DOI: 10.1002/cbic.200900721

The Identification of a Novel Natural Activator of p300Histone Acetyltranferase Provides New Insights into theModulation Mechanism of this EnzymeFabrizio Dal Piaz,*[a] Alessandra Tosco,[a] Daniela Eletto,[a] Anna L. Piccinelli,[a]

Ornella Moltedo,[a] Silvia Franceschelli,[a] Gianluca Sbardella,[a] Paolo Remondelli,[a]

Luca Rastrelli,[a] Loredana Vesci,[b] Claudio Pisano,[b] and Nunziatina De Tommasi[a]

Introduction

The acetylation of proteins is a dynamic event that involvesthe enzymatic activity of histone acetyltransferases (HATs) andhistone deacetylases (HDACs). HATs primarily acetylate (andprevent positive charges from forming on) the e-amino groupsof specific lysines in histones, as well as in transcription factors(i.e. , p53) and other nuclear proteins (i.e. , a-tubulin). As a con-sequence, HATs modulate gene transcription, nucleosome as-sembly, and DNA repair. Altered HAT and HDAC function leadsto several diseases, ranging from cancer to neurodegenerativedisorders ;[1–3] because not all such diseases involve hypoacety-lation, but also hyperacetylation, compounds able to enhanceor repress histone acetyltransferase (HATs) activity could bepromising therapeutic agents. Several members of the HATfamily have been identified so far, and many of them, that is,p300/CBP,[4, 5] TAFII250,[6] and SRC-1,[7] function as transcriptionalcoactivators. p300 plays a pivotal role in a variety of cellularprocesses including cell cycle control, differentiation, andapoptosis. It is a potent histone acetyltransferase that can acet-ylate core histones within nucleosomes as well as free histoneforms, and mutations in the p300 gene are associated withdifferent human cancers and other human diseases.[8, 9] Thisprotein is recruited to the chromatin template through directinteractions with the transcriptional activator proteins, thus en-hancing transcription by acetylation of nucleosomal histoneslocated at the promoter regions of target genes.[10]

Significant progress in the field of cancer therapy has beenmade by the use of HDAC inhibitors as antineoplastic agents;in fact, a number of HDAC inhibitors are currently undergoingclinical evaluation for efficacy in the treatment of human

tumors.[11–15] In contrast, HAT enzymes have been less investi-gated as therapeutic targets and, as a matter of fact, only asmall number of HAT modulators have been reported so far,the first being Lys-CoA and H3-CoA-20, selective for p300 andPCAF, respectively.[16] Their main disadvantages were low cellpermeability and metabolic instability.

Some natural compounds, such as anacardic acid, curcumin,and garcinol were described lately as PCAF and p300 inhibi-tors.[17] Garcinol, a polyisoprenylated benzophenone isolatedfrom Garcinia indica fruit rind, was found to affect both in vitroand in vivo enzymatic activity of HATs, and inhibits the HATactivity-dependent chromatin transcription and downregulatesglobal gene expression.[18] Moreover, garcinol was also report-ed as a cell-permeable HAT inhibitor and a potent antiapoptot-ic agent in tests with the HeLa cell line.[19]

These observations prompted us to study the effects thatseveral polyisoprenylated benzophenone derivatives (PBDs)containing garcinol-like structural features isolated from natu-ral sources have on p300 HAT activity. Structurally, PBDs feature

Many severe human pathologies are related to alterations ofthe fine balance between histone acetylation and deacetyla-tion; because not all such diseases involve hypoacetylation,but also hyperacetylation, compounds able to enhance orrepress the activities of histone acetyltransferases (HATs) couldbe promising therapeutic agents. We evaluated in vitro and incell the ability of eleven natural polyisoprenylated benzophe-none derivatives to modulate the HAT activity of p300/CBP, anenzyme that plays a pivotal role in a variety of cellular process-es. Some of the tested compounds bound efficiently to the

p300/CBP protein: in particular, guttiferone A, guttiferone Eand clusianone inhibit its HAT activity, whereas nemorosoneshowed a surprising ability to activate the enzyme. The abilityof nemorosone to penetrate cell membranes and modulatehistone acetylation into the cell together with its high affinityfor the p300/CBP enzyme made this compound a suitable leadfor the design of optimized anticancer drugs. Besides, the stud-ies performed at a cellular and molecular level on both theinhibitors and the activator provided new insights into themodulation mechanism of p300/CBP by small molecules.

