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NEUROPHARMACOLOGY

Novel Isoquinolinone-Derived Inhibitors of Poly(ADP-ribose) Polymerase-1: Pharmacological Characterization and Neuroprotective Effects in an in Vitro Model of Cerebral Ischemia

Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Firenze, Italy (A.C., E.M., M.C., R.P., R.B., D.E.P.G., F.M.); and Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Perugia, Italy (E.C., G.C., M.M., R.P.)

Received January 8, 2003; accepted February 18, 2003.

	Abstract

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Excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme catalyzing the transfer of ADP-ribose units from NAD to acceptor proteins, induces cellular energy failure by NAD and ATP depletion and has been proposed to play a causative role in a number of pathological conditions, including ischemia/reperfusion injury. In this study, we used an in vitro enzyme activity assay to characterize a series of newly synthesized isoquinolinone derivatives as potential PARP-1 inhibitors. Several compounds displayed powerful inhibitory activity: thieno[2,3-c]isoquinolin-5-one (TIQ-A) displayed a submicromolar IC₅₀ of 0.45 ± 0.1 µM, whereas the 5-hydroxy and 5-methoxy TIQ-A derivatives had IC₅₀ values of 0.39 ± 0.19 and 0.21 ± 0.12 µM, respectively. We then examined the neuroprotective effects of the newly characterized compounds in cultured mouse cortical cells exposed to 60 min of oxygen and glucose deprivation (OGD). When PARP-1 inhibitors were present in the incubation medium during OGD and the subsequent 24-h recovery period, they significantly attenuated neuronal injury. TIQ-A provided neuroprotection even when added to the culture 30 min after OGD and was able to reduce the early activation of PARP induced by OGD as detected by flow cytometry. When the IC₅₀ values observed in the PARP-1 activity assay for selected compounds were compared with their IC₅₀ values for the neuroprotective activity, a significant correlation (r = 0.93, P < 0.01) was observed. Our results suggest that TIQ-A and its derivatives are a new class of neuroprotectants that may be helpful in studies aimed at understanding the involvement of PARP-1 in physiology and pathology.

To study the biological role of PARP-1, a range of strategies such as antisense oligonucleotides, gene deletion, expression of dominant-negative forms (D'Amours et al., 1999), and RNA interference (Gan et al., 2002) have been used. In addition, several classes of competitive PARP-1 inhibitors have been reported. Relevant examples include benzamide (Banasik et al., 1992), 3,4-dihydro-5-hydroxy-1(2H)-isoquinolinone (5OH-DIQ) (Arundel-Suto et al., 1991), 3,4-dihydro-5-[4–1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone (DPQ) (Suto et al., 1991), and 6(5H)-phenantridinone (PND) (Banasik et al., 1992) (Fig. 1). Recently, it has been proposed that adenosine, inosine, or hypoxanthine may act as endogenous PARP-1 inhibitors (Virag and Szabo, 2001). The disclosure of a number of high-resolution crystallographic structures of the PARP-1 catalytic site complexed with various inhibitors has represented a major breakthrough in the understanding of the molecular basis of PARP-1 inhibitor binding (Ruf et al., 1996, 1998). Indeed, all the crystallized inhibitors show a conserved pattern of interactions which include: 1) a hydrogen-bonding interaction between the mandatory amido group of inhibitors in an anti disposition and Gly863; and 2) a - interaction between an aromatic ring of inhibitors and Tyr907-Tyr896. To extend the available information on the structure-activity relationship for competitive PARP-1 inhibitors, we have recently reported a thorough quantitative structure-activity relationship/docking study based on the analysis of as many as 46 competitive PARP-1 inhibitors (Costantino et al., 2001). As a continuation of this work and with the aim of characterizing new PARP-1 inhibitors, we now report the effects of a new series of isoquinolinone derivatives on PARP-1 enzymatic activity and in an in vitro model of cerebral ischemia [oxygen and glucose deprivation (OGD) in primary cultures of mixed cortical cells].

View larger version (15K): [in this window] [in a new window]   Fig. 1. Molecular structures of the known PARP-1 inhibitors benzamide, 5OH-DIQ, DPQ, and PND.

