HistoneacetyltransferaseinhibitorsandpreclinicalstudiesFabio Manzo, Francesco Paolo Tambaro, Antonello Mai & Lucia Altucci†
Seconda Università degli Studi di Napoli, Dipartimento di Patologia generale, Vico L. De Crecchio 7, 80138, Napoli, IT
Background: Drugs able to regulate the histone modifier enzymes are very promising tools for the treatment of several diseases, such as cancer. Histone acetyltransferase (HAT) inhibitors are compounds able to inhibit the cata-lytic activity of HATs reported to be active in cancer, or in several other diseases, such as Alzheimer (AD), diabetes and hyperlipidaemia. Objectives: Here we review the status and the rationale for the use of HAT inhibitors in the treatment of various diseases. Methods: Patents have been found on the espacenet database; the clinical trials have been reported as in the clinical-trial.gov website. Results and conclusion: Despite the fact that other drugs able to regulate the histone modifier enzymes (such as histone deacetylase inhibitors) have been already approved for the treatment of cancer, HAT inhibitors seem promising for the treatment of human diseases such as AD and diabetes, although side effects and toxicity need to be investigated.
Keywords: Alzheimer disease, cancer, clinical trials, epigenetics, HAT inhibitors, patents
Expert Opin. Ther. Patents (2009) 19(6):761-774
1. Introduction
In the past century, advances in molecular and cellular biology have led to the discovery of new families of proteins able to regulate the DNA folding and, consequently, transcription. In the nucleus of eukaryotic cells, DNA is highly com-pacted and organised into chromatin, whose basic unit is the nucleosome, com-posed by DNA and an octamer of core histones (H2A, H2B, H3, H4). The histones expose their N-terminal tails out of the octamer. These tails can be highly post-translationally modified, leading to the transcription regulation. Many are the post-translational modifications known at the present, acetylation, methylation, phosphorylation, ubiquitylation and sumoylation, can be taken as main examples. Generally, histone acetylation gives rise to DNA relaxation with a positive influence on transcription [1]. Histone methyltransferases (HMT) [2,3], histone acetyltrans-ferases (HAT) [4] and histone deacetylases (HDAC) [5] are some of the enzymes that modify histones. Also, HATs include a diverse set of enzymes, classified according to their catalytic domain and the multiprotein complexes in which they may reside. Mainly, HATs can be divided into three groups (Figure 1A), the GNATs (Gcn5 N-acetyltransferases) [6], the 60 kDa Tat interactive protein (MYSTs) [6] and the orphan HATs. P300/CBP-associated factor (PCAF), Elp3, Hat1, Hpa2 and Nut1 belong to the first group [6], with the founding member, GCN5. Morf, Ybp2, Sas2 and Tip60 represent the second group. Not containing a precise consensus HAT domain, the third group is called ‘orphan’, although these enzymes show an intrinsic HAT activity. p300/CBP, for example, belongs to this group together with Taf1 and several nuclear receptor (NR) coactivators. HATs participate in several complexes that may regulate their function. Depending on the complexes, HATs can be divided into several groups: i) the SAGA complex [7], able to modify H3K9 and H3K14 [8] in a specific manner; ii) the NuA3 complex [9], more specific for H3K14 modification; and iii) the Elongator complex [10], with similar function
as the SAGA complex, but in the gene-coding region. Mainly, the HAT domains include the bromodomain, the chromodo-main, the WD40 repeats, the Tudor domains and the PHD fingers, able to recognise modified histones (Figure 1A). According to the presence of each domain, the HATs interact with different histone modifications. GCN5, in fact, is able to bind the acetylated lysine residues through its bromodomain [11] thereby mediating the recruitment of the SAGA complex. More recently, the chromodomain of Eaf3 was shown to be important for recognition of and binding to methylated
H3K36 [12]. Its loss increased the acetylation in transcribed regions and the formation of spurious transcripts that are initiated within open reading frames. Furthermore, the WD40 domain presence in WDR5 mediates its binding on dimethylated H3K4 [13], in vivo. Interestingly enough, the crystal structure explained the specificity of the binding on the dimethylated form, with respect to the tri-methylated one. HATs containing the WD40 domain and the Tudor domain are present in several complexes, such as SAGA, interacting with the methylated H3K4 [14]. HAT functions
GCN5
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Figure1.HATstructureanddomains.A. Representation of the HAT domain, GCN5, ESA-1, CBP/P300, TAFII250. B. Tertiary structure of GCN5(PDBID: 1PU9) bound to the histone H3 (305 – 328) in presence of AcCoA. GCN5 is shown as ribbons in dark grey, H3 is represented as spheres in light grey and AcCoA as white sticks. The figure was remodelled by using Pymol. C. Tertiary structure of P300 (PDBID: 3BIY) in complex with the bi-substrate inhibitor Lys-CoA. P300 is shown as ribbons in light grey, bromide ions as spheres and Lys-CoA as white sticks. The figure was remodelled by using Pymol.HAT: Histone acetyltransferase.
