Identification of 5-Methoxyflavone as a Novel DNA Polymerase-Beta

Oct 30, 2015 - Cell-cycle reactivation is a core feature of degenerating neurons in Alzheimer's disease (AD) and Parkinson's disease (PD). A variety o...
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Identification of 5‑Methoxyflavone as a Novel DNA Polymerase-Beta Inhibitor and Neuroprotective Agent against Beta-Amyloid Toxicity Sara Merlo,† Livia Basile,‡ Maria Laura Giuffrida,§ Maria Angela Sortino,† Salvatore Guccione,‡,# and Agata Copani*,‡,§,# †

Department of Biomedical and Biotechnological Sciences and ‡Department of Drug Sciences, University of Catania, Catania, Italy § Institute of Biostructure and Bioimaging, National Research Council (CNR), Catania, Italy ABSTRACT: Cell-cycle reactivation is a core feature of degenerating neurons in Alzheimer’s disease (AD) and Parkinson’s disease (PD). A variety of stressors, including β-amyloid (Aβ) in the case of AD, can force neurons to leave quiescence and to initiate an ectopic DNA replication process, leading to neuronal death rather than division. As the primary polymerase (pol) involved in neuronal DNA replication, DNA pol-β contributes to neuronal death, and DNA pol-β inhibitors may prove to be effective neuroprotective agents. Currently, specific and highly active DNA pol-β inhibitors are lacking. Nine putative DNA pol-β inhibitors were identified in silico by querying the ZINC database, containing more than 35 million purchasable compounds. Following pharmacological evaluation, only 5-methoxyflavone (1) was validated as an inhibitor of DNA pol-β activity. Cultured primary neurons are a useful model to investigate the neuroprotective effects of potential DNA pol-β inhibitors, since these neurons undergo DNA replication and death when treated with Aβ. Consistent with the inhibition of DNA pol-β, 5methoxyflavone (1) reduced the number of S-phase neurons and the ensuing apoptotic death triggered by Aβ. 5-Methoxyflavone (1) is the first flavonoid compound able to halt neurodegeneration via a definite molecular mechanism rather than through general antioxidant and anti-inflammatory properties.

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fail to complete cell division, resulting in a genetic imbalance that is likely to be the ultimate cause of neuronal death.5,6 AD is not the only neurodegenerative pathology linked to neuronal cell-cycle events, as many others, including Parkinson’s disease (PD),7,8 frontotemporal dementia,9 amyotrophic lateral sclerosis,10 and temporal lobe epilepsy,11 are now known to share this same pathogenic mechanism. Experimental evidence has shown ectopic neuronal expression of the typical molecular machinery necessary for the G1/S phase transition in at-risk areas of the AD brain,12 as well as in AD mice models13,14 and Aβ-challenged in vitro models.15,16 The peculiarity of neuronal DNA replication during cell-cycle re-entry, however, is that it is carried out by DNA polymerase-β (DNA pol-β), a DNA repair enzyme that is known to perform de novo DNA synthesis only under some circumstances.17,18 In primary neurons, induction and activation of DNA pol-β have been shown to be CDKdependent and to have a causal role in Aβ-induced DNA replication and neuronal death.19,20 On the basis of these premises, DNA pol-β targeting appears as a novel, intriguing strategy against AD. As opposed to classical cytostatic drugs, selective DNA pol-β inhibitors might in fact represent a key to neuronal-specific cell-cycle inhibition.

