MAY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved

Stem cell An unspecialized precursor cell with the capacity to self-renew (continuously produce unaltered progeny) and to differentiate into more mature specialized cell types. Haploinsufficiency A disease mechanism in which one of two copies of a gene is mutated, resulting in insufficient activity of the gene products (typically a protein) to bring about a functional, wild-type condition. Brachydactyly mental retardation syndrome A disorder that presents with a range of features, including intellectual disabilities, developmental delays, behavioural abnormalities, sleep disturbance, craniofacial and skeletal abnormalities, and autism spectrum disorder. Presenilins A family of related multipass transmembrane proteins that function as part of the γ-secretase intramembrane protease complex. They were first identified in screens for mutations causing early-onset forms of familial Alzheimer’s disease. agents are non-selective within their target protein families and have substantial side effects. Although it remains to be demonstrated in the clinic, agents that target specific HDACs with greater selectivity may be beneficial in certain cancers. For example, treatment of neuroblastoma cell lines with a selective inhibitor of HDAC8 mimicked genetic knockdown of HDAC8 as well as inhibiting cellular proliferation and triggering differentiation29,30. Second-generation HDAC inhibitors — several of which are more selective — are currently in clinical trials for multiple types of cancer (TABLE 2). Deregulation of epigenetic regulatory proteins and their signalling networks can occur via several mechanisms, including direct inactivating or activating mutations, gene amplification, indirect upregulation or inactivation of enzymes, and translocations that lead to the expression of gain-of-function fusion proteins that contain reader domains31. Well-known examples include overexpression of the key developmental histone lysine N-methyltransferase EZH2 in several types of leukaemia and in various solid tumours32. The gene encoding the protein methyltransferase MLL is also subject to many chromosomal translocations that lead to the expression of chimeric fusion proteins and inappropriate recruitment of other epigenetic factors such as the methyltransferase DOT1-like protein (DOT1L)33. Inhibition of DOT1L was recently shown to selectively kill cells and tumour xenografts that contained MLL translocations34. EZH2 can be aberrantly upregulated by the overexpression of dominant mutations that increase its trimethylation activity, offering the possibility of selective therapy targeting the mutant protein35. A recent example of a potential epigenetic targeted therapy was shown in a model of midline carcinoma. In this cancer, carcinogenesis is driven by chromosomal translocation, which results in the expression of a fusion protein containing the bromodomain of bromodomain-containing protein 4 (BRD4) or BRD3 and a testis-specific transcription factor (NUT) that drives carcinogenesis. A selective antagonist of the BET family of bromodomains (which includes BRD2, BRD3, BRD4 and bromodomain testis-specific protein (BRDT)) resulted in the selective killing of BRD4– NUT-positive midline carcinoma xenografts36. Modulation of epigenetic mechanisms also offers the potential for overcoming the genetic changes that drive cancer — especially oncoproteins that may not be druggable. For example, with the exception of nuclear hormone receptors, it is recognized that it is extremely challenging to inhibit most sequence-specific transcription factors using small molecules37. This includes the transcription factor MYC, whose pathological activation is among the most common genetic events observed in cancer genomes38. Although MYC was one of the first known and most common oncoproteins39, over 30 years of research have failed to identify compounds that can directly inhibit the activity of the MYC protein. However, several recent exciting reports indicate that MYC may be effectively inhibited in several haematological malignancies through pharmacological inhibition of one of its regulatory partners, BRD4. BRD4 binds acetylated histones via its bromodomain and mediates chromatin-dependent signalling and transcription at MYC target loci40. Inhibition of the interaction between BRD4 and acetylated histones results in reduced levels of MYC target genes and inhibition of transcription of the MYC gene itself 41,42. Similarly, overexpression of the bromodomain-containing nuclear cofactor ATPase AAA domain-containing protein 2 (ATAD2) is crucial for the proliferation and survival of triple-negative/basal-like breast cancer cells and controls the expression of the oncogene MYB43. The bromodomain of ATAD2 has a key role in tumorigenesis44. These results highlight the potential for targeting ‘undruggable’ oncogenic transcription factors by inhibiting the catalytic or chromatin-interaction activities of druggable epigenetic cofactors that drive the expression of oncogenic transcription factors. There are numerous other cancer-linked alterations in the genes coding for (and the activity of) readers, writers and erasers of histone marks. Many of these alterations occur in key developmental genes and are associated with cancers that derive from stem cell-like early progenitors of a given tissue type, such as many haematological malignancies45–48 and medulloblastoma49,50. Thus, these self-renewing cells may be locked in an epigenetic state that prevents them from undergoing differentiation. Inhibition of mutated epigenetic proteins or inhibition of the transcriptional programme of other oncogenic signalling factors could be an attractive strategy for overcoming the block to differentiation in these types of cancers. Similarly, the oxygen-independent glycolytic metabolism that is observed in rapidly proliferating cancer cells (known as the Warburg effect) may be orchestrated and maintained by epigenetic signalling networks51. Genomic instability is also a hallmark of cancer, and inactivation of epigenetic proteins that contribute to DNA damage checkpoints (such as the HAT 60 kDa Tatinteractive protein (TIP60; also known as KAT5)52 or the tumour protein p53 binding protein 1 (TP53BP1; a Tudor domain-containing protein)53 appears to contribute to oncogenesis. Although TIP60 and TP53BP1 act as tumour suppressors and are not likely to