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can be regulated by protein–protein interactions, and influence the maintenance of the DNA folding and the acetylation state of several non-histone targets. In this way, HATs directly modify the chromatin state, influencing fundamental pro-cesses such as the genome stability and the DNA repair. Several inhibitors have been synthesised and seem to display biological effects, inducing apoptosis in cancer cells, degenera-tive disorders regression, cancer preventive effects and so on. One of the best models to summarise the genomic effects of the HAT inhibitors is the H4K16 regulation. Indeed, loss of H4K16 acetylation has been highly correlated with tumori-genesis [15] and an increase of its acetylation induces chroma-tin decondensation, mainly related to telomeric regions [16]. H4K16 acetylation has been shown to destabilise nucleosomes and to correlate with non-condensed chromatin regions [16]. This, in turn, could regulate the access of transcription factors and chromatin remodelling enzymes to specific regions of the genome, inducing chromatin condensation owing to the Sir proteins recruitment at yeast telomeres. Although all charac-terised H4 acetyltransferases can modify H4K16, two HAT complexes, the SAS (something about silencing) complex in yeast and the MSL (male-specific lethal) complex in flies and humans [17,18] modify this residue for very specific functions. In yeast, the SAS complex was originally identified as a com-plex that is involved in gene silencing (hence the name ‘something about silencing’) [19,20]. It was subsequently shown that the SAS complex works antagonistically with the NAD-dependent HDAC Sir2 to create a silencing boundary at telomeres through the regulation of H4K16 acetylation. In this scenario, Sir2 spreads from the telomere and encounters the SAS complex and H4K16 acetylation, which prevents further spreading of Sir2, setting up a boundary of heterochromatin. Esa1, the catalytic subunit of the NuA4 HAT complex, can also acetylate H4K16 and other lysine residues [21], and is required for H4K16 acetylation at the INO1 gene promoter. However, the loss of Esa1 does not have the same effect at telomeres as the loss of Sas2, which is the catalytic subunit of the SAS complex [22,23]. Additionally, genome-wide chroma-tin immunoprecipitation (ChIP-on-chip) analysis clearly demonstrates that Sas2 is the telomere-specific H4K16 acetyl-transferase, because deletion of Sas2 results in a specific decrease in H4K16 acetylation at the telomere.
2. Histoneacetyltransferasederegulationandcancer
As described earlier, HATs are part of five main complexes with several functions. TRRAP is a member of the GNAT complexes and of the ATM/PIKK family of proteins. It was isolated as a binding partner of the oncogenic protein c-myc, with whom it interacts in the presence of mitogens [24]. Recently, it was shown that the myc/max heterodimer can recruit the 2-MDa GCN5-containing HAT co-activator complex. Interestingly, a naturally occurring truncated form of Myc, which does not interact with purified STAGA complex,
shows reduced transcription activation potential and cannot transform primary cells. This result clearly suggests that oncogenic transformation carried out by Myc is not a result of the protein expression per se, but rather depends on its physical and functional contacts with multisubunit GCN5-containing complexes [25]. PCAF has been described as a HAT acetylating E2F1, which leads to an increase in its DNA binding activity, stability and transactivation [26,27]. E2F1 and E2F4 interact directly with TRRAP and GCN5, suggesting that E2Ffamily of factors stimulate transcription by recruiting the essential cofactors GCN5 and TRRAP [28], probably as subunits of the endogenous STAGA/TFTC complexes. These results provide a mechanism for E2F-transcription factors to overcome dominant repression of transcription. Note, however, that on certain E2F-regulated gene promoters the STAGA/TFTC-type complexes may synergise with other HAT complexes (TIP60/NuA4) as H4 acetylation mark, and TIP60 have been also detected at E2F-responsive genes [29]. Also BRCA1 and BRCA2 are able to bind the hGCN5 and TRRAP containing complexes, which seem to be indispensable for the function of the co-regulator complex in both BRCA1-mediated gene regulation and DNA repair [30]. However, natural mutants for the binding site of BRCA1 with TRRAp occur in the C-terminal transactivation domain, causing the loss of interaction with this complex. HATs are involved in leukemogenesis. Chromosomal translocation of the genes that encode p300 or CBP with those for other HAT proteins [such as monocytic leukaemia zinc finger (MOZ) and MOZ-related factor (MORF) proteins] as well as the HMT mixed lineage leukae-mia (MLL) induces leukaemia through the formation of MOZ-, MORF- and MLL-p300/CBP fusion proteins. Such translocation-derived fusion proteins can cause either loss-of-function or gain-of-function in gene expression, resulting in aberrant cell cycle regulation and cancerogenesis [31]. Mutation in CBP is the genetic basis of the Rubinstein-Taybi syndrome, a complex disease that includes a high incidence of neoplasia. These bio-clinical observations highlight the importance to target HAT enzymes in cancer therapy.
3. Histoneacetyltransferaseinhibitors
Considerable efforts have been made in recent years to identify HAT modulators, both for mechanistic studies and for anticancer properties. The HAT inhibitors described so far can be classified into bi-substrate inhibitors, natural products and synthetic small molecules.
3.1 Bi-substrateinhibitorsA direct structural insight into small molecule mediated inhibition of HAT proteins comes from a crystal structure of the Tetrahymena Gcn5 (tGcn5) HAT domain bound to a modified H3-CoA-20 inhibitor (Figure 1B) [32]. The bi-substrate inhibitor used in this study was prepared with an isopropionyl bridge between CoA and a peptide to more closely mimic
the Ac-CoA-lysine intermediate (Figure 2A). With an IC50 of 300 nM for tGCN5, this is the most potent reported inhibitor of the Gcn5/PCAF HAT family identified so far. A superposition of the inhibited complex with the ternary tGcn5/CoA/H3 peptide complex shows that although the CoA and lysine substrate superimpose well, the rest of the peptide residues go in different directions and all but four of the residues that flank the lysine substrate are disordered in the inhibited complex. This result suggests that the inhibitor complex represents a late intermediate of the reaction just before the peptide release. The analysis of the tGcn5/inhibitor interface reveals that the pantetheine arm and pyrophosphate of CoA mediate the most extensive protein contacts, consistent with the CoA contacts in several other CoA-using GNAT enzymes [33,34]. These interactions likely provide a large degree of binding energy, but would be expected to show a lower degree of enzyme specificity. The lysine side chain and the isopropionyl linker of the inhibitor also make extensive van der Waals interactions with the protein, but the peptide region of the inhibitor only makes a handful of interactions with the protein. Together, the structure of the tGCN5/H3-(Me)CoA-20 complex reveals that small molecule compounds that optimise the interactions in the CoA pantetheine arm and pyrophosphate, and the lysine and acetyl region, combined with enhanced interactions at the peptide–protein surface, would provide a reasonable starting point for elaborating analogues of H3-(Me)CoA-20 with enhanced Gcn5/PCAF HAT specificity and potency (Figure 1B). In early 2008, a high-resolution X-ray crystal structure of a semi-synthetic heterodimeric p300 HAT domain in complex with Lys-CoA (Figure 1C) has been reported [35]. The structural data show that the inhibitor lies in a narrow tunnel, with the Lys portion surrounded by the enzyme. The authors propose for p300/CBP an unusual ‘hit-and-run’ (Theorell-Chance) catalytic mechanism, a specialised case of an ordered sequential mechanism with the ternary complex having a very short lifetime, distinct from other characterised HATs [35].