lzheimer’s disease (AD) is a neurodegenerative pathology representing the leading cause of dementia in the Western world. The characterizing clinical manifestation is a progressive cognitive impairment that goes from mild in prodromal disease to severely disabling in advanced stages. From a molecular point of view, AD is a multifaceted pathology; thus multiple steps in the road that leads to disease onset and progression may represent potential pharmacological targets for preventive or therapeutic intervention. However, to date no diseasemodifying drugs are available for AD treatment, and current therapeutic interventions (i.e., acetylcholinesterase inhibitors donepezil, rivastigmine, and galantamine and NMDA receptor antagonist memantine) are aimed at providing only symptomatic relief. The key player in the pathogenesis of AD is recognized to be the amyloid beta protein (Aβ), a 39−42 amino acid peptide derived from the enzymatic cleavage of a type I membrane protein, the amyloid precursor protein (APP).1 The accumulation of Aβ(1−42) is believed to favor aggregation of monomeric Aβ(1−42) into neurotoxic oligomeric species that eventually lead to neuronal apoptosis.2 In the past decade a novel mechanism of Aβ-induced neuronal apoptosis has emerged: induction of an aberrant cell-cycle re-entry of differentiated neurons.3,4 According to what is known as the “cell-cycle hypothesis of AD”, postmitotic neurons that resume cell cycle are able to carry out only partial DNA replication but © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 27, 2015

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DOI: 10.1021/acs.jnatprod.5b00621 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and carboxyl and hydrogen bonding groups that matched the proposed pharmacophore. All of the nine compounds selected by virtual screening were first tested for potential pol-β inhibitory activity by the methylmethanesulfonate (MMS) sensitization assay, according to the protocol described in detail in the Experimental Section. Concentrations used for each of the tested compounds were 5, 10, 30, and 60 μM. As shown in Figure 1, when cells were exposed to alkylating agent MMS alone, cell viability as determined by the MTT assay was significantly reduced in both wild-type 92TAg and pol-β KO 88TAg fibroblasts. Three out of nine compounds tested showed intrinsic toxicity in the range 10−60 μM, with a reduction of cell viability up to 90% at the highest concentration (Figure 1A,B). These compounds were thus excluded as candidate molecules of interest. Five other compounds were instead devoid of intrinsic toxicity, yet inactive, as they exhibited no amplification of MMS toxicity in wild-type cells even at the highest nontoxic concentration used (Figure 1C,D) and were also excluded. Finally, among those screened, 5-methoxyflavone (1) was the only active compound able to significantly enhance toxicity of MMS on 92TAg cells, but not on 88TAg cells (Figure 1E,F). This effect was present in the range 10−30 μM and comparable to that of the reference compound dideoxycytidine (DDC) (Figure 1G). A slight but not significant reduction of cell viability appeared to be present in 88TAg cells treated with 30 μM 5methoxyflavone (1) alone, whereas at 60 μM intrinsic toxicity of 5-methoxyflavone (1) became significant on both 92TAg and 88TAg (Figure 1E,F). Thus, 10 μM was chosen as the maximal concentration for subsequent experiments. Selective inhibition of DNA pol-β by 5-methoxyflavone (1) was further tested in a cell-free assay developed to measure the enzyme’s gap-filling activity on a given DNA template. As shown in Figure 1H, 5-methoxyflavone (1; 1 or 10 μM) significantly reduced polymerase activity on a gapped substrate. The plant-derived triterpenoid oleanolic acid (OA) was used as a positive control for pol-β inhibition at a concentration of 50 μM, as suggested by the assay manufacturer, and its effects are reported in Figure 1H. A more detailed study of the interaction between the identified active compound and DNA pol-β was carried out by docking 5-methoxyflavone (1) into the 8 kDa domain of DNA pol-β, on the site enclosed between helix C and helix D, which represents the binding site of many natural and synthetic inhibitors of DNA pol-β.23 Docking scores, expressed in arbitrary units, are reported in Table 2 along with specific residues involved and type of interactions. Compound best poses (selected out of 50) were evaluated and compared to those of lithocolic acid (LCA) and OA, known to bind to the 8 kDa lyase domain of DNA pol-β and indirectly inhibit polymerase activity at the 31 kDa catalytic site.3,24 Interestingly, docking simulations generated low-energy binding modes and favorable binding interactions. 5-Methoxyflavone (1) showed a good affinity for DNA pol-β, as reflected by the MolDock score, although reference compounds seemed to bind the 8 kDa domain of DNA pol-β more tightly, likely as a result of their bigger, bulky structure and/or long-range electrostatic interactions (Table 2). In more detail, the chromone ring of 5methoxyflavone (1) intercalated into the hydrophobic cage formed by the side chain of residues Ala57, Ala59, Ala70, Gly56, and Gly66 between helix C and helix D of the inspected domain, as did the carbonaceous skeletons (or multiring scaffolds) of the reference compounds LCA and OA. Moreover,