[a] Dr. F. Dal Piaz, Dr. A. Tosco, Dr. D. Eletto, Dr. A. L. Piccinelli, Dr. O. Moltedo,Dr. S. Franceschelli, Prof. G. Sbardella, Prof. P. Remondelli, Prof. L. Rastrelli,Prof. N. De TommasiDipartimento di Scienze Farmaceutiche, Universit� degli Studi di SalernoVia Ponte Don Melillo 1, 84084 Fisciano (SA) (Italy)Fax: (+ 39) 089969602E-mail : fdalpiz@unisa.it

[b] Dr. L. Vesci, Dr. C. PisanoResearch and Development DepartmentSigma-Tau, Via Pontina Km 30,400, 00040 Pomezia, Roma (Italy)

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a highly oxygenated and densely substituted bicyclo-[3.3.1]nonane-2,4,9-trione core decorated with C5H9 (prenyl) orC10H17 (in several isomeric forms, geranyl, etc.) side chains.They can be divided into three classes: type-A PBDs, whichhave a C(1) acyl group and an adjacent C(8) quaternary center;type-B PBDs, which bear a C(3) acyl group; and the rare type-CPBDs, which have a C(1) acyl group and a distant C(6) quater-nary center.[20, 21] In our study, four type-B PBDs (compounds 1,3, 4 and 10) and eight type-A PBDs (compounds 2, 5–9, 11and 12) were studied. Seven of them (compounds 5–9, 11 and12) showed a secondary cyclization involving the hydroxylatedenolic group of the core and pendant olefinic groups, afford-ing furan- or pyrano-fused structures.

Our studies demonstrated that all the type-B PDBs wereable to inhibit the HAT activity of p300 with a potency compa-rable to that of garcinol. Surprisingly, nemorosone, the onlytype-A compound tested thatdoes not have secondary cycliza-tion, enhanced the acetyltrans-ferase activity of p300 both invitro and into the cells. The iden-tification of a new HAT activatorrepresents an important discov-ery for its potentiality as an anti-cancer drug, considering thatvery few small compounds en-hancing the p300 activity havebeen described.[22, 23] Therefore,the ability of nemorosone topenetrate cell membranes andenhance histone acetylation to-gether with its high affinity forp300 enzyme made this com-pound a suitable lead to designnew drugs. Moreover, a compari-son between the interaction ofinhibitory and activating com-pounds with the p300 HATdomain by means of a panel ofchemical and biological ap-proaches provided new insightsinto the modulation mechanismof p300 by small molecules.

Results and Discussion

The binding affinity of elevenisoprenylated molecules (com-pounds 2–12, Scheme 1) to-wards the human histone acetyltransferase (HAT) domain ofp300 (p300/HAT) as well as theirability to modulate the biologi-cal activity of the enzyme wereevaluated by SPR experiments,mass spectrometry (MS) analy-ses, enzymatic assays and in

vitro tests. The achieved results were then compared withthose obtained on garcinol (compound 1), which was selectedas positive control.

Kinetic and thermodynamic study of the interaction withp300/HAT

Surface plasmon resonance allows the measurement of kineticand thermodynamic parameters of ligand–protein interactions.An SPR-based binding assay as implemented with Biacoretechnology was used to investigate the binding between theHAT domain of p300 and compounds 1–12. Human recombi-nant p300/HAT was immobilized on a biosensor chip and thetwelve PBDs were injected at different concentrations (from0.05–10 mm) over the protein surface; the binding of eachcompound was read out in real time as the change in mass at

Scheme 1. Structures of the polyisoprenilated benzophenones. 1 Garcinol, 2 Nemorosone, 3 Guttiferone A, 4 Gut-tiferone E, 5 Propolone A, 6 Propolone B, 7 Porpolone C, 8 Propolone D, 9 Porpolone D peroxide, 10 Clusianone,11 Garcinielliptone, 12 Hyperbone B.

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the sensor surface. After injection, running buffer was flowedover the surface and dissociation of the compounds from thesurface was observed. This assay allowed us to assess howcompounds 1–12 associate and dissociate from the protein inreal time and gave detailed information on their interactionwith p300/HAT (Figure 1). Five out of the twelve tested benzo-phenones (compounds 1, 2, 3, 4, and 10) interacted efficientlywith the immobilized protein, as demonstrated by the concen-tration-dependent responses and the clearly discernible expo-nential curves during both the association and dissociationphases. SPR experiments carried out on compounds 5–9, 11and 12 also produced good sensorgrams, but showed verylow and/or concentration-independent responses; this sug-gests negligible interactions of such molecules with p300/HAT.To evaluate possible unspecific bindings, compounds 1–12were also injected on an immobilized bovine serum albumin(BSA), and no interaction was observed (data not shown).

SPR data were used to evaluate kinetic and thermodynamicparameters for compounds 1–12/enzyme complex formation.The good quality of the acquired sensorgrams allowed the

curves to be fit to a single-site bimolecular interaction model(A + B = AB); this indicates a 1:1 small molecule: p300/HAT stoi-chiometry for all the observed complexes. Each constant wascalculated by fitting at least 15 curves, which were obtainedby injecting the investigated benzophenones at six differentconcentrations, ranging from 0.05–10 mm (Table 1). On thebasis of these data, some structure–activity relationship analy-sis on the twelve PBDs was performed.