	Materials and Methods

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Materials. Glutamate and PND were purchased from Sigma-Aldrich (Milan, Italy). [Adenine-2,8-³H]NAD (1–5 Ci/mmol) was obtained from PerkinElmer Life Sciences (Milan, Italy). Lactate dehydrogenase (LDH) activity was quantified using the cytotoxicity detection kit (LDH) from Roche Diagnostics (Monza, Italy). DPQ and partially purified (90%) bovine PARP-1 were purchased from Alexis Corporation (Vinci, Italy). The polyclonal antibody directed against poly(ADP-ribose) (PAR; LP96-10) was from Alexis Corp. (Vinci, Italy). The anti-glial fibrillary acidic protein (GFAP) fluorescein isothiocyanate (FITC)-conjugated antibody and the phycoerythrin-conjugated goat anti-rabbit IgG were purchased from Molecular Probes Europe (Leiden, The Netherlands).

5OH-DIQ and the compounds shown in Fig. 2, i.e., 3,4-dihydro-5-mercapto-isoquinolin-1(2H)-one (compound 1), [3,4-dihydro-5-oxoisoquinolin-1(2H)-one]-benzoic ester (compound 2), 3,4-dihydro-5-ethynyl-isoquinolin-1(2H)-one (compound 3), 3,4-dihydro-5-hydroxy-isoquinolin-2(1H)-one (compound 4), [3,4-dihydro-5-oxo-isoquinolin-2(1H)-one]-benzoic ester (compound 5); 6-hydroxy-2,3,4,5-tetrahydro-benzo[c]azepin-1-one (compound 6); 5-(4-piperidin-1-yl-but-2-ynyloxy)-3,4-dihydro-2H-isoquinolin-1-one (compound 7), 5-(5-piperidin-1-yl-pent-1-ynyl)-3,4-dihydro-2H-isoquinolin-1-one (compound 8); 6-(4-piperidin-1-yl-butoxy)-2,3,4,5-tetrahydro-benzo[c]azepin-1-one (compound 9), 2-methyl-8-(4-piperidin-1-yl-butoxy)-3H-quinazolin-4-one (compound 10), thieno[2,3-c]isoquinolin-5-one (TIQ-A), 9-hydroxythieno[2,3-c]isoquinolin-5(4H)-one (compound 11), and 9-methoxythieno[2,3-c]isoquinolin-5(4H)-one (compound 12) were synthesized as described elsewhere (Pellicciari et al., 2003). The structures of the new compounds were determined by ¹H NMR and ¹³C NMR with a Bruker AC 200 MHz spectrometer (Newark, DE). All derivatives displayed a high-performance liquid chromatography purity >95%, as detected with a Shimadzu LC-10 workstation (Kyoto, Japan) equipped with an SPD-10A UV-Vis detector and using a LiChrospher Si60 (Merck KGaG, Darmstadt, Germany) (250 x 5 mm, 5 µm) column for compounds 5OH-DIQ and 1 through 10 or a LiChrospher 100 RP-18 (250 x 5 mm, 5 µm) column for compounds TIQ-A, 11, and 12. The flow rate was 0.8 ml/min, the detection was carried out at 210 and 254 nm, and the mobile phase was a mixture of CH₂Cl₂/MeOH (LiChrospher Si60) or H₂O/MeCN + 0.1% trifluoroacetic acid (LiChrospher 100 RP-18).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Molecular structures of newly synthesized isoquinolinone, DPQ, and PND derivatives.

Assay of PARP-1 Activity and Inhibition Studies. PARP-1 activity was evaluated as previously described (Moroni et al., 2001). Briefly, the enzymatic reaction was carried out in a final volume of 100 µl consisting of 20 mM Tris-HCl (pH 8.0), 20 mM MgCl₂, 5 mM dithiothreitol, 20 µg of sonicated calf thymus DNA, 0.2 µCi of [adenine-2,8-³H]NAD, and 0.13 U of partially purified bovine PARP-1. To evaluate the inhibitory potency of the tested compounds, different concentrations were dissolved in 50% dimethyl sulfoxide, and then 10 µl were added to the enzymatic reaction. An equal amount of the vehicle was also added to the control samples. The mixture was incubated at 37°C for 1 h, and the reaction was terminated by adding 500 µl of 50% trichloroacetic acid followed by brief centrifugation. After two gentle washes of the pellet with 1 ml of distilled water, the radioactivity incorporated from [adenine-2,8-³H]NAD into proteins was evaluated by liquid scintillation spectrometry.