The common drawback is that most current inhibitors are not selective in regard to other polymerases or to primase.3,21 We here performed a virtual screening of a database containing more than 20 000 natural and millions of drug-like compounds and selected nine compounds for their best scores. Compounds were validated by docking on the 8 kDa lyase domain of the enzyme, conferring higher probability of selectivity over other pols. Among the tested compounds, 5methoxyflavone (1) was identified as the only candidate compound endowed with the ability to inhibit DNA pol-β in multiple in vitro assays and to prevent cell-cycle initiation and subsequent neuronal apoptosis in Aβ-challenged primary neuronal cultures. Our results provide strong evidence that 5methoxyflavone (1) may represent a novel bioactive entity able to yield neuroprotection in AD.

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RESULTS AND DISCUSSION A virtual screening (VS) of natural and drug-like compounds was performed using the ZINC database to identify potential inhibitors of DNA pol-β at the 8 kDa domain of the enzyme. Such methodology is based on a condensed representation of the electrostatic and van der Waals fields surrounding a molecule, as described by the XED (extended electron distribution) force field.22 Namely, field points are placed in correspondence with the field extrema and used as a 3D query to search a multiconformational database for novel chemotypes with different molecular frameworks whose fields match the template. This field-based approach frees the chemist from working within structural constraints, enabling scaffold hopping and retrieval of nonobvious analogues. The list of the best matching hits is ranked using a similarity score (Sim) calculated as the average of shape (SSim) and field (FSim) similarity scores, each weighted at 50%. The similarity score ranges from 1 (perfectly aligned, virtually identical analogues) to 0 (misaligned, totally different molecules). In Table 1 similarity scores for the top nine ranked compounds are reported. Selected compounds are part of the TimTec Natural Compounds and Historical Collection. Substructures common to all molecules include a planar condensed aromatic system Table 1. Similarity Scores for Ligand-Based Virtual Screening compound

similarity score

ST046159 ST066909 ST5213293 ST000822 ST056000 ST5227035 ST069360 (1) ST5269668 ST011502

0.754 0.741 0.741 0.730 0.729 0.726 0.722 0.721 0.719 B

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Figure 1. MMS sensitization assay on wild-type 92TAg and pol-β-null 88TAg fibroblasts. Compounds were added 2 h before pulsing with MMS for 2 h and applied again after washing of the alkylating agent. Cell survival was measured 24 h after the beginning of the experiment. Compounds with intrinsic toxicity, in both 92TAg and 88TAg cells, are graphed in A and B. Compounds devoid of intrinsic toxicity, yet inactive, are graphed in C and D. 5-Methoxyflavone (1) was active on 92TAg cells (E), but not on 88TAg cells (F), similarly to the reference compound, dideoxycytidine (DDC) (G). In H direct DNA pol-β inhibition by 5-methoxyflavone (1) and reference compound, oleanoic acid (OA, 50 μM), in a cell-free assay. Values are the means ± SEM of 3−9 determinations. In E, #p < 0.05 vs MMS, and *p < 0.05 vs 1 μM 1; in F, *p < 0.05 vs 1 μM 1; in G, *p < 0.05 vs the respective control condition (as indicated by brackets); in H,*p < 0.05 vs reaction mixture alone (one-way ANOVA + Newman−Keuls test). C

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Table 2. MolDock Docking Scores ligand

MolDock scorea

H bond

LCA

−86.48

Asp74 (CO···OH)

OA 1 a

hydrophobic interactions

van der Waals Glu75, Thr67

−82.77

Ala57, Ala59, Ala70, Ala78, Gly56, Gly66, Leu77 Ala57, Ala59, Ala70, Val65

−68.25

Ala57, Ala59, Ala70, Ala78

Asp74, Glu71, Glu75, Lys60, Lys68, Thr67

Asp74, Glu71, Glu75, Ser55

long-range electrostatic interactions (r > 4.5) Lys60, (salt bridge), Lys35, Lys41, Lys61, Lys68, Lys72 Lys60, Lys61, Lys68, Lys72, Lys81, Thr67

