BIOTECNOLOGIE FARMACOLOGICHE2008/09

LEZIONE 2

CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO

Adriana Maggi

I PROCESSI DI METILAZIONE GIOCANO UN RUOLONELLA PATOGENESI UMANA

The mechanism(s) by which the native MeCP2 protein operates in the cell are not well understood. Historically, MeCP2 has been characterized as a proximal gene silencer with 2 functional domains:1. a methyl DNA binding domain and 2.a transcription repression domain.

However, several lines of new data indicate that MeCP2 structure and function relationships are more complex: an analysis of cell-based experiments suggesting MeCP2 is a regulator, rather than a strict silencer, of transcription. The new data establish MeCP2 as a multifunctional nuclear protein, with potentially important roles in chromatin architecture, regulation of RNA splicing, and active transcription..

methyl DNA binding protein 2 (MeCP2)

Andreas Rett, che aveva osservato un comportamento insolito, tragico e affascinante insieme, in alcune bambine sedute nella sala d'aspetto del suo studio. Correva l'anno 1966.

Nel 1999, il team di Huda Zoghbi del Baylor College of Medicine di Houston scoprì che la sindrome di Rett è associata a una mutazione del gene MeCP2, localizzato sul cromosoma X. Il gene MeCP2 è un repressore della trascrizione genica.

La proteina MeCP2 è espressa in modo predominante nei neuroni maturi. MeCP2 modula le connessioni sinaptiche e quindi la comunicazione cellulare attraverso la regolazione di alcuni geni, tra cui il brain-derived neurotrophic factor (BDNF).

SINDROME DI RETT

Guy J, Gan J, Selfridge J, Cobb S, Bird A. Reversal of neurological defects in a mouse model of Rett syndrome. Science 2007; 315:1143-1147

Guy et al., con un sistema murino molto sofisticato hanno silenziato la sintesi della proteina MeCP2 causando la patologia, poi ne hanno reinstaurato la sintesi con una remissione della sintomatologia

Più recentemente, si è visto che le MeCP2 interagisce con la proteina YB-1 che è coinvolta nella matutazione degli RNA e in particolare della subunità NR1 dei recettori per il glutammato NMDA

MeCP2 gene

The alterations of DNA methylation level and patterns are a common feature of human cancer cells. A global DNA hypomethylation has been observed in many cancers, without obvious sequence specificity

Recently, an extensive study of about 1200 CpG islands has indicated that hypermethylated CpG islands are not randomly distributed and the patterns of the hypermethylation might be specific of subclasses of cancers.

The methylation status of tumor suppressor genes has been extensively investigated and such alterations have been reported in many human tumors (Robertson and Jones, 2000).

METILAZIONE DEL DNA E CANCRO

Several reports link genome hypomethylation to genome instability. In particular, it was shown recently that strongly reduced DNMT activity in a transgenic mouse model caused demethylation of centromeric satellite and other repeat sequences, which resulted in a variety of chromosome defects and concomitant tumorigenesis

METILAZIONE DEL DNA E CANCRO

Figure 1. Epigenetic cancer therapy. DNA methyltransferases (DNMTs) can cause ectopic methylation and gene silencing. These events are called epimutations and promote tumorigenesis if directed to tumor suppressor genes. Importantly, the maintenance of epimutations requires the continuous activity of DNMTs. This accounts for the principal reversibility of epimutations by DNMT inhibitors.

Il mantenimento della metilazione del DNA (da parte di DNMT1) è indispensabile nella duplicazione del DNA per mantenere metilati specifiche porzioni del DNA.

Nel topo la mutazione di DNMT1 è letale

Human DNA methyltransferases (DNMTs) and their functionally important domains. All known DNMTs share a highly conserved C-terminal catalytic domain. The N-terminal domains differ strongly between DNMT1 and DNMT3 enzymes and contain several motifs for regulatory functions: a PCNA binding domain (PBD), a replication foci targeting domain (RFTD), a CXXC domain implicated in DNA binding, a PWWP domain linked to protein targeting and an ATRX domain implicated in histone deacetylase interactions.

The DNMT3A and DNMT3B proteins are similar and are probably the products of a recent gene duplication event.

5-Azacytidine (Vidazae) has been approved for the treatment of myelodysplastic syndrome.