Oxygen-Glucose Deprivation in Cortical Cell Cultures. Cultures of mixed cortical cells containing both neuronal and glial elements were prepared, used at 14 days in vitro, and exposed to OGD as previously described in detail (Pellegrini-Giampietro et al., 1999a,b). Briefly, culture medium was replaced by a glucose-free balanced salt solution saturated with 95% N₂/5% CO₂ and heated to 37°C. Multiwells were then sealed into an airtight incubation chamber equipped with inlet and outlet valves, and 95% N₂/5% CO₂ was blown through the chamber for 10 min to ensure maximal removal of oxygen. The chamber was then sealed and placed into the incubator at 37°C for 60 min. OGD was terminated by removing the cultures from the chamber, replacing the exposure solution with oxygenated medium, and returning the multiwells to the incubator under normoxic conditions. The extent of neuronal death was assessed 24 h later. In this system, 60 min of OGD induced a neuronal damage that was approximately 75% of the maximal degree of neuronal injury achieved by exposing the cultures for 24 h to 1 mM glutamate. OGD-induced cell injury was quantitatively evaluated by measuring the amount of LDH released from injured cells into culture media 24 h following exposure to OGD, as previously described (Pellegrini-Giampietro et al., 1999a,b). The LDH level corresponding to complete neuronal death (with no glial death) was determined for each experiment by assaying sister cultures exposed to 1 mM glutamate for 24 h. Background LDH release was determined in control cultures not exposed to OGD and subtracted from all experimental values. The resulting value correlated linearly with the degree of cell loss estimated by observation of cultures under phase-contrast microscopy or under bright-field optics following 5 min of incubation with 0.4% trypan blue, which stains debris and nonviable cells.

Measurement of PARP Activity by Flow Cytometry. PARP activity was evaluated in cortical cells exposed to OGD by cytofluorimetric measurement of PAR formation according to Affar et al. (1999), with minor modifications. Briefly, 1 h after OGD neuronal cultures were detached using 0.05% trypsin for 5 min at 37°C in PBS, washed with cold PBS, and fixed with 4% paraformaldehyde at room temperature. Fixed cells were permeabilized with 0.2% NP40, washed with PBS, saturated with PBS-MT (PBS containing 5% nonfat powdered milk and 0.1% Tween 20) for 1 h, and then incubated overnight at 4°C with anti-PAR (1:100) and FITC-conjugated anti-GFAP (1:100) antibodies diluted in PBS-MT. After several washes with PBS-MT, cells were incubated with a phycoerythrinconjugated anti-rabbit IgG (1:50) for 30 min. The cell suspension (containing both neurons and astrocytes) was analyzed using a flow cytometer (Coulter XL; Beckman Coulter, Inc., Fullerton, CA). By means of appropriate electronic gates, neuron-related events (PAR-positive neurons) were sorted by subtracting FITC-labeled cells (GFAP-positive astrocytes) from phycoerythrin-positive cells.

Statistical Analysis. Concentration-response curves of PARP inhibitors were analyzed and IC₅₀ values were calculated with the Prism software package (GraphPAD Software, Inc., San Diego, CA). Statistical significance of differences between results was evaluated by performing ANOVA followed by Tukey's w test for multiple comparisons. Spearman's r correlation coefficient was computed between IC₅₀ values of experimental compounds on the inhibition of PARP activity and the IC₅₀ values on OGD-induced LDH release. The P value was calculated using a two-tailed test.