be therapeutic targets, the actions of these proteins underscore the extensive role of epigenetic proteins in oncogenesis, both positive (driving tumour growth) and negative (suppressing tumour growth). This dichotomy also raises important safetyrelated issues for potential epigenetic therapy (see below). Neuropsychiatric disorders. Several studies have shown that levels of epigenetic proteins are altered in clinical neurodisease states, especially in intellectual disability syndromes. Haploinsufficiency of HDAC4 causes brachydactyly mental retardation syndrome, developmental delays and behavioural problems54. Moreover, haploinsufficiency of the HAT CREB binding protein (CREBBP) causes Rubinstein–Taybi syndrome, a genetic disorder that results in cognitive dysfunction. In a mouse model of this disorder — neonatal Crebbp+/– mice — the mice exhibit behavioural impairments, and this phenotype can be reversed by inhibition of histone deacetylation55. CREBBP might also be a key target for presenilins in the regulation of memory formation and neuronal survival56. In addition, mutations in epigenetic proteins can result REVIEWS NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | MAY 2012 | 387 © 2012 Macmillan Publishers Limited. All rights reserved

Amyotrophic lateral sclerosis A progressive neurological disease that is associated with the degeneration of central and spinal motor neurons. This neuron loss causes muscles to weaken and waste away, leading to paralysis. in neuropsychiatric disorders: for example, mutations in the gene encoding the euchromatic histone-lysine N-methyltransferase 1 (EHMT1; also known as G9A-like protein 1 (GLP1)) result in a complex intellectual disability syndrome that is mirrored following deletion of this gene in the adult mouse brain57–59. X-linked mental retardation (XLMR) is an inherited disorder mostly affecting males, and is caused by genetic abnormalities of the X chromosome, including many transcriptional co-activator proteins60. For example, the XLMR protein PHF8 (PHD finger protein 8) catalyses the demethylation of H3K9me2 and H3K9me1 (REF. 61). The PHD of PHF8 binds to H3K4me3, and colocalizes with H3K4me3 at transcription initiation sites. Furthermore, PHF8 interacts with another XLMR protein, zinc finger protein 711 (ZNF711), which binds to a subset of PHF8-regulated proteins including the histone demethylase lysine-specific demethylase 5C (KDM5C; also known as JARID1C). These results functionally connect the XLMR-linked gene PHF8 to two other XLMR-linked genes, ZNF711 and JARID1C, indicating that genes linked to intellectual disability may be genetically associated within pathways that cause the complex phenotypes that are observed in patients who develop intellectual disability61. Sirtuin 1 (SIRT1) is ubiquitously expressed in areas of the brain that are especially susceptible to age-related neurodegenerative states in rats and humans. Therefore, activation of endogenous sirtuin pathways may offer a therapeutic approach to delay and/or treat human agerelated diseases62. Reduced levels of HDAC11 mRNA and increased levels of HDAC2 mRNA are observed in the brain and spinal cord of patients with amyotrophic lateral sclerosis63. The functional and therapeutic implications of these findings will be realized once more selective inhibitors of HDAC2 are available. Despite the lack of such tools, studies with currently available, partially selective HDAC inhibitors such as vorinostat (Zolinza; Table 1 | Components of the epigenome* Family Activity Number of proteins Major classes and function Writers Histone acetyltransferases K K 18 • MYST family (MOZ, SAS2, YBF2/SAS3, TIP60) proteins: involved in DNA damage and oncogenic translocation • GNAT: involved in EGF signalling and cell cycle progression • EP300: promiscuous (involved in a range of cellular events) Protein methyltransferases K K R R 60 • SET domain: methylates both histone and non-histone lysines • PRMTs: methylate both histone and non-histone arginines • PRDMs: SET domain-like tissue-specific factors Erasers Histone deacetylases K K 17 • Classes I, IIb and IV enzymes: have both histone and non-histone substrates, involved in gene silencing • Class IIa enzymes: scaffolding proteins • Sirtuins (class III): NAD-dependent, have deacetylation and ADP-ribosylation activity Lysine demethylases K K 25 • Lysine-specific demethylases: flavin-dependent enzymes that regulate transcription during development • Jumonji domain: 2-oxoglutarate-dependent Readers Bromodomain-containing proteins K 61 • Targeting of chromatin-modifying enzymes to specific sites, often physically linked to PHD fingers and the catalytic domain of histone acetyltransferases Methyl-lysine- and/or methyl-arginine-binding domain-containing proteins (for example, Tudor domains, MBT domains, chromodomains and PWWP domains) K R 95 • Tudor domains: bind dimethylated lysine, trimethylated lysine and dimethylated arginine • MBT domains: bind monomethylated and dimethylated lysine with low sequence specificity • Chromodomains: bind trimethylated lysine with sequence specificity • PWWP domains: bind to both trimethylated lysine and DNA PHD-containing proteins K R K K 104 • A large and diverse family that acts on multiple substrates EGF, epidermal growth factor; EP300, E1A-associated protein p300; GNAT, glycine-N-acyltransferase-like protein 1; MBT, malignant brain tumour domain; MYST, histone acetyltransferase MYST; PHD, plant homeodomain; PRDM, PR domain-containing protein; PRMT, protein arginine methyltransferase. *The major protein families that form the epigenome deposit (‘write’), bind to (‘read’) or remove (‘erase’) methyl marks (orange squares) or acetyl marks (blue circles) on specific lysine or arginine side chains of histones, as summarized in this table. Histone acetyltransferases and protein methyltransferases are the enzymes responsible for writing acetyl and methyl marks, respectively. Histone deacetylases and lysine demethylases erase the marks. Bromodomains bind acetylated lysines (shown by beige shape), whereas Tudor domains, MBT domains, chromodomains and PWWP domains bind methyl marks on lysine or arginine residues (shown by beige shape). PHD fingers are present in a large number of proteins and read either methyl or acetyl marks on lysine or arginine side chains, as well as unmodified lysines. REVIEWS 388 | MAY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved