Revisión de Literatura

Histone deacetylase (HDAC) is a multi-protein complex that suppresses gene expression. In cooperation with its counterpart histone acetyltrans-ferase (HAT), it regulates the acetylation of histone proteins and other substrates, influencing chromatin structure and regulating gene availability for transcription.³⁰

HDAC catalyses the cleavage of the acetyl group from lysine residues on histones, leaving the residues with a positive charge. The negatively charged DNA strands are then attracted to the positively charged histones.

This close interaction between the histones and the DNA can prevent gene transcription by limiting transcription factors’ spatial access to the DNA. In this way, HDACs can regulate the availability of genes for transcritption.³¹

The balance of histone acetylation is maintained by two groups of enzymes, HDAC and HAT. The process of lysine acetylation and deacetylation is post-translational and reversible.³⁰The acetylation of his-tone lysine residues is important both in normal and cancer cells because it causes active and inactive gene segments to develop. The 11 classical human HDACs are classified by sequence and domain relationships into

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four classes according to their homology to yeast proteins. Histone deacety-lases participate in various protein and co-repressor complexes.³⁰

Class I histone deacetylases (HDACs 1, 2, 3 and 8) are homologous to the yeast protein RPD3. They are expressed in many tissues, including heart, liver and skeletal muscle.^32,33

Class II HDACs (4, 5, 6, 7, 9 and 10) are homologous to the yeast pro-tein HDA1 (histone deacetylase 1). They are expressed in a tissue-specific manner.³⁴ HDAC4 and HDAC5 are expressed mainly in brain, heart and skeletal muscle, while the expression of HDAC6 is high in heart, liver, kidney and pancreas.

Class III, includes SIRT1-7, which differs structurally from other HDACs. It is named after its yeast-homolog SIR2 (silencing information regulator 2), and requires NAD⁺as a cofactor. Classical HDAC inhibitors cannot inhibit SIR2.³⁵

Class IV consists of HDAC11, which regulates interleukin-10 expres-sion in antigen-presenting cells.

Increased HDAC activity is observed in cancer cells, which leads to changes in local chromatin structure, alterations in gene transcription and impaired differentiation. Therefore, HDAC inhibitors (HDI) are novel can-didates for cancer treatment. They lead to an increase in histone acetylation and thus convert the DNA in a more open and transcriptionally active con-formation. This in turn increases gene activation, ultimately resulting in cell differentiation and/or apoptosis.

By crystallographic methods, it was found that the active site of his-tone deacetylases contains a zinc ion.³⁶All HDAC structures have a long, hydrophobic binding pocket channel with the zinc ion at one end. The zinc is coordinated by two aspartate residues and a histidine residue.³⁶It is directly involved in the catalytic mechanism of deacetylation and chelation of the zinc ion plays an essential role in the binding of inhibitors.³⁷Apart from coordinating the zinc ion, HDIs can also spatially block the binding pocket channel so that the natural substrate cannot be bound. HDIs thus inhibit HDACs reversibly via a competitive mechanism.^38,39

The already known HDAC inhibitors have been categorized into five different groups based on their chemical structure: hydroxamic acids, epox-ides (cyclic tetrapeptepox-ides with AOE (2-amino-8-oxo-9,10-epoxydecanoic

NH

NH OH

O

O

Figure 22 Structure of trichostatin A.

acid) group), cyclic tetrapeptides without AOE radical, short-chain fatty acids and benzamides.

Trichostatin A (TSA) is a member of the hydroxamic acid (Fig. 22), the largest group of HDIs. TSA is a product of Streptomyces hygroscopicus and was initially developed as a fungicide in 1990. It acts at nanomolar concentrations and is considered to be one of the most potent HDIs.^40,41

TSA induces cell cycle arrest both in G1 and in G2 phases via the follow-ing mechanisms: HDAC inhibitors induce expression of the CDKN1A gene, which codes for the cyclin-dependent kinase inhibitor p21 (cdk= cyclin dependent kinase).⁴²p21 causes cell cycle arrest in the G1 phase. SP1 binds directly to HDAC1 and co-mediates suppression of gene expression. Sp1 enhances its own expression by participating in a NFκB/HDAC complex.⁴³ The fact that TSA induces p21 indicates that the binding of TSA directly activates the CDKN1A promoter.^44,45 SP1 is also necessary for transac-tivation of hTERT, a gene that encodes catalytic subunits of telomerase.

The enzyme telomerase protects the ends of chromosomes (telomeres) from degradation. The inhibition of transcription of hTERT promoter by HDACs completely abolishes expression of telomerase. TSA induces telom-erase activity in normal cells but not in cancer cells. The HDAC inhibitor activates the hTERT promoter in normal cells, in which Sp1 plays a key role.⁴⁶ SP1 interacts not only with HDACs, but also with other transcrip-tion activators. For example, recent studies have shown that SP1 interacts with p300, a histone acetyltransferase.⁴⁷ SP1-dependent gene regulation is thus controlled by a competition between HDACs and other activating factors.

Trichostatin A has a number of limitations as an HDAC inhibitor. It cannot inhibit class III HDACs, since they have a different mechanism of action. For clinical use, TSA is not suitable because of its toxicity and low specificity. It is used rather as a reference in the pre-clinical research.

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Amide analogues of TSA, such as MD85, oxamflatin or scriptaid are also inducers of differentiation and/or apoptosis in low micromolar concentrations.

Suberoylanilide hydroxamic acid (SAHA) is another member of the hydroxamic acids (Fig. 23). SAHA inhibits HDACs at micromolar concen-trations and causes G1 and G2 cell cycle arrest.^48,45The replacement of the hydroxamic acid structure of SAHA by a carboxylic acid or an amidoxime structure results in an inactive compound. N-methylation or the introduc-tion of a methyl group leads to a loss of activity. The optimal length of the spacer was determined to be six methylene groups.

The natural product trapoxin B is an isolate from Helicoma ambiens (Fig. 24). It is an epoxy and irreversibly inhibits histone deacetylases in a nanomolar range leading to cell cycle arrest in the G1 and G2 phases.⁴⁰The effect of this cyclic tetrapeptide is attributed to the epoxy-keto structure at the end of the hydrophobic chain, which covalently binds to HDAC.

HDAC6 is resistant to trapoxin B.⁴⁹

NH

NH OH

O

O

Figure 23 Structure of suberoylanilide hydroxamic acid.

O N

NH NH

NH O

O O

O

O

Figure 24 Structure of trapoxin B.

Trapoxin B has several analogues that inhibit HDAC in a different way.⁵⁰ HC-toxin, a natural product from Cochliobolus carbonum, inhibits reversibly despite its epoxy-keto structure. The epoxy-keto structure was initially considered to be essential for inhibition because compounds with-out it were ineffective. Apicidin from Fusarium pallidoroseum and Apicidin A, which have a simple keto-enol function, are also potent inhibitors. In these compounds, the tryptophan residue is important for the inhibitory effect.

FK228, better known as romidepsin or depsipeptide, belongs to the group of cyclic tetrapeptides without an AOE radical (Fig. 25). It is a natural product of Chromobacterium violaceum. FK228 nitiates cell cycle arrest in the G1 and G2-phases.⁵¹ Garlic ingredients and their metabolites such as diallyl sulfide and allylmercaptan are also HDAC inhibitors, which suggest that the zinc ion in the active site of an HDAC is complex after the opening of the disulfide bond by thiol groups.

Butyrate belongs to the group of short-chain fatty acids and is a reversible HDI (Fig. 26). It induces cell cycle arrest in the G1 phase, a hyper-acetylation of histone H4⁵² In addition, butyrate induces apoptosis.^53,54

The therapeutic value of butyrate is limited by its low activity (in mil-limolar concentrations) and the rapid in vivo metabolism of the compound.

O

NH

O

NH O NH

NH O

O

O

S S

H

Figure 25 Structure of FK228.

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COO^- Na⁺ Figure 26 Structure of butyrate.

Administration of prodrugs, such as tributyrin or pivaloyl oxymethyl butyrate, may circumvent this problem.