	Results

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Effects of Isoquinolinone Derivatives on PARP-1 Activity. In a previous report, we showed that the widely used PARP-1 inhibitor benzamide (Fig. 1) was able to reduce the enzyme activity with an IC₅₀ of 30 ± 4 µM when tested in an in vitro assay system (Moroni et al., 2001). 5OH-DIQ can be considered as a constrained benzamide analog, where the 5-position corresponds to the 3-position of benzamide and the amido moiety is restrained in an anti disposition (Fig. 1). In agreement with previous observations (Arundel-Suto et al., 1991), 5OH-DIQ was a moderately potent PARP-1 in vitro with an IC₅₀ of 17 ± 4 µM (Table 1; Fig. 3A). A thiol (compound 1 in Fig. 2) or a benzoyloxy (compound 2) group inserted in 5-position reduced the inhibitory potency of 5OH-DIQ, whereas an acetylene group (compound 3) did not modify its activity (Table 1; Fig. 3A). Compounds 4 and 5, which are endowed with an "inverse" syn disposition of the amido group, had no significant PARP-1 inhibitory activity up to a concentration of 100 µM. Compound 6, characterized by having the amido group constrained into a seven-membered ring, displayed a reduced potency with respect to the corresponding 3,4-dihydroisoquinolinone derivative (Table 1; Fig. 3A), thus suggesting that the incorporation of the amido moiety into a seven-membered ring may result in a nonoptimal interaction with the active site residues. It should be noted, however, that a different series of potent PARP-1 inhibitors incorporating a seven-membered lactame moiety into a tricyclic structure has recently been reported (Canan Koch et al., 2002). The different conformation induced on the seven-membered ring by the tricyclic with respect to the bicyclic scaffold may explain the difference.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Antagonist activity of isoquinolinone, DPQ, and PND derivatives on PARP-1 activity in vitro. Values are expressed as dpm/h and were obtained by incubating the inhibitor with 0.2 µCi [adenine-2,8-3H]NAD and 0.13 U partially purified bovine PARP-1 for 1 h at 37°C. A, isoquinolinone derivatives with no switch into the syn conformation of the amide group of nicotinamide (compounds 1, 2, and 3) are effective in inhibiting PARP-1 activity in the µM range. B, DPQ derivatives such as compounds 7, 8, and 10 are effective in reducing PARP-1 activity. C, PND derivatives with a thiophene group (TIQ-A, compounds 11 and 12) are very potent PARP-1 inhibitors, with IC50 values between 1 and 10 µM. Each point represents the mean ± S.E.M. of at least three experiments performed in triplicate.

Effects of DPQ Derivatives on PARP-1 Activity. DPQ (Fig. 1) is another potent PARP-1 inhibitor (Banasik et al., 1992; Eliasson et al., 1997; Moroni et al., 2001), with an IC₅₀ of 2.2 ± 0.5 µM (Table 1; Fig. 3B). A series of analogs was synthesized by modifying either the buthoxy-piperidine side chain or the dihydroisoquinolinone ring (Fig. 2). Compounds having the buthoxy-piperidine side chain conformationally constrained with a triple bond inserted in the 2'-position (compound 7) or in the place of the phenolic oxygen (compound 8) were synthesized, but their PARP-1 inhibitory activity was lower than that of DPQ (Table 1; Fig. 3B). As described for compound 6, when the six-membered ring structure of dihydroisoquinolinone of DPQ was changed into a seven-membered ring (compound 9), the inhibitory potency decreased. Conversely, when it was changed into a quinazolin-4-(3H)-one structure (compound 10), PARP-1 inhibitory activity was not affected (Table 1, Fig. 3B).

Effects of PND Derivatives on PARP-1 Activity. PND (Fig. 1) is a PARP-1 inhibitor significantly more potent than benzamide (Banasik et al., 1992; Moroni et al., 2001). We evaluated the effect of the isosteric replacement of ring C of PND with a five-membered thiophene ring. TIQ-A (Fig. 2) turned out to be a very potent PARP-1 inhibitor, with an IC₅₀ of 0.45 ± 0.1 µM (Table 1; Fig. 3C). Further manipulation of TIQ-A led to the 5-hydroxy and 5-methoxy derivatives (compounds 11 and 12), two molecules exhibiting a similar potent inhibitory activity (IC₅₀ 0.39 ± 0.19 and 0.21 ± 0.10 µM, respectively).