Arbitrary units.

the same enzymatic residues (Asp74, Glu71, and Glu75) were involved in van der Waals interactions with the best scored configurations of both 5-methoxyflavone (1) and the reference compound OA.25 On the other hand, LCA showed also a Hbond and additional hydrophobic contacts with the enzyme. In the case of 5-methoxyflavone (1), differently from the best scored pose reported in Table 2, some lower-scored poses of the compound displayed the presence of H-bonds (not shown). This evidence suggests that an initial hydrogen bond network could intervene to selectively fit the molecule into the 8 kDa domain of DNA pol-β before a final enzyme/compound complex is stabilized by hydrophobic and van der Waals interactions. Evidence that LCA is a competitive inhibitor of template DNA binding to the 8 kDa domain25 suggests that 5methoxyflavone (1) might occupy its same site on the lyase region and compete with the template for interaction to the ssDNA-binding region of the pol-β. Once the activity of 5-methoxyflavone (1) as a DNA-pol-β inhibitor was established, we moved on to examine the biological effects of 5-methoxyflavone (1) as a neuroprotectant agent against Aβ toxicity. The Aβ(1−42) peptide has been shown to be toxic depending on its aggregation state, and oligomeric preparations have been demonstrated to induce neuronal death at very different concentrations, ranging from 5 nM to 5 μM.26 In our hands, oligomerized Aβ(1−42) yielded a consistently reproducible toxicity to primary neuronal cultures at 1 μM. For the experiments, 10 μM 5-methoxyflavone (1) was added to pure rat cortical neurons for 45 min prior to addition of 1 μM oligomeric Aβ(1−42) and maintained during the entire treatment. 5-Methoxyflavone (1) did not show any toxicity on neuronal cells up to a concentration of 10 μM by the MTT assay (not shown); see also Figure 3 A. In these experiments, OA proved, in our hands, to be unsuitable as a control, due to significant toxicity to primary neuronal cultures at the concentration necessary to inhibit pol-β in our cell-free assay (50 μM; data not shown). On the other hand, lower concentrations of OA (1−5 μM), not active on pol-β inhibition, have been previously shown to be protective against Aβ toxicity in cultured cortical neurons via antioxidant and antiexcitatory mechanisms.27 In order to determine if the ability of 5-methoxyflavone (1) to protect neurons from Aβ-induced death does indeed rely on inhibition of pol-β-dependent replication, apoptosis and cellcycle induction were evaluated by cytofluorimetric analysis of propidium iodide (PI)-labeled samples. Cell-cycle analysis appears as a necessary step in order to exclude that antioxidant and anti-inflammatory properties of flavones28 could exert antiapoptotic effects against Aβ with mechanisms other than pol-β inhibition itself. Neuroprotection against Aβ(1−42) toxicity was determined by analysis of apoptosis in the presence of 5 or 10 μM 5-methoxyflavone (1). The lower concentration

Figure 2. Best scored configurations of 5-methoxyflavone (1), LCA, and OA docked into the 8 kDa lyase domain of the rat DNA-pol-β (PDB code: 1BNO). Backbone of lyase domain is shown in a cartoon representation. Amino acid residues involved in the interaction are depicted in light blue stick format. Ligands are represented in stick format. H-bonds are shown as yellow dotted lines.

was chosen based on our previous experiments showing that although the compound was active at 1 μM in a cell-free system, it was ineffective on fibroblasts (see Figure 1E,F,H). Results show that the percentage of apoptotic neurons was not significantly affected by 5-methoxyflavone (1) alone (5 and 10 μM) compared to untreated control (Figure 3A). Apoptosis induced by oligomeric Aβ(1−42) (1 μM for 48 h) was significantly reduced in the presence of 10 μM 5-methoxyflavone (1) (Figure 3A), whereas 5 μM 5-methoxyflavone (1) showed a trend toward neuroprotection that did not reach statistical significance (Figure 3A). Further experiments were thus performed using 10 μM of the compound. Analysis of Sphase distribution showed that reduced apoptosis was paralleled by inhibition of Aβ-triggered cell-cycle re-entry in the presence of 10 μM 5-methoxyflavone (1), which did not affect cell-cycle distribution per se (Figure 3B). These data were D