Compound 2 was the only type-A PBD that interacted withp300; it showed a high affinity for the immobilized protein(0.25 mm) and a low kinetic dissociation constant (3.2 10�4 s�1).No complex formation was observed upon injection of com-pounds 5–9, 11–12 onto p300/HAT surface: these compoundsare structurally related to compound 2, the main differenceconsisting of the presence of a secondary cyclization on 5–9,11–12, whereas compound 2 shows the free enolic functionon the bicyclo[3.3.1]nonane-2,4,9-trione core. This finding indi-cated a critical role for the core hydroxylated enol involvingC(2), C(3) and C(4) in the interaction with p300, because nega-tive results were obtained for compounds carrying a furan-

Figure 1. SPR interaction analysis of compounds 1–12 binding to immobilized p300/HAT. SPR response data (sensorgrams). Each compound was injected atfive different concentrations (0.05, 0.25, 1, 5 and 10 mm).

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fused (7–9, 11–12) or a pyrano-fused (5–6) structure, regard-less of the core carbon atoms involved.

The four type-B benzophenones tested (1, 3–4, 10) showedsimilar affinities for immobilized p300/HAT, and generatedcomplexes with KD values ranging from 0.1–0.8 mm and kd

values of approximately 4 � 10�3 s�1. Compound 3 was the bestligand for the protein; thus, this suggests that the presence ofa further isoprenyl chain on the core increases its affinity forp300/HAT. On the other hand, the higher KD measured for com-pound 10 demonstrated the importance of the phenolic func-tion in the interaction of these compounds with the protein.These results are in agreement with the hypothesis of Kunduand co-workers ; this suggests that the critical role played by C-13 and C-14 -OH groups of garcinol in its recognition of HATdomain of p300.[24, 25] Observing the kinetic constants, the kd

value measured for compound 2 was clearly lower then thoseobserved for the other interacting compounds. This differencewas possibly dependent on the different PBD classes to whichthese compounds belong.

A direct comparison between the sensorgrams obtained inthe SPR experiments performed on different compounds (Fig-ure 2 A) leads to further considerations: responses generatedby the interaction between p300/HAT and 1 or 3 are very simi-lar at any concentration, both in the association and in the dis-sociation phase; this suggests that these compounds interactwith the protein through a comparable mechanism. On theother hand, sensorgrams obtained for 2 showed a clearly dif-ferent curve shape, particularly concerning the dissociationphase. 1 and 3 completely dissociated from the p300 surfacewithin minutes after the end of the association phase, whereasthe complex between the protein and compound 2 was verystable and a separate injection of 50 mm glycine was requiredto completely regenerate the chip surface. The peculiar kd cal-culated for the interaction of compound 2 with p300/HAT sug-gested a binding mode different from that of 1 and 3.[26] Inter-estingly, injection of different concentrations of N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide(CTPB), a well-characterized semisynthetic activator of HATs,onto the p300/HAT chip surface[22] produced a sensorgramcomparable to that acquired for 2 (Figure 2 A); these results

suggested that the same protein region can be involved in theinteraction of p300 with these two compounds.[27] Besides, SPRanalyses were performed by injecting compound 1, 2 or 3 onp300/HAT chip surface in presence of 0.5 mm acetyl coen-zyme A (AcCoA). Under such conditions, interaction of 1 and 3with the immobilized protein was significantly reduced(Table 2) and some association was observed only through in-jection of the two compounds at high (>1 mm) concentrations.This result is in agreement with the competitive inhibitionmechanism previously described for garcinol.[25] On the otherhand, kinetic and thermodynamic constants measured for 2 inthe presence of the enzyme cofactor were only slightly differ-ent for those described above (Table 2); thus this suggests dif-

Table 1. Thermodynamic (KD) and kinetic (kd) dissociation constants mea-sured for the interaction of compounds 1–12 with p300/HAT.

Compound KD [mm] kd � 10�4 [s�1]

1 0.40�0.05 36.9�5.12 0.25�0.03 3.2�1.03 0.10�0.04 39.0�8.24 0.39�0.08 40.0�6.35 no binding6 no binding7 no binding8 no binding9 no binding

10 0.81�0.03 43.4�6.311 no binding

Data are reported as mean values �SEM. The mean was calculated usingat least 15 independent values.

Figure 2. Thermodynamic analysis of small molecules-p300/HAT interactions.A) High resolution interaction analysis of compounds 1, 2, 3 and CTPB, asemisynthetic p300 activator, binding to immobilized p300. Each compoundwas injected at five different concentrations (0.05, 0.25, 1, 5 and 10 mm).B) Thermodynamic dissociation constants for the interaction of 2 and 3 withimmobilized p300 were measured at different temperatures in order to eval-uate the enthalpy and entropy changes involved in complexes formation.

Table 2. Thermodynamic (KD) and kinetic (kd) dissociation constants mea-sured for the interaction of compounds 1–3 with p300/HAT in the pres-ence of 0.5 mm AcCoA.

Compound KD [mm] kd � 10�4 [s�1]

1 12. 3�1.6 (6) 108.3�15.1 (6)2 0.36�0.15 (15) 3.8�1.7 (15)3 15.8�2.7 (6) 125.6�21.3 (6)

Data are reported as mean values �SEM. The number of independentvalues used to calculate the mean are in parenthesis.

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ferent binding sites for AcCoAand compound 2 on the protein.