3.2 NaturalcompoundsIn addition to bi-substrate analogues, several natural products have been reported as HAT inhibitors including anacardic acid from cashewnut shell liquid [36], a polyisoprenylated benzophenone derivative from Garcinia indica fruit rind called garcinol that inhibits the HAT activity of both p300 and PCAF [37] with a mild-to-low micromolar IC50 value and EpigalloCathenin (EGCG), highly present in tea. This com-pound showed a strong anti-inflammatory action and seems to be useful in treating rheumatoid arthritis [38], suppressing osteoclast differentiation and arthritis in vivo. Moreover, curcumin, a polyphenolic compound from Curcuma longa rhizome, was shown to be a specific inhibitor of p300/CBP HAT activity with an IC50 of 25 μM [39]. Interestingly, this inhibitor was shown to be non-competitive with histone or Ac-CoA [39], indicating that it can directly inhibit p300/CBP through a covalent bond. Indeed, curcumin
contains two Michael acceptor motifs in its structure, so that it can covalently associate with p300 and CBP, cause enzyme inhibition in vitro and promote recognition by proteasome targeting enzymes in vivo. To confirm, at least in part, this hypothesis, [3H]curcumin was incubated with purified p300 and with CBP and PCAF immunoprecipitated (IP) by SKBr3 cells. After incubation, the purified or IP HATs were separated by SDS-PAGE, and a radioactive band corre-sponding to both p300 and CBP but not PCAF was clearly detected [40]. Moreover, the reduced form of curcumin, tetra-hydrocurcumin, when tested at the same conditions and concentrations as curcumin, failed in inhibiting p300 both in enzyme and functional tests (increase of acetylation levels of histone H3 and p53) [40]. These all data clearly demon-strate that the Michael acceptor activity of curcumin is required for its effect on p300.
3.3 SmallmoleculesyntheticinhibitorsIn addition to natural substances, a limited number of small molecules have been described as HATi. The γ-butyrolactone MB-3 has been recently discovered as a small, cell-permeable GCN5 inhibitor [41], and a series of isothiazolones have been found to inhibit p300 and PCAF activity, also showing antiproliferative properties against a panel of human colon (HCT116, HT29 and KM12) and ovarian (A2780, cisplatin resistant A2780, CH1, 41M and SKOV-3) tumour cell lines (50% growth inhibition = 0.8 to > 50 μM) [42]. Recently, a series of garcinol analogues (the LTK compounds) has been repor-ted as p300-specific HAT inhibitors (IC50 values = 5 – 7 μM) and inactive for PCAF [43]. In addition, a group of quinoline derivatives has been described by our group as HATi (Figure 2C) [42,44-48]. The first compounds have been discovered through a phenotypic screening in yeast, then validated by a GCN5-dependent gene transcription assay and by the determination of the H3 acetylation levels in Saccharomyces cerevisiae. When tested on the human leukaemia U937 cell nuclear extracts to evaluate their HAT inhibitory action, the quinoline derivatives were able to reduce the enzyme activity when used at 25 – 500 μM. In the U937 cell line, these compounds displayed some effects on cell cycle, apoptosis and granulocytic differentiation, being able to reduce the acetylation levels of histone H3 and/or α-tubulin. Differently from HDAC inhibitors [49-51], for HAT inhibitors a pharmacophore model has not been reported. By surveying the chemical structures of both natural product and syn-thetic molecule HAT inhibitors, we can suggest some chemical features that can play a role in HAT inhibition. In particu-lar, an acidic (carboxyl or enolizable ketone) function and/or an hydroxy group supported on an aromatic ring, and a lipophylic moiety (from the C3 chain of MB-3 to the C15 chain of anacardic acid, or to the isoprene moieties of garcinol and its analogues, including the alkyl substituents at the C2 position of our quinolines) seem to be key features useful for designing new HAT inhibitors. In comparison to our knowledge of the anticancer effects of HDACi [52-54], the
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OH
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Figure2.ChemicalstructuresofknownHATinhibitors.A. Bi-substrate analogues. B. Natural compounds. C. Small molecules.HAT: Histone acetyltransferase.