This has been a major milestone in the field of cancer epigenetics and provides an important validation for the concept of an epigenetic cancer therapy

Deacetilasi istoniche: una nuova classe di farmaci nella terapia antitumorale

Generalmente agiscono causando apoptosi di cellule tumorali, ma non di cellule non tumorali; le HDAC sono generalmente associate a fattori di trascrizione oncogeni.

The effects of HDAC inhibitors on gene expression are highly selective, leading to transcriptional activation of certain genes such as the cyclin-dependent kinase inhibitor p21WAF1/CIP1 but repression of others.

HDAC inhibition not only results in acetylation of histones but also transcription factors such as p53, GATA-1 and estrogen receptor-alpha. The functional significance of acetylation of non-histone proteins and the precise mechanisms whereby HDAC inhibitors induce tumor cell growth arrest, differentiation and/or apoptosis are currently the focus of intensive research.

Several HDAC inhibitors have shown impressive antitumor activity in vivo with remarkably little toxicity in preclinical studies and are currently in phase I clinical trial.

HDACs are classified in four groups based on their homology to yeast histone deacetylases:

Class I which includes HDAC1, -2, -3 and -8 are related to yeast RPD3 gene;

Class II which includes HDAC4, -5, -6, -7, -9 and -10 are related to yeast Hda1 gene;

Class III, also known as the sirtuins are related to the Sir2 gene and include SIRT1-7, and

Class IV which contains only HDAC11 has features of both Class I and II.

Classificazione delle deacetilasi istoniche

The “classical” HDIs act exclusively on Class I and Class II HDACs by binding to the zinc containing catalytic domain of the HDACs. These classical HDIs fall into several groupings, in order of decreasing potency:(i) hyroxamic acids, such as Tricostatine A, (ii) cyclic tetrapeptides (such as trapoxin B), and the depsipeptides, (iii) benzamides, (iv) electrophilic ketones, and (v) the alophatic acid compounds such as phenylbutyrate and valproic acid.

"Second generation" HDIs include SAHA/Vorinostat, Belinostat/PXD101, MS275, LAQ824/LBH589, CI994, and MGCD0103.

The sirtuin Class III HDACs are NAD+ dependent and are therefore inhibited by nicotinamides, as well derivatives of NAD, dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.[4]

Classificazione degli inibitori delle istone deacetilasi

VORINOSTAT (Zolinza) è il primo inibitore delle acetilasi istoniche approvato per il trattamento di neoplasie Nel 2006 il VORINOSTAT è stato approvato per il trattamento del linfoma a cellule T cutaneo

Esistono studi preclinici che indicano una attività antinfiammatoria del vorinostat

Nel 2007 ricerche presso la Mayo Clinics hanno dimostrato che il Vorinostat è efficace nel glioblastoma ricorrente

NH

O

OHO

HN

N-hydroxy-N'-phenyl-octanediamide

HDAC inhibitors can activate both the death-receptor and intrinsic apoptotic pathways

Histone deacetylase inhibitors as therapeutics for polyglutamine disorders Rachel Butler and Gillian P. BatesNature Reviews Neuroscience 7, 784-796 (October 2006)

P53 puo’ agire a diversi livelli:

Puo’ attivare proteine per il riparo del DNA

Puo’ regolar e la duplicazione cellulare a livello G1/S

Puo’ iniziare processi apoptotici

P53

MODULAZIONE DELLA TRASCRIZIONE DA PARTE DI RECETTORI NUCLEARI

Il ruolo degli INSULATORS nella regolazione dellaespressione genica

Es. i geni globinici e LCR HS4,scs di Drosophila che isolano hsp70)

In genere sono sequenze ricche di isole CpG non metilateCi sono proteine che legano gli insulators

Transcription factors are associated with the nuclear matrix. It has been proposed that the nuclear matrix recruits transcription factors, facilitating their interaction with regulatory DNA elements.

ESPRESSIONE GENICA IN EUCARIOTE: SEQUENZA DI EVENTI

3’ TRASCRIZIONE

MODIFICAZ.TP

TRADUZIONE

RNA, trascritto primario

Capping e poliadenilazione

Splicing o maturazione

Fuoriuscita dal nucleo

riconoscimento da parte dei ribosomi

traduzione

Modificazioni post-traduzionali