Neuroprotective Effects of Isoquinolinone Derivatives against OGD Neurotoxicity in Murine Cortical Cell Cultures. PARP-1 inhibitors provide neuroprotection in models of postischemic neurodegeneration (Eliasson et al., 1997; Ha and Snyder, 2000; Moroni et al., 2001). To investigate whether the potency of the newly characterized PARP-1 inhibitors correlated with their neuroprotective effects, we used cultured murine cortical cells exposed to OGD as an in vitro model of cerebral ischemia. Phase-contrast microscopy revealed that exposure to a high concentration (1 mM) of glutamate for 24 h induced complete neuronal cell death, but no injury in the underlying glial layer, and a substantial (approximately 4-fold the basal levels) release of LDH into the bathing medium (data not shown; see Pellegrini-Giampietro et al., 1999a for details). Exposure to OGD for 60 min produced an intermediate level of neuronal damage; the release of LDH was approximately 75% that observed by exposing the cultures to 1 mM glutamate. None of the tested PARP-1 inhibitors produced any significant increase in the release of LDH when added alone into the incubation media (not shown).

A number of PARP-1 inhibitors displaying an IC₅₀ < 100 µM in the in vitro enzymatic assay were added to the incubation medium during OGD exposure and the subsequent 24 h recovery period. As previously shown (Moroni et al., 2001), benzamide, PND, and DPQ reduced OGD injury in a concentration-dependent manner, with IC₅₀ values of 103 ± 12, 10.6 ± 2, and 4 ± 0.6 µM, respectively. In a similar manner, 5OH-DIQ attenuated neuronal death following OGD with an IC₅₀ of 71 ± 14 µM, whereas compound 8 displayed an IC₅₀ of 63 ± 12 µM. The thieno-derivatives TIQ-A and compound 11 markedly reduced OGD-induced neuronal death with IC₅₀ values of 0.15 ± 0.01 and 0.2 ± 0.1 µM, respectively. Figure 4A reports the neuroprotective effects of these PARP-1 inhibitors at a concentration of 100 µM. Although compounds 3 and 12 inhibited PARP at concentrations lower than 100 µM, they were not tested against OGD because they could not be completely dissolved in the lower concentration of dimethyl sulfoxide (0.1 versus 5%) required for cell culture studies as compared with the in vitro enzymatic assay. When the IC₅₀ values observed in the PARP-1 activity assay for these compounds were compared with their IC₅₀ values for the reduction of OGD-induced LDH release, a significant correlation was found (r = 0.89, P < 0.0014; Fig. 4B). In keeping with the relative inhibitory potencies on PARP-1, TIQ-A and compound 11 were significantly more potent than their parent compound PND in reducing OGD-induced LDH release (Fig. 5A). Importantly, TIQ-A at 10 µM proved to be neuroprotective also when added immediately or up to 30 min after the termination of OGD (Fig. 5B).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Neuroprotection by isoquinolinone, DPQ, and PND derivatives against OGD neurotoxicity in murine cortical cultures and correlation with their PARP-1 inhibitory potency. A, selected PARP-1 inhibitors reduce OGD-induced neuronal death. OGD was applied for 60 min, and neuronal damage was assessed by measuring the release of LDH in the medium 24 h later. Each PARP-1 inhibitor was added at a concentration of 100 µM during OGD and the subsequent 24-h recovery period. Data are expressed as percent of glutamate-induced neuronal damage. Each bar represents the mean ± S.E.M. of at least five experiments. *, P < 0.05 and **, P < 0.01 versus OGD (ANOVA + Tukey's w test). B, positive correlation (P < 0.001) between the IC50 values of selected compounds for the inhibition of PARP-1 activity and their IC50 values for the reduction of OGD-induced LDH release. Pearson's r correlation coefficient was calculated between the two IC50 values, correlation was verified by nonparametric (Spearman's) rank correlation, and the P value was calculated using a two-tailed test.