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Figure 3. Effects of 5-methoxyflavone (1) on Aβ-triggered apoptosis and cell-cycle induction. Pure neuronal cultures were exposed to 5 μM (A) or 10 μM (A−C) 5-methoxyflavone (1) for 18 h (C) or 48 h (A, B) in the presence of 1 μM oligomeric Aβ(1−42). The percentage of apoptotic neurons (A) and the percentage of neurons in the S phase (B) were scored by cytofluorimetric analysis of propidium iodide-labeled samples. Alternatively, S phase neurons were scored after immunostaining for cyclin A2 followed by cytofluorimetric analysis (C). Values are means ± SEM of 3−8 (A) and 3 determinations (B, C). *p < 0.05 vs control (C); **p < 0.05 vs Aβ(1−42) (one-way ANOVA + Fisher’s LSD test).

further corroborated by analysis of expression of cyclin A2, a cell-cycle regulator synthesized at the onset of S phase and implicated in the control of DNA replication.29 5-Methoxyflavone (1) (10 μM for 18 h) was able to prevent the significant increase of cyclin A2-positive neurons induced by Aβ(1−42) alone (1 μM for 18 h; Figure 3C). Moreover, cell-cycle analysis experiments were carried out on actively cycling cells to exclude that 5-methoxyflavone (1) could yield non-DNA pol-βdependent inhibition of DNA replication. In fact, the lyase domain of DNA pol-β, to which 5-methoxyflavone (1) binds, shares structure similarity with the p58 subunit of the primase/ polymerase α complex.3 The 92TAg and 88TAg cell lines were synchronized in low-serum medium for 72 h (0.2% fetal bovine serum, FBS), followed by transfer to 10% FBS-supplemented medium for 15 h in the presence of 10 μM 5-methoxyflavone (1) or 8 mg/mL aphidicolin, a selective DNA pol-α inhibitor,30 as a positive control of cell-cycle inhibition. Results show that in both 92 TAg (Figure 4A) and 88 TAg (Figure 4B) cells, 5methoxyflavone (1) did not modify the percentage of S-phase cells compared to control conditions, as opposed to aphidicolin, which in both cell lines prevented DNA replication and stopped cells in the G0/G1 phase. This confirms that 5-methoxyflavone (1), at least at the concentration of 10 μM, which is neuroprotective against Aβ toxicity, does not interfere with replicative polymerases, including the primase/pol-α complex, nor does it prevent canonical cell-cycle progression through alternative mechanisms. 5-Methoxyflavone (1) belongs to the family of flavones, naturally occurring low molecular weight polyphenolic phytochemicals constituting a major subclass of a family of ubiquitous plant products known as flavonoids.28 The threering molecular scaffold of flavones is particularly amenable for development of nature-inspired compounds with different functionalities that confer selective pharmacological properties,28,31 with improved stability and safety and the advantage of an easier availability from large-scale chemical synthesis.33 Flavones have been previously shown to be capable of a variety of biological activities including anti-inflammatory, antimicrobial, antiallergic, antioxidant, antitumor, and cytotoxic actions.28 Structurally related flavone compounds have also been involved in neuroprotection, but with undefined mechanisms mainly involving antioxidant properties and radical scavenging.32

Figure 4. Lack of effects of 5-methoxyflavone (1) on cell cycle in murine fibroblasts. Wild-type 92TAg and pol-β-null 88TAg fibroblasts were synchronized in low serum (0.2% FBS) for 72 h, then returned to 10% FBS medium and exposed to 1 (10 μM) or aphidicolin (8 μg/ mL) for 15 h. The percentages of 92TAg (A) and 88TAg (B) cells in the different phases of the cell cycle were scored by cytofluorimetric analysis of propidium iodide-labeled samples. Values are means ± SEM of n = 4. *p < 0.05 vs other groups (one-way ANOVA + Newman−Keuls test).