To better understand the driv-ing forces of the interactionsbetween the protein and com-pounds 2 or 3, the temperaturedependence of each interactionwas studied by using SPR at sixdifferent temperatures, rangingfrom 5–30 8C (Figure 2 B). In thecase of compound 2, a least-squares linear fit (correlation co-efficient R = 0.98) yielded aDHvan’t Hoff of 31�4 kJ mol�1 fromthe slope and a DS of 240�20 J mol�1 K�1 from the y-axisintercept. For compound 3, aDHvan’t Hoff of 62�5 kJ mol�1 anda DS of 346�28 J mol�1 K�1 (cor-relation coefficient R = 0.98)were calculated. Therefore, inboth the cases the favorable DSovercomes the unfavorable DHand drives the association be-tween p300 and the small mole-cules; this demonstrates that hy-drophobic interactions playdominant roles in the formationof both the complexes. This finding is consistent with thechemical structure of those compounds. However, entropy andenthalpy changes involved in the two cases were dissimilar,suggesting again different binding sites for compounds 2 and3 on p300/HAT.

Enzymatic assays

In order to evaluate the ability of compounds 1–12 to modu-late the enzymatic activity of p300/HAT, colorimetric assayswere performed: the acetyltransferase activity of human re-combinant p300 was measured in the presence of three differ-ent concentrations (1, 5 and 10 mm) of each compound andthen compared to that measured in the presence of the vehi-cle, set as 100 % (Figure 3). Garcinol (1) inhibited HAT enzymat-ic activity in a concentration-dependent manner: again, our re-sults were comparable with those reported.[25] As expected, allthe type-B benzophenones showed a similar inhibitory activity ;interestingly, a good correlation between the measured SPR af-finities and the inhibitory potencies of these compound wasobserved. On the other hand, compound 2 exhibited a clearlydifferent effect, and the incubation of recombinant HAT withthis compound increased the acetyltransferase activity of theenzyme, that was almost doubled in the presence of 10 mm ofcompound 2. As expected, compounds 5–9 and 11–12, whichgave negative results in the SPR experiments, showed negligi-ble effects on p300 biological activity.

Assessment of histone acetylation levels

To directly evaluate changes in the acetylation levels of H4 andH3 into the cells following incubation with compounds 1–3,LC/MS analyses were performed on histone proteins extractedfrom control and treated HeLa cells.[28, 29] Representative spectraused for analysis of histones extracted from control and treat-ed cells are reported in Figure 4 A and B: seven differentlymodified forms were detected both for H4 and H3, and wereidentified on the basis of their respective molecular masses.The analysis of H4 in the control cells revealed the presence ofapproximately 69 % of monoacetylated (including the mono-and dimethylated) forms, 27 % of the diacetylated forms, and4 % of the triacetylated forms. For H3, approximately 65 % ofmonoacetylated, 32 % of the diacetylated, and 3 % of the tria-cetylated forms were detected. A 24 h incubation of HeLa cellswith 10 mm of compounds 1–3 affected the acetylation levelsof the two proteins (Figure 5 A and B). Incubation of the cellswith compound 1 decreased the acetylation levels of both pro-teins; specifically, relative abundance of the triacetylated H3and H4 decreased by 44 % and 45 % respectively, whereas diac-etylated species showed less evident variations (�4 % for H3and �10 % for H4). On the other hand, higher amounts of themonoacetylated forms were detected for both proteins (about

+10 %). Similar data were gathered after treatment with com-pound 3 : also in this case, lower amounts of the di and triace-tylated species were detected (�3 % and �38 % for H3, and�10 and �37 % for H4) and increments in the relative abun-dances of the mono-acetylated proteins were observed (+5 %for H3 and +7 % for H4). These variations were significant and

Figure 3. Enzymatic activity of p300/HAT was tested in the presence of compounds 1–12. The effect of each mole-cule on the enzyme was evaluated at three different concentration (1, 5 and 10 mm) and compared with the activ-ity shown by the pure enzyme in the presence of 2 % DMSO (100 %).* p<0.05 vs DMSO.

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comparable to those observed for other HAT inhibitors.[29] Onthe other hand, incubation of HeLa cells with 10 mm com-pound 2 produced a decrement in relative abundance ofmonoacetylated H3 and H4 (�8 and �11 %, respectively) andincrements of di and triacetylated forms of the proteins (+6and +53 % for H3, and +21 and +32 %, respectively).

Because the effect of the treatment with 1–3 was affectedby the deacetylation events occurring in the cell, similar experi-ments were performed on cells coincubated with suberoylani-lide hydroxamic acid (SAHA), a potent HDAC inhibitor.[30] Underthese experimental conditions, highly acetylated H3 and H4forms were observed (Figure 4 C and D); however, the acetyla-tion level variations observed following the treatments withcompounds 1–3 were more pronounced than those detectedin the previous experiment (Figure 5); this clearly confirmedour hypothesis. The variations in Figure 5 C and D were calcu-lated considering the acetylation of cells incubated with SAHA(control). In particular, 1 and 3 again produced similar inhibito-ry effects on histone acetylation, as inferred by the lower ace-tylation levels measured for both H3 and H4. Conversely, incu-bation of SAHA-treated cells with 2 produced an evident incre-ment of highly acetylated species (+95 % H3-4Ac, +75 % H3-3Ac; +120 % H4-5Ac), even if a lower compound concentrationwas used, if compared to the previous experiment. In fact, inthis experiment we were forced to use 5 mm of compound 2instead of 10 mm, owing to the additive effect played by HDACinhibition and HAT activation that led to very high histone ace-tylation levels that seriously perturbed cells.