View larger version (11K): [in this window] [in a new window]   Fig. 5. PND, TIQ-A, and compound 11 protect against OGD neurotoxicity in murine cortical cultures. A, neuronal death was significantly reduced by addition of 0.1 to 100 µM TIQ-A or compound 11 and of 10 to 100 µM PND to the incubation medium during OGD exposure and the subsequent 24-h recovery period. Each point represents the mean ± S.E.M. of at least five experiments. B, TIQ-A was neuroprotective also when added during or at 0, 15, and 30 min after OGD. Each point represents the mean ± S.E.M. of at least four experiments. *, P < 0.05 and **, P < 0.05 versus OGD alone.

By means of a flow cytometric assay, we also investigated the effects of TIQ-A on poly(ADP-ribosyl)ation in neurons exposed to OGD. As shown in Fig. 6, the neuronal content of PAR, the product of PARP-1 activity, was dramatically increased 1 h after OGD. This effect was almost completely prevented by the addition of 10 µM TIQ-A to the incubation medium.

View larger version (16K): [in this window] [in a new window]   Fig. 6. TIQ-A (10 µM) reduces the early formation of PAR induced by 60 min OGD in cortical neurons. One hour after OGD, neuronal cultures were fixed, labeled with antibodies directed against PAR and GFAP, and processed for flow cytometry analysis. The GFAP-positive contribution to PAR fluorescence was subtracted from all samples. The graph plots the relative number of cells displaying increasing levels of PAR fluorescence and is representative of four analyses.

	Discussion

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PARP-1 is a 113-kDa dimeric protein characterized by three major domains: an N-terminal DNA-binding domain containing the nuclear localization sequence, an internal automodification region bearing a BRCA1 C-terminal domain, and a C-terminal that includes the catalytic domain (D'Amours et al., 1999; Smith, 2001). Although this complex structural organization might be used to develop enzymatic inhibitors targeting different functional domains, all the known chemical inhibitors of PARP-1 have been designed as substrate analogs. Among these, 3,4-dihydroisoquinolinone derivatives are potent inhibitors that have been extensively investigated in in vitro (Arundel-Suto et al., 1991; Suto et al., 1991; Banasik et al., 1992; Zhang et al., 1994) and in vivo (Takahashi et al., 1997; Takahashi and Greenberg, 1999) models.

In the present study, we examined the pharmacological profile of new 3,4-dihydroisoquinolinone derivatives that were structurally designed as potential PARP-1 inhibitors and correlated their inhibitory activity with the neuroprotective effects on cultured cortical cells exposed to OGD. The IC₅₀ values observed for 5OH-DIQ, DPQ, and PND were somewhat higher than those previously reported (Suto et al., 1991; Banasik et al., 1992). Although a comparison of IC₅₀ values from different laboratories must be undertaken with caution, it should be noted that this is the first study comparing the inhibitory potencies of these three molecules using a commercially available pure bovine PARP-1 preparation.

The evaluation of our novel compounds extends the existing information on the structure-activity relationship of PARP-1 inhibitors. In particular, the need of an anti disposition of the amido moiety of the 3,4-dihydroisoquinolinone derivatives involved in a hydrogen bonding with the amido counterpart of the enzyme was confirmed by the lack of activity of compounds 4 and 5, endowed with a constrained syn disposition of the amido group. The weak inhibitory activity of the seven-membered derivative (compound 6) may tentatively be ascribed to a shift in the orientation of the amido group, which prevented the formation of a productive hydrogen bonding with the Gly863 site of the enzyme. We also investigated the effect of changing the substituent in the 5-position of dihydroisoquinolin-1(2H)one. Whereas the 5-hydroxy derivative (5OH-DIQ) was confirmed to have PARP inhibitory activity (Arundel-Suto et al., 1991), substitution with both the larger thiol or the benzoyloxy groups (as in compounds 1 and 2) caused a marked reduction in the potency, thus delineating a size-limited binding pocket. The more directional and electron-rich acetylene group (compound 3) on the other hand showed a potency similar to 5OH-DIQ.