antitumour effects of HATi have been clearly less studied. This is probably owing to the fact that i) HAT inhibitors are still less efficient than HDAC is; ii) the molecular basis of their inhibition are far from understood; iii) the inhibitory doses are still high to be well adapted to cellular and biological models. However, several HAT inhibitors have been patented for many applications. Mainly garcinol, curcumin and anacardic acid are present in the espacenet database, curcumin (Figure 2B) being the most studied. Indeed, for this compound there are 487 patents and several inventions (US2008260695 [55]; WO2008096343 [A1] [56]) providing new strategies to treat and/or prevent cancer. The first invention includes fermented soy extract, oligomeric proanthocyanidin, epigallocatiechin gallate, spirulina, curcumin and Antrodia camphorata. A weight ratio of soy extract, oligomeric proanthocyanidin, epigallocatiechin gallate, spirulina, curcumin and A. camphorata is ∼ 12 – 30:1:4:2:1:1, more preferably, 12 – 20:1:4:2:1:1, most preferably, 12:1:4:2:1:1. The composition of the invention adhering to the described conditions shows high efficacy in the prevention and treatment of cancer. In addition, the weight ratio may be adjusted to meet different conditions. The composition is very safe and does not cause biological damage; hence, it can also be used as a food supplement. It is reported to be very efficient for prevention and treatment of cancers, including breast, prostate, blood, colorectal, uterine, ovarian, endometrial, cervical, testicular, malignant lymphoma, rhabdomyosarcoma, neuroblastoma, pancreatic, lung, brain, skin, gastric, liver, kidney and nasopharyngeal cancers. Preferably, it is used in the prevention and treatment of colorectal, lung, ovarian, breast, cervical and liver cancers. The second invention is related to the use of a composition comprising ethanol, curcumin and dissolved whole turmeric powder for the preparation of an alcoholic curcumin and turmeric liquid pharmaceutical composition for treating proliferative and other clinical disorders in human patients, the composition evidencing enhanced bioavailability vis-à-vis a comparable dosage of curcumin in dry powder form. This new composition will enhance the absorption of the curcumin to the intestine, leading to a greater bioavailability and efficacy of the compound in treating patients. To acknowledge there is a new application to treat Alzheimer disease
(AD; WO 2008131059 [A2] [57]). Codman, Shurtleff and Di Mauro proposed a new way to administrate curcumin prod-rugs intranasally and curcumin hybrids in a bolus of helium gas to the brain. Because curcumin is able to effectively act against many targets of AD, it has been hypothesised that the 4.4-fold lower incidence of AD in the Indian population between the ages of 70 and 79 is owing to the high dietary consumption of curcumin [58]. In those aged ≥ 80 years, age-adjusted AD prevalence in India is roughly one-quarter the rates in the US (4 versus 15.7%) [59]. Furthermore, several curcumin analogues have been also pat-ented (US2008234320 [A1]) [60]. Interestingly enough, these compounds have been reported to show anti-angiogenic activities [61] on several types of cancers and they seem to be useful in the treatment of rheumatoid arthritis [62] and other inflammatory states [63].
Anacardic acid (Figure 2B) is a low-permeable compound, and this limits its applications. At present, there are 72 patents, none of them dealing with therapeutic issues. However, there are several industrial applications involving this com-pound in the production of plastic materials, as presented by Durite Plastics (US2251547 [A] [64]). Actually, there are only 12 patents involving garcinol (Figure 2B), 3 for the reduction of body fat and 9 on its use as an antitumour agent or as an anti-inflammatory drug (Table 1). To obtain a lipase inhibitor with a high lipid absorption–inhibitory effect that is also useful as an anti-obesity drug or as an hyperlipidaemia inhibitor, a isoprenylated benzophenone derivative was synthesised, contained in the organic solvent extract of the body of a Garcinia plant or garcinol, preferably in the amount of 0.0001 – 5% (w/w). The benzo-phenone derivative showed a strong lipase-inhibiting activity. It can be extracted easily, processed or converted into derivatives. It results to be highly safe being preferably administered at a dose of ∼ 10 – 2,000 mg/day taken as polyisoprenylated benzophenone derivative. Extracts from the plants of the Hypericaceae family showed high anti-inflammatory and anti-carcinogenic activity (JP2000355536 [A] [65]; JP2000351728 [A] [66]). The extract is obtained by properly grinding fruit, pericarp, tree or bark of the plant and extraction treating the ground material or the sap of the
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Figure2.ChemicalstructuresofknownHATinhibitors(continued).A. Bi-substrate analogues. B. Natural compounds. C. Small molecules.HAT: Histone acetyltransferase.
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Table1.Garcinolpatentsasinespacenetdatabase.
Title:patentsforgarcinol Inventor Patentnumber Year
Composition for reduction of body fat Majeed Muhammed; Madmaev Vladimir
JP2008110996 (A) 2008
Bioavailable composition of natural and synthetic hca
Majeed Muhammed; Hadmaev Vladimir
US2002187943 (A1) 2002
Compositions comprising keratolin, a lipogenesis inhibitor and excipient for topical application, have a slimming effects on the face
Anti-mrsa active substance Iinuma Munekazu JP8259493 (A) 1996
A process for the extraction of garcinol hydroxycitric acid and anthocyanins that are useful in food industry as colouring additives from the plant kokum (Garciniaindica)
plant with an organic solvent of 1 – 100 times as much weight ratio as the raw material under several conditions at the boiling point of the organic solvent for 1 min – 8 weeks. Although several patents are present in literature, most of them deal with the enhancement of patient treatment. We hope that in future new and more active and specific analogues will be synthesised.