Methoxylated flavones, in particular, display chemopreventive properties superior to their nonmethylated counterparts33 together with the advantage of a potentially greater degree of blood−brain barrier penetration due to their less polar/more lipophilic structure.34 Diverse biological actions have been identified for 5-methoxyflavone (1), including modulation of TRAIL-induced apoptosis in human leukemic cell lines,35 E

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vasodilatory activity on in vitro isolated rat aortic rings,36 gastroprotective activity,37,38 and antiproliferative activity in a number of cancer cell lines, such as prostate cancer LNCap cells.39 These latter effects are however obtained with EC50’s in the range 25−80 μM, i.e., much higher concentrations than the one we used here to selectively inhibit pol-β.39 Moreover, tumor cells like LNCap exposed to 5-methoxyflavone (1) appear to accumulate in G2/M phase, an effect typically related to genotoxicity and not yet characterized in detail,39 while cellcycle progression was not affected, in our conditions, in actively cycling murine fibroblast cell lines. The effort to identify and develop highly selective pol-β inhibitors is of even more critical importance, as recent evidence has shown pol-β-mediated cell-cycle induction to be a shared pathogenic mechanism in different neurodegenerative diseases. In this regard, of particular relevance is the recent work by Zhang et al.,7 confirming a causal role between pol-β induction and neuronal damage, as already fully characterized in AD3 and in Parkinson’s disease.7 DNA pol-β is a member of the X family of DNA pols, which is specialized in base excision repair (BER) processes.40 Lack of pol-β has been proven to lead to increased susceptibility to genotoxic agents,41 including AD-related stress.42 Nevertheless, absence of pol-β is not associated with a significant increase in spontaneous rate of DNA damage,41 granting the opportunity to explore DNA polβ inhibitors as drugs without expecting worrisome DNA damage. In support of this hypothesis is the evidence that BER can be carried out by at least two other human polymerases, DNA pol-λ, more closely related to pol-β,43 and DNA pol-ι.44 Altogether, our results identify and confirm for the first time 5-methoxyflavone (1) as a hit nature-inspired molecule endowed with biological activity as a DNA pol-β inhibitor and able to halt pol-β-mediated neurodegeneration.