The results obtained with DPQ derivatives showed that compounds 7 and 8, prepared with triple bonds in the side chain to constrain its binding orientation, were less potent than DPQ itself, suggesting that these changes do not favor their insertion in the enzyme active site. These results, however, confirmed the hypothesis that the hindrance of the side chain orientation affected DPQ potency, thus leaving open the possibility that different arrangements of the side chain group might allow us to obtain DPQ derivatives with increased inhibitory activity. Studies on compound 9 confirmed that a seven-membered ring inserted in the DPQ moiety causes a marked reduction in the inhibitory potency on PARP-1. Furthermore, the chimera made by combining the buthoxy-piperidine chain of DPQ with a quinazolinone moiety, a structure previously reported to be endowed with PARP-1 inhibitory activity (Griffin et al., 1998), led to compound 10, an inhibitor with an IC₅₀ comparable with that of DPQ. Much more intriguing were the results obtained with the isosteric replacement of the PND benzene ring with a thiophene, which led to TIQ-A and to compounds 11 and 12, which were all potent PARP-1 inhibitors.

Because PARP-1 inhibitors are neuroprotective in experimental models of ischemic brain injury (Eliasson et al., 1997; Ha and Snyder, 2000; Moroni et al., 2001) we studied the effects of the new compounds on neuronal death after OGD in vitro. Our results show that selected PARP-1 inhibitors effectively attenuated OGD neurotoxicity and that the degree of neuroprotection correlated with their inhibitory potency on PARP-1 activity. TIQ-A and compound 11 were significantly more potent than DPQ in inhibiting both PARP-1 activity and OGD-induced neuronal death. Their IC₅₀ values against OGD injury were 0.15 ± 0.01 µM for TIQ-A, 0.20 ± 0.05 µM for compound 11, and 4.0 ± 0.8 µM for DPQ. Considering the need for drugs able to exert neuroprotection when given after the onset of cerebral ischemia in the clinical setting, the finding that TIQ-A is active in a post-treatment paradigm supports its potential therapeutic relevance.

The correlation between the neuroprotective properties and the potency in inhibiting PARP-1 activity of these compounds, along with the evidence that the formation of PAR is triggered by OGD and efficiently suppressed by TIQ-A, confirms the pivotal role of this enzyme in the pathophysiology of ischemic brain damage and underscores the therapeutic significance of these experimental compounds. Unfortunately, the rate of discovery of new PARP family members has exceeded that of PARP subtype-specific inhibitors. Therefore, we cannot rule out that the neuroprotective effects of the compounds reported here may be due to inhibition of family members other than PARP-1. Indeed, a very recent study demonstrates that, akin to PARP-1 inhibition, suppression of PARP-2 activity exerts anti-inflammatory effects (Popoff et al., 2002). However, whether PARP-2 and/or other PARP family members may participate in the pathogenesis of ischemic neuronal death is yet to be determined. In this scenario, the forthcoming development of PARP subtype-specific inhibitors (Perkins et al., 2001) will undoubtedly help to elucidate the role of the various members of the family in physiology and pathology.

	Footnotes

 
This work was supported by grants from GlaxoSmithKline (Harlow, UK) (to F.M. and R.P.) and by University of Florence grants (to F.M. and D.E.P.G.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.048934.

ABBREVIATIONS: PARP, poly(ADP-ribose) polymerase; DPQ, 3,4-dihydro-5-[4-(1-piperidinyl)buthoxyl)]-1(2H)-isoquinolinone; LDH, lactate dehydrogenase; OGD, oxygen-glucose deprivation; 5OH-DIQ, 3,4-dihydro-5-hydroxy-1(2H)-isoquinolinone; PND, 6(5H)-phenanthridinone; PAR, poly(ADP-ribose); GFAP, glial fibrillary acidic protein; FITC, fluorescein isothiocyanate; TIQ-A, thieno[2,3-c]isoquinolin-5-one; PBS, phosphate-buffered saline; ANOVA, analysis of variance.

Address correspondence to: Prof. Flavio Moroni, Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Viale Pieraccini, 6, 50139 Firenze, Italy. E-mail: flavio.moroni{at}unifi.it

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