These data confirmed the results of in vitro enzymatic tests,and demonstrated that compounds 1 and 3 inhibit enzymaticacetylation of histones also into the cell, whereas compound 2acts as an efficient enhancer of histone acetylation.

Antiproliferative tests on human tumor cell lines

The antiproliferative activity of 1–12 was evaluated on differ-ent human tumor cell lines (NB4 promyelocytic leukemia, NCI-H460 nonsmall cell lung carcinoma, HCT-116 colon cancer cells,HeLa cervical cancer cells) ; obtained results are summarized intable 3: compounds 1–5 and 10 showed IC50 values rangingfrom 3.2–11.6 mm towards all the tested cell lines, and no selec-tivity was found toward a specific tumor hystotype (seeTable 3). It is remarkable that five out of the six cytotoxic com-pounds were the same, giving positive results in both SPRanalyses and enzymatic assays.

Effect on cell cycle

The effects of treatments with compounds 1–3 on HeLa cellcycle were evaluated by flow cytometry analyses. Cell cycle dis-tributions observed after 48 h of incubation with such com-pounds are reported in Figure 6. While compound 1 and 3 donot cause any significant change on HeLa cell cycle, compound2 induced a sensible increase in G1 phase population, with aconsequent decrease of proliferating cells, in a concentration-dependent manner.

Figure 4. Mass spectrometry analysis of histones from differently treatedcells. Deconvoluted ESI-MS spectra of: A)–B) H3 and H4 isolated from HeLacells untreated (black line) or treated with 10 mm 1 (grey line) or 10 mm 2(broken line); C)–D) H3 and H4 isolated from HeLa cells treated with 5 mm

SAHA (black line), with 5 mm SAHA and 10 mm compounds 1 (grey line), orwith 5 mm SAHA and 5 mm 2 (broken line). Ac: acetylation; Me: methylation.

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Conclusion

Cellular homeostasis is regulated by a fine balance betweenhistone acetylation and deacetylation. Any imbalance in thisprocess leads to aberrant acetylation patterns, with hypoacety-lation at loci that should be transcriptionally active and hyper-acetylation at genes that should be repressed, both effectsresult in disease manifestation. Cancer, diabetes, asthma andcardiac hypertrophy are some of the pathologies related to analteration of the fine balance between histone acetylation anddeacetylation; because not all such diseases involve hypoace-tylation, but also hyperacetylation, compounds able to en-hance or repress HATs activity could be promising therapeutic

agents.[31] Therefore, there is anincreasing interest in moleculesable to modulate HATs activity,both activating or inhibitingthese enzymes. The first knowncell-permeable small-moleculeHAT inhibitor is garcinol, an iso-prenylated benzophenone deriv-ative isolated from Garciniaindica fruit rind.[18, 19] Some semi-synthetic derivatives of garcinolhave been tested that show asimilar inhibitory potency and, insame cases, better selectivity.However, all the tested com-pounds were B-type PDB.[32, 33] Inthe light of the interesting inhib-itory activity reported for garci-nol toward p300/HAT activity, wetested eleven PDBs in order toidentify new modulators of thisenzyme. We investigated their

ability to interact with p300/HAT and modulate its enzymaticactivity. In order to explore biologically relevant chemicalspace, we selected type-A and -B PDBs that had significantstructural differences. Moreover, many complementary experi-mental approaches have been used to investigate the interac-tion between selected compounds and p300, as well as theeffect of these PDBs on biological activity of the enzyme invitro and into the cells.

Our findings demonstrated a high affinity of the three B-type PBDs tested (compounds 3, 4 and 10) towards p300/HAT;structural differences between them only slightly influencedtheir interaction with the enzyme, even if some role is playedby the presence of the phenolic groups and by the position ofisoprenyl group, as inferred by the KD values measured by SPRexperiments. Concerning biological effects, the four B-type

Figure 5. Variations of the abundance of differently acetylated forms of H3 and H4 histones after various treat-ments, in respect to that of the control cells which was set to zero, as determined by LC/MS analyses. A)–B) H3and H4 acetylation levels variations in cells incubated with compounds 1–3 (10 mm), compared with cells incubat-ed with vehicle. C)–D) H3 and H4 acetylation levels variations in cells incubated with 5 mm SAHA (S) and com-pounds 1 or 3 (10 mm) or 2 (5 mm), compared with cells incubated with 5 mm SAHA.

Figure 6. Effects of compounds 1–3 on HeLa cell cycle. Relative percentagesof the cells in G1, S and G2/M phases following 48 h incubation with 5 and10 mm of compound 1, 2 or 3 are shown and compared with untreatedcells. * p<0.05 vs control cells.