4. Curcumin:thedissectionofmoleculareffects
Among the listed HATi, curcumin, the most known inhibitor of p300, has been reported to display antitumour activities [67]. Suppression of cyclin D1, activation of caspase-8 and suppres-sion of the activity of NF-κB [68,69] have been reported to be key aspects of its anticancer effects. Moreover, numerous studies in animals have demonstrated that curcumin displays potent chemopreventive and therapeutic activities against a wide variety of tumours [70,71]. Also anacardic acid has been reported to sensitise cancer cells to ionising radiations [72] and to display antioxidant effects useful for anticancer effects [73]. Several reports describe many intracellular path-ways regulated by curcumin, including NF-KB [59,74-76], MMPs [77], TNF-A, AKT [78], oncogenic ras [79], src [80] and myc [81], the extracellular signal-regulated kinase (ERK) and the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathways. A key target point for antitumour ability of curcumin seems to be the
reactive oxigen species (ROS) production and the NF-KB inhibition. Notably, ROS activation could be responsible for inhibition of NF-KB transcriptional factor activity, and they seem causally related since that precedes NF-KB inhibition. In cancer models, the co-treatment of curcumin with antioxi-dants or ROS-inhibitors blocked the apoptosis induction, thus validating the ROS role in the chemotherapeutic ability of curcumin. Interestingly, Fang et al. [82] related the ROS production with the TrxR regulation, defining its ‘in vitro’ modification (IC50 = 3.6 μM) and the ‘ex vivo’ strongly induced NADPH oxidase activity that resulted in an increased generation of ROS. This report indicated a dual activity of curcumin, at lower concentrations as antioxidant (chemo-preventive drug) and at higher concentrations as pro-oxidant (chemotherapeutic drug). Most of its actions are related to the direct binding of the curcumin molecule to some proteins, as MD-2 [83], GSK-3β [84] and the curcumin molecular structure shows a per se antioxidant activity not related to the HAT enzymatic inhibition. Furthermore, cur-cumin showed enzymatic inhibitory activity on other pro-teins differently by HATs. Recently, it was reported that curcumin inhibits the catalytic activity of glyoxalase1 [85], affecting the anti-inflammatory and tumorigenic process in such a way that it can directly affect the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1), which regulates the stability of the tumour suppressor WT p53. Several side targets are known, but the complete picture about the effects
of curcumin is still incomplete. However, although curcumin is considered a promising drug for its anti-inflammatory and antitumour properties, few reports that describe some tum-origenic activities have also to be considered. Animals [86] were fed diets containing the turmeric extract at different concentrations for a period of 3 months (0.1, 0.5, 1, 2.5 and 5%) and 2 years (0.2, 1 and 5%). Hyperplasia of the mucosal epithelium was observed in the colon of rats that received 5% of the turmeric extract for 3 months in the diet. Despite this unfavourable effect and a significant increase in liver weight in rats and mice fed with concentra-tions of ≥ 0.5%, no signs of carcinogenic lesions were observed in these 3-month studies. However, toxic and car-cinogenic effects were observed when animals were fed with the turmeric extract for a period of 2 years. Thus, male or female rats that received turmeric extract had ulcers, chronic active inflammation, hyperplasia of the cecum or forestom-ach, or increased incidences of clitoral gland adenomas; these effects were mainly observed in the group fed with 5% turmeric extract. Likewise, mice fed with different concen-trations of the turmeric extract had increased incidences of hepatocellular adenoma (1% group) or carcinomas of the small intestine (0.2 and 1% groups). In the 2-year study of mice, a 0.2% turmeric extract in the diet was estimated to deliver aver-age doses of curcumin of ∼ 200 mg/kg/day body weight. Furthermore, in vitro studies revealed the induction of DNA damage after curcumin treatment, due both to the increase of ROS production and to the binding of curcumin on Topoisomerase I and II [87-89]. Indeed, curcumin has two elec-trophilic α,β-unsaturated ketones in its structure, which may react with nucleophilic groups through a covalent reaction (Michael addition, see earlier). These α,β-unsaturated ketones can react covalently with the thiol (SH) groups of cysteine residues of different proteins. Through this reaction, cur-cumin enhances the TOPO II–DNA complexes in leukae-mia cells. If high doses of this drug could be interesting for treating cancer cells, low concentrations could lead to the formation of secondary leukaemias as for the topoisomerase inhib-itors. In the same fashion, curcumin has the ability to suppress the activity of p53 [90], inducing its proteosomal degradation. Although these findings seem to indicate the action of cur-cumin against cancer, the possibility of certain pro-carcinogenic effects do exist. In lung cancer it was shown that the CBP gene is genetically altered in almost 15% of lung cancer cell lines and 5% of primary lung tumours [91]. Thus, point mutations and homozygous deletions of the CBP gene might be involved in the pathogenesis of a subset of lung carcinomas. Whereas these data fully confirm the role of HATs in cancer, it is clear that further studies will have to clarify the beneficial effects of HATi against cancer to allow their potential use in treatment of human diseases, and probably the generation of more specific inhibitors will avoid the formation of the shown in vivo side effects. These could be dependent on the binding of curcumin to other proteins and hence are not related to the HAT enzymatic inhibition.