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lithocolic and nervonic acid, the binding interactions of which were previously reported.23 Materials. Aβ(1−42) was purchased from Bachem Distribution Service (Weil am Rhein, Germany) and subjected to oligomerization as previously described.26 Briefly, Aβ(1−42) peptide was suspended in dimethyl sulfoxide (DMSO) to 5 mM and then diluted to 100 μM in ice-cold Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12; Gibco, Grand Island, NY, USA). The suspension was allowed to oligomerize overnight at 4 °C and was used at the final concentration of 1 μM in the presence of the ionotropic glutamate receptor antagonist MK-801 (1 μM; Sigma-Aldrich, St. Louis, MO, USA) to avoid the potentiation of endogenous glutamate.48 Control experiments were conducted under the same conditions except for the addition of the peptide. Natural compounds ST069360 (5methoxyflavone; 1), ST046159, ST00822, ST56000, ST001502, ST066909, ST5227035, and ST5269668 are included in the Natural Compounds and Historical Collection and were purchased from TimTec LLC (Newark, DE, USA). 5-Methoxyflavone (1)’s purity was 97%, as determined by NMR and mass spectrometry (TimTec catalog number 42079-78-7). Oleanolic acid and aphidicolin were purchased from Sigma. All screened compounds were solubilized in DMSO. Cell Lines. The 92TAg and 88TAg cell lines used for survival experiments were both from American Type Culture Collection (ATCC, Manassas, VA, USA). Both are mouse embryonic fibroblast lines. 88TAg is DNA polymerase beta null [−/−] derived from DNA pol-β null embryos at day 14.5 of gestation and established by transfection with an expression vector for SV40 large T antigen.49 The cells are transgenic for lambda LIZ (LacI/cII). This matched pair is wild type for DNA pol-ι.44 All cells were maintained at 5% CO2 and 37 °C in DMEM supplemented with 10% FBS and penicillin/ streptomycin (Invitrogen, Milan, Italy). For MTT experiments, cells were plated on 48-well microplates at a density of 5 × 103/well and serum-deprived at the time of treatment. Pure Neuronal Cultures. Neuronal cultures were obtained from Sprague−Dawley rats (Harlan Laboratories, Udine, Italy) at embryonic day 15 as previously described.48 Briefly, cortices were dissected in a Ca2+/Mg2+-free buffer, centrifuged at low speed, and mechanically dissociated in plating medium consisting of DMEM/ Ham’s F12 (1:1) supplemented with 10 mg/mL bovine serum albumin, 10 μg/mL insulin, 100 μg/mL transferrin, 100 μM putrescine, 20 nM progesterone, 30 nM selenium, 2 mM glutamine, 6 mg/mL glucose, 50 units/mL penicillin, and 50 μg/mL streptomycin. Cortical cells were plated at a density of 400 × 103 on 24-well Nunc microplates precoated with 0.1 mg/mL poly-D-lysine. To prevent non-neuronal cell proliferation, cytosine-β-D-arabinofuranoside (10 μM) was added to the cultures 18 h after plating and kept for 3 days before medium replacement. This method yields >99% pure neuronal cultures.48 All animal experimental procedures were carried out in accordance with the directives of the Italian and EU regulations for care and use of experimental animals and were approved by the Institutional Animal Care and Use Committee of the University of Catania. Human DNA Pol-β Assay. DNA pol-β inhibition was evaluated with a specific DNA pol-β assay kit (Profoldin, Hudson, MA, USA). The assay was carried out strictly following the manufacturer’s instructions. Briefly, a reaction mixture containing a gapped DNA template, dNTPs (both supplied with the kit), and DNA pol-β (Trevigen, Gaithersburg, MD, USA) was incubated for 30 min at RT with or without the compound to be tested. The formation of repaired duplexes, which, unlike gapped DNA, selectively incorporate a fluorescent dye in the presence of Reagent U (both supplied by the kit) was determined by measuring at 535 nm with the excitation wavelength at 485 nm in a fluorimeter. OA (50 μM) was used as a positive control for inhibition. Activity was evaluated as fluorescent signal to no-enzyme-background ratio. Methylmethanesulfonate Sensitization Assay. Selective DNA pol-β inhibition was assessed as the ability of tested compounds to amplify toxicity induced by the alkylating agent MMS by preventing DNA repair, an effect present in wild-type but not in pol-β-null cells. The wild-type 92TAg and the pol-β knockout 88TAg cell lines were

EXPERIMENTAL SECTION

Ligand-Based Virtual Screening. A field-point-based virtual screening from the ZINC database was carried out to identify potential inhibitors of DNA pol-β at the 8 kDa domain of the enzyme using the software Forge 10.0.1.45 For consistency with results obtained with a previous version of the software,3 the XED Force Field version 2 was used;22 all other settings were left as default. A 3D reference template to query the database was built by aligning the putative bioactive conformations of three known DNA pol-β inhibitors, namely, palmoic acid (PA), koetjapic acid (KJA), and Mordant Blue (MB).23 The reference template was validated using a customized data set of reported DNA pol-β inhibitors: Mordant Blue, naphthochrome green (NCG), carbenoxolone (CBX), 3-(4-carboxyphenyl)-2,3-dihydrotrimethyl-1-indene-5-carboxylic acid (CPIC), biquinoline-dicarboxylic acid (BQD), glycyrrhizic acid (GA), 4,4′-biphenyl dicarboxylic acid (BPDC), 4,4′-(hexafluoroisopropylidene)bis(benzoic acid) (HFPB).23 Putative bioactive conformers of PA, KJA, and MB as carboxylates were obtained by docking into the 8 kDa domain of DNA pol-β using the Molegro Virtual Docker software (MVD; version 6.0); then the ZINC database was searched. ZINC is a free database of commercially available compounds for virtual screening that contains over 35 million purchasable compounds in ready-to-dock, 3D formats, inclusive of many drug-like molecules.46 Docking Analysis. Docking was carried out by the molecular docking algorithm MolDock Optimizer, using the MolDock Grid scoring function as implemented in Molegro Virtual Docker software, version 6.47 3D structures of ligands were obtained and minimized by Sybyl-X 2.0. The crystal protein structure of the 8 kDa domain of rat DNA pol-β was downloaded from Protein Data Bank (http://www. rcsb.org/pdb, PDB code: 1BNO). An Intel Core i7, ram memory of 16 GB operating under Linux/Ubuntu 10.04, was used to perform all calculations. The docking software performance was checked using F