Table 3. Antiproliferative activity measured for compounds 1-12 towardsdifferent cell lines.

Compound NB4[a] NCI-H460[a] HCT-116[a] HeLa[a]

1 9.2�1.3 8.5�0.9 10.5�2.8 9.8�1.52 4.8�0.5 5.0�0.3 6.8�0.3 5.2�0.53 5.7�1.4 4.2�0.3 5.9�0.5 11.6�1.14 10.4�0.2 5.4�1.3 6.4�0.6 11.3�1.55 6.8�0.2 6.3�1.1 4.4�0.3 10.8�1.86 >20 23�4.7 16.4�2.3 >207 >20 >20 16.3�1.3 >208 >20 >20 >20 >209 >20 >20 >20 >20

10 4.4�0.7 8.3�0.6 3.2�0.8 9.6�2.011 >20 >20 >20 >2012 >20 >20 >20 >20

[a] NB4, promyelocytic leukaemia cells ; NCI-H460, nonsmall cells lung car-cinoma; HCT-116, colon cancer cells ; HeLa, cervical cancer cells. Data arereported as mean IC50 (mM) �SEM, in which IC50 is compound concentra-tion required to afford 50 % reduction in cell growth after 24 h incuba-tion.

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PDBs showed a similar inhibitory potency towards p300 bothin vitro or into the cells. Interestingly, the only A-type PDB thatefficiently interacts with p300 (compound 2) acts as an activa-tor of the enzyme and enhances histone acetylation also intothe cells. Because during the process of transcription, p300 isrecruited on the chromatin template and enhances the tran-scription acetylating promoter proximal nucleosomal histones,the identification of a new p300 activator represents a very in-teresting finding for its potentiality as an anticancer drug. Ac-tually, very few small compounds that enhance p300 activityhave been described.[22, 23] Therefore, the ability of nemorosoneto penetrate cell membranes and modulate histone acetylationin the cell, together with its high affinity for the p300/CBPenzyme, make it a suitable lead compound to design opti-mized drug.

The different behavior of structurally related compounds isonly partially surprising: a previously published paper demon-strated that compounds that differ in the methylation of aphenol or in the cyclization of an isoprenyl group showed adifferent substrate selectivity, possibly changing their bindingmode to the enzyme.[25, 34] Analogously, we observed that A-type PDBs carrying both a furan-fused (compounds 7–9, 11-12) or a pyrano-fused (5, 6) structure were completely inactivetowards p300. Kundu and co-workers suggested that garcinoland related compounds can modulate HAT activity by destabi-lizing its three-dimensional structure;[24] on this basis, the acti-vating effect showed by compound 2 could be explained interms of stabilization of the enzyme conformation. This hy-pothesis is inferred by our SPR data: the low kinetic dissocia-tion constant measured for compound 2 is diagnostic for ahardly dissociable complex, consistent with a stable tertiarystructure. Further conformational studies will be required tocompletely elucidate this point. However, these data shed newlights on the molecular mechanism underlying p300 modula-tion by hydrophobic small molecules.

Finally, our data demonstrate that compounds 2 and 3 canpenetrate cell membranes and modulate histone acetylationinto the cell ; besides, these compounds show antiproliferativeproperties and it is worth noting that compound 2, the onlyp300/HAT activating molecule, blocks cells in G1 phase; thissuggests a molecular mechanism involving cell cycle check-points. These observations, together with the high affinity to-wards p300/HAT observed for compounds 2 and 3 make themsuitable lead compounds to design new optimized drugs.

Experimental Section

Materials: Amine coupling reagents N-hydroxysuccinimide (NHS),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride(EDC), and sodium ethanolamine hydrochloride, (1 m, pH 8.5) werepurchased from Biacore AB (Uppsala, Sweden). Ampoules of ster-ile-filtered dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO). Recombinant p300/HAT was purchasedfrom Tebu-bio (Magenta, Italy). HAT Activity Colorimetric Assay Kit(JM-K322–100) was purchased from MBL (Milan, Italy). CTPB waspurchased from Alexis Biochemicals (Vinci, Florence, Italy). Com-pound 1 (garcinol) was purified from Garcinia Indica rind. In brief,G. indica dried fruit rind (kind gift of Hillgreen Herbals, Bangalore,

India) (250 g) was extracted with ethanol (70 g). Part of the ethanolextract (6.0 g) was submitted to silica gel column chromatography(4 � 1 m, 50 g of silica gel for 1 g of crude extract), eluting withchloroform followed by increasing concentrations of MeOH (be-tween 1–40 %) in CHCl3. Fractions (25 mL) were collected, analyzedby TLC (silica gel plates, in CHCl3 or mixtures CHCl3/MeOH 99:1,98:2, 97:3, 9:1), and grouped into 16 fractions. Fraction 3 (200 mg)was subjected to RP-HPLC on a C18 m-Bondapak column (30 cm �7.8 mm, flow rate 2.0 mL min�1) with MeOH/H2O (4:1, v/v) to yieldpure compound 1 (35 mg, tR = 35 min). Compounds 2–12 werepurified from Cuban propolis as described elsewhere.[35] Suberoyla-nilide hydroxamic acid (SAHA) was a kind gift from Prof. A. Mai.[36]