5. TheuseofpatentedHATiinclinicaltrials
The epigenetic modulators are very promising enzymes to treat several type of human pathologies. One of the best example is related to HDAC inhibitors. Interestingly enough, even HAT inhibitors show antitumour effects, although they seem to be more effective as anti-inflammatory agents. As described in the previous section, curcumin is one of the most promising HAT inhibitors already in clinical trial against several pathologies, such as the AD, delirium, some mental disorders, leukaemia and haematological disorders, several type of neoplasms such as adenocarcinoma, sarcoma, osteosarcoma and so on (Table 2A). Of a total of 31 clinical trials, 10 have been already completed while the most part of the others is recruiting. Two clinical trials on AD (Table 2B) have already been completed but results are not yet available. However, in the study NCT00099710, 33 patients have been treated with the curcumin C3 complex (curcumin, demethoxycurcumin and bisdemethoxycurcumin) in two different concentrations. Curcumin effect seems to avoid directly the formation of amyloid placks, reducing AD insurgence in animal models [92]. Interestingly, in the NCT00164749 study 30 subjects with age > 50 have been treated with two concentrations of curcumin, 1 and 4 g/day, for 6 months. Although the complete results for this study are still not available, recently the authors made known a plasmatic increase of cholesterol. This finding, if supported by other studies, could mean that the effect of curcumin on AD is independent of cholesterol. Besides the analysis of the effects of curcumin on AD, two other studies, at present actively recruiting, will analyse its effects on rheumatoid arthritis and psoriasis (NCT00752154; NCT00235625). Lifestyle, humidity and stress are among the main factors causing or increasing dysregulation of the immune system. Essentially, cortisones excluded, there are no efficient drugs. In the NCT00752154 study, 20 patients will be treated with curcumin for 4 months with an acute dose of 12g/day. A similar dose was used to treat patients with psoriasis vulgaris in the NCT00235625 study for 16 weeks. This study is in Phase II clinical trial. However, curcumin and its derivates display interesting anti-inflammatory effects, most likely mediated by the inhibition of NF-KB and reduction of cytokines content. It is, thereby, possible that a combina-tion with TNF modulators, already present in clinical trials (trasduzumab), might result in a more efficient clinical outcome. All the other clinical trials that were found were on the treatment of cancer. Most of them are still recruiting volunteers, even if three are on colon cancer and pancreatic cancer (NCT00094445; NCT00027495; NCT00176618). In all these studies no results have been posted so far. In the first study 50 patients were enrolled and treated with curcumin (the concentration was not specified) [69]. The other two studies were completed and whereas one was on colon cancer prevention the other was on its treatment. A cohort of 24 patients was enrolled and treated for 24, 36, 48 and 72 h
at escalation doses. In the second study an acute dose of curcumin 250 mg p.o. b.i.d. was used to treat 60 patients. We hope that some results are posted soon.