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incubated for 2 h with the tested compound prior to a 2 h pulse with MMS (500 μM for 92TAg and 250 μM for 88TAg cells). Medium was changed to remove MMS, and the tested compound was added for further incubation for 24 h prior to MTT viability assay. MTT Assay. Cell viability was assessed by the 3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Cultures were incubated with MTT (0.9 mg/mL) for 2 h at 37 °C, then lysed by incubation with DMSO for 15 min at 37 °C. Formazan production was evaluated in a plate reader (absorbance = 570 nm). Aβ-Induced Neurotoxicity Experiments. Pure neuronal cultures were subjected to medium change 24 h before the experiment. Aβ(1− 42), oligomerized as described in the Materials paragraph of this section, was added to the medium at a final concentration of 1 μM. Cells were incubated for 18−48 h, based on the assay to be performed. Apoptosis and Cell-Cycle Analysis. Neuronal cells were harvested with mild trypsinization and immediately fixed with icecold 70% ethanol in phosphate-buffered saline (PBS). Cells were incubated for at least 24 h at −20 °C, then washed with PBS and incubated with RNase (100 μg/mL; Sigma) for 1 h at 37 °C. Propidium iodide (50 μg/mL) was added 10 min prior to simultaneous cytofluorimetric analysis of apoptosis and cell-cycle distribution. DNA content and ploidy were assessed by using a Beckman-Coulter FC500 flow cytometer, and cell-cycle distribution profiles were analyzed with the ModFit LT software program. Apoptotic cells were scored from the area of hypoploid DNA preceding the G0/G1 DNA peak. Immunostaining and Flow Cytometric Analysis. Neuronal cells were harvested with mild trypsinization and immediately fixed with 4% paraformaldehyde for 30 min at 4 °C. Cells were permeabilized with 0.1% Triton-X100 solution in PBS for 10 min on ice and blocked with 3% BSA solution in PBS for 30 min. Cells were then processed for immunostaining by overnight incubation at 4 °C with rabbit anti-Cyclin A2 (1:200; Abcam, Cambridge, UK), followed by incubation for 1 h at RT with Alexa-Fluor 488-conjugated anti-rabbit secondary antibody (1:400; Invitrogen). Positive cells were scored by analysis on a Beckman-Coulter FC500 flow cytometer. Statistical Analysis. All experiments were run at least in triplicate, and data were analyzed using SigmaPlot 12.5 statistical software. Normal distribution was determined with the Shapiro−Wilk test, and mean comparison was performed with one-way ANOVA. Statistical significance level was always set to p < 0.05.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (39) 095 7384212. E-mail: [email protected]. Notes

The authors declare no competing financial interest. # Co-senior authors.

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ACKNOWLEDGMENTS The authors thank Cresset, New Cambridge House, Bassingbourn Road, Litlington, Cambridgeshire, SG8 0SS, UK (www.cresset-group.com), for the software Forge 10.0.1; Dr. P. Tosco and Dr. M. Mackey (Cresset, New Cambridge House) for technical support and helpful discussions. This work was supported by MIUR project 105/04 (A.C.).

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DOI: 10.1021/acs.jnatprod.5b00621 J. Nat. Prod. XXXX, XXX, XXX−XXX

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