Kinetic and thermodynamic study of the interaction with p300/HAT: SPR analyses were conducted by using a Biacore 3000 opticalbiosensor equipped with research-grade CM5 sensor chips (BiacoreAB).[37] Using this platform, two separate p300/HAT surfaces, oneBSA surface and one unmodified reference surface were preparedfor simultaneous analyses. Immediately after chip docking, theinstrument was primed with water and the sensor chip surfaceswere preconditioned by applying two consecutive injections(20 mL) of each of the following solutions (at a flow rate of100 mL min�1): NaOH (50 mm), HCl (10 mm), SDS (0.1 %, w/v), andH3PO4 (10 mm). Prior to each interaction analysis, the biosensordetector response was normalized (automated procedure) and theinstrument was primed five times with running buffer (10 mm

NaH2PO4, 150 mm NaCl, 0.5 % DMSO, pH 7.4). Data were collectedat 2.5 Hz. Proteins (100 mg mL�1 in 10 mm sodium acetate, pH 5.0)were immobilized on individual sensor chip surfaces at a flow rateof 5 mL min�1 by using standard amine-coupling protocols[38] toobtain densities of 8–12 kRU. Compounds 1–12 were dissolved inDMSO (100 %) to obtain 4 mm solutions, and diluted 1:200 (v/v) inPBS (10 mm NaH2PO4, 150 mm NaCl, pH 7.4) to a final DMSO con-centration of 0.5 %. Compounds concentration series were pre-pared as twofold dilutions into running buffer: for each sample thecomplete binding study was performed using a 6 points concen-tration series, typically spanning 0.05–10 mm, and triplicate aliquotsof each compound concentration were dispensed into single-usevials, capped, and randomized in the instrument’s auto samplerrack. Included in each analysis were multiple blank samples of run-ning buffer alone. Five of these blanks were analyzed at the begin-ning of the analysis and the remaining blanks were interspersedthroughout the analysis for double-referencing purposes.[39] Bind-ing experiments were performed at 25 8C, by using a flow rate of50 mL min�1, with 60 s monitoring of association and 200 s monitor-ing of dissociation. Regeneration of the surfaces was performed bya 140 s injection of 50 mm glycine (pH 9.5) followed by an Extra-Clean wash command that automatically flushes the sample deliv-ery system with running buffer. SPR response data (sensorgrams)were zeroed on both the response and time axes at the beginningof each injection and double referenced.[39] First, bulk refractiveindex changes were corrected for by subtracting the responsesgenerated over an unmodified reference surface from the bindingresponses generated over the albumin surfaces. Any systematic ar-tefacts observed between the p300/HAT and reference flow cellswere corrected by subtracting the response generated by an aver-age of the buffer injections from the binding responses generatedby compounds injections. Hallmarks that make this data set agood one include concentration-dependent responses, overlappingof replicate injections, and clearly discernible exponential curvatureduring both the association and dissociation phases.[40] Simple in-teractions were adequately fit to a single-site bimolecular interac-tion model (A+B = AB), yielding a single KD. Sensorgram elabora-tions were performed using the Biaevaluation software provided

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Modulation of p300 Activity by Small Molecules

by Biacore AB. To perform SPR analyses in the presence of AcCoA,experiments were carried out as previously described but usingPBS containing 0.5 mm AcCoA as running buffer. To determine theenthalpy change and the entropy change of the p300/HAT interac-tions with compounds 2 and 3, the equilibrium dissociation con-stants provided by the ratios of the kinetic rate constants weremeasured at 5, 10, 15, 20, 25, and 30 8C. Van’t Hoff analysis was car-ried out assuming DH and DS of the interaction vary negligiblywith temperature, using the following equation: lnKD =DHvan’t Hoff/RT-DS/R where R is the universal gas constant (8.31 J mol�1 K�1) andT is the absolute temperature.

Enzymatic activity assays: The p300 activity assays were per-formed by a colorimetric kit (JM-K322–100, MBL) using active re-combinant p300/HAT as positive control and acetyl-CoA as a cofac-tor. Acetylation of peptide substrate by active HAT produced thefree form of CoA, which serves as an essential coenzyme for NADHproduction; developed NADH was detected spectrophotometrically(440 nm) upon reaction with a tetrazolium dye. Activity assayswere performed in a 96 well plate and the reactions were conduct-ed at 37 8C for 3 h, after which a colorimetric reading at 440 nmwas performed. Each compound was dissolved in DMSO andtested at three different concentration (1, 5 and 10 mm) in tripli-cate, in the presence of 2 % DMSO; because some benzophenonesare colored, blank wells containing all the components except theenzyme were used as specific background well, and the readingsobserved were subtracted from those obtained for respective test-ing compound; calculated O.D. were compared with the value ob-served in the absence of any testing compound and in the pres-ence of DMSO (2 %; 100 % of enzymatic activity), subtracted of itsblank well reading.