6. Expertopinion
Drugs based on epigenetics are promising agents for the treatment of several human diseases. The inhibitors of the HDAC catalytic activity are in clinical trials, and one of them, vorinostat, is already used in the treatment of cutaneous T-cell lymphoma (CTCL). However, several other compounds, able to inhibit the enzymatic activity of histone modifier enzymes with a different range of action are now available and may turn useful for clinical development. The growing evidence that more enzymes participate in chromatin equilib-rium suggests that we are still far from understanding their role and the chemical targeting of their action. Also, novel enzymes get discovered daily and add levels of complexity to this already incomplete scheme.
Histone acetyltransferase inhibitors may find application in the treatment of several diseases such as cancer, diabetes and neurological disorders such as AD. On analysing the inhibitory activities of this class of compounds, two points have emerged: i) their specificity; and ii) the synthesis and potential availability of new inhibitors to cover the missing enzymes. One main criticism that might be addressed is related to the specificity issue and to the link between the inhibition of the catalytic HAT function and the biological effects. As described in the review, curcumin has the ability to bind other proteins and is not specific for HAT catalytic inhibition. Generally, this is owing to the molecular structure that has antioxidant activity per se, thus affecting the ROS content. To better define these effects, a global approach would be necessary to analyse the whole proteome and ‘enzymome’ affected by these inhibitors. Which effects are really mediated by HAT inhibition? Which HAT(s) have to be necessarily inhibited? These questions may find a solution in the near future. Despite these open questions, a main part of these compounds are in advanced clinical trials. Moreover, assuming that the link of dependence between the catalytic inhibition and the biological effects are true, a principal fact remains that only CBP/P300 and PCAF inhibitors have been reported and are available. Very recently, new compounds able to act on TIP60 have been synthesised [93]. TIP60 is known to be involved in several processes and bound to NRs, such as retinoic acid receptor (RAR). Probably, the analysis of TIP60 will help to identify possible inhibitors able to dissect its functions and be exploited in the clinic.
A third point for reflection is that many inventions tend to modify only the administration of the compound or its
chemical structure and do not deal with new chemical structures. This reflects the need of novel chemical and biochemical approaches to define innovative compounds. Indeed, often derivatives of known inhibitory compounds do not display improved biological capacities. Also, despite the presence of new anacardic acid analogues with improved effects in cellular systems, no patents have been applied for analogues of anacardic acid with improved permeability.
Finally, not knowing the complete picture of chromatin regulating enzymes complicates the definition of their functions and possible exploitation for the treatment of human diseases. We get the impression to unreveal a part of the picture, still missing the general view. A better view on the enzymatic function and on the role of known or novel inhibitors will certainly increase our knowledge and influence a focused use of HAT and chromatin enzyme modifiers. The knowledge that HAT inhibitors are part of a ‘growing’ field is the clear impression obtained from the present literature. There is the need for new chemical and biochemical approaches to select novel and more specific modulators to be applied for clinical purposes. Only a multidisci- plinary approach where the molecular understanding is synchronised and synergises with the biomedical efforts will pave the way for the finding of new drugs to cure human disease.
Epi-drugs show an interesting activity for the treatment of several pathologies. Histone acetyltransferase inhibitors are promising as anticancer or anti-inflammatory drugs. However, the decryption of their activities will certainly need time. For example, their effects on normal cells should be better investigated. The relationship between the use of curcumin in the Indian community and the low incidence of AD indicates the importance of the HAT modulatory compounds. The molecular events that underlie these (and other) capacities still need to be better defined and novel studies with combined molecular and biomedical strategies will be needed.
Acknowledgements
In memory of Ettore M. Schiavone, colleague and friend.
Declarationofinterest
The work in the authors’ laboratory has been supported by AIRC (Associazione Italiana per la Ricerca contro il Cancro), Ministero dell’Istruzione Università e Ricerca (PRIN2006), EU (LSHC-CT2005-518417; HEALTH-F4-2007-200767; HEALTH-F2-2007-200620), FIRB RBIP067F9E and RETI FIRB RBPR05NWWC_006.
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AffiliationFabio Manzo1, Francesco Paolo Tambaro1,2, Antonello Mai3 & Lucia Altucci†1
†Author for correspondence1Seconda Università degli Studi di Napoli, Dipartimento di Patologia generale, Vico L. De Crecchio 7, 80138, Napoli, IT Tel: +39 081 566 7569; Fax: +39 081 224 4840; E-mail: [email protected] of Houston, MD Anderson Cancer Center, Texas, USA3Sapienza Università di Roma, Istituto Pasteur, Fondazione Cenci-Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, P.le A. Moro 5, 00185 Roma, IT