Histone acetylation levels measurements: LC/MS analyses of his-tone proteins extracted from control and treated HeLa cells wereperformed as reported elsewhere.[41] HeLa cells were cultured inDMEM medium (Sigma, St. Louis, USA) supplemented with fetalbovine serum (10 % (v/v) ; Cambrex, East Rutherford, USA), penicillin(100 U mL�1) and streptomycin (100 mg mL�1, Cambrex) at 37 8C ina CO2 (5 %) atmosphere. In the first experiment cells were incubat-ed with compound 1, 2 or 3 (10 mm) and control cells with DMSO,in the second experiment cells were incubated with 5 mm SAHAplus 10 mm compound 1 or 3, or with 5 mm SAHA plus 5 mm com-pound 2. After 24 h cells were harvested and washed three timeswith PBS (1 � , Sigma), then suspended in lysis buffer (10 mm TrispH 8, 1 mm KCl, 1.5 mm MgCl2 and 1 mm DTT) supplemented withprotease inhibitor cocktail (Sigma) and with sodium butyrate(10 mm) to prevent histone deacetylation. All subsequent manipu-lation were performed at 4 8C. After incubation for 30 min on arotator, nuclei were collected by centrifugation for 10 min at10 000 g. The nuclear pellets were suspended in H2SO4 (0.4 m) and,after incubation for 1 h, they were centrifuged for 15 min at12 000 g. Histones contained in the supernatant were precipitatedby adding TCA (33 %) followed by overnight incubation. After cen-trifugation at 10 000 g, for 30 min, hystones pellets were washedtwice with acetone, air-dried and finally dissolved in water (50 mL).LC/MS analyses were carried out using a Q-TOF mass spectrometerequipped with an electrospray ion source (Q-TOF premier-Waters)coupled with an Alliance HPLC system (Waters). Reversed-phasechromatographic separation of histones was performed on a C4Jupiter column (2.1 � 50 mm id, 5 mm, 300 �, Phenomenex, Tor-rance, CA, USA) using a gradient elution from 30:70 to 50:50 A/B(v/v), in which A is water/TFA (0.1 %) and B is acetonitrile/TFA(0.1 %), over 90 min, at a flow rate of 0.2 mL min�1. The columnwas equilibrated with the initial mobile phase for 10 min before

the next injection. The ESI system employed a 4.5 kV spray voltage(positive polarity), a capillary temperature of 180 8C and a conevoltage of 50 V. The mass chromatograms were recorded in totalion current (TIC), within 650 and 2000 m/z. Data were acquired byusing the MassLinx software (Waters). Mass calibration was per-formed by means of multiply charged ions from a separate in-jection of horse heart myoglobin (average molecular mass:16 951.5 Da). All masses are reported as average values. Analyseswere performed injecting a 1 mg mL�1 solution (20 mL) of purifiedhistone proteins. Each population of differently modified histoneswas accurately quantified by measuring the total ion current pro-duced by the respective species.[42] Each set of quantitative datawas obtained as means of three analyses of two independent his-tones preparations.

Antiproliferative tests on cancer cell lines: To assess the cytotox-icity of compounds 1–12, three different cancer cell lines wereused: HCT-116 colon carcinoma cells grown in McCoy’s 5 A con-taining FCS (10 %), NB4 promyelocytic leukemia and NCI-H460 non-small cell lung carcinoma cells grown in RPMI with FCS (10 %),HeLa cells grown as described above. Chemicals were dissolved indimethylsulphoxide (DMSO) and added for different times to thecells in exponential phase of growth (1.0 � 105 cells/plate). Cytotox-icity was determined by using the trypan blue dye exclusion test.To analyze the cytotoxic potency of the drugs, IC50 values wereevaluated by using the ALLFIT program. Each determination wasrepeated three times.

Cell cycle analysis: HeLa cells were seeded in 12-well plasticplates at 1,5 � 105 cells per well. After 24 h, 5 and 10 mm of eachcompound was added to the cells for 24 h and 48 h. At the end oftreatments cells were collected with a hypotonic buffer (0.1 %sodium citrate, 0.1 % Triton X-100 and 50 mg trypan blue ml�1 pro-pidium iodide; 300 mL) and incubated in the dark for 30 min at4 8C. Samples were analysed by a FACS-Calibur flow cytometerusing the Cell Quest software (Becton Dickinson, North Ryde, NSW,Australia) and analysed with standard procedures using the ModFitLT software. All the experiments were performed in triplicate.

Statistical analysis: All experiments were performed at least intriplicate and the results were expressed as means � standarderror of the mean (SEM) or as means � standard deviation (SD).Differences between treatment groups were analyzed by the Stu-dent t test. Differences were considered significant when p <0.05.

Acknowledgements

The authors thank Prof. A. Mai for SAHA for MS based histoneacetylation levels measurements.

Keywords: histone acetyltransferase activators · massspectrometry · natural products · polyisoprenylatedbenzophenones · surface plasmon resonance

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Received: November 25, 2009

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Modulation of p300 Activity by Small Molecules