Biochimica et Biophysica Acta 1844 (2014) 1765–1772

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Investigating the allosteric reverse signalling of PARP inhibitors with microsecond molecular dynamic simulations and fluorescence anisotropy Jean-Rémy Marchand a,1,2, Andrea Carotti a,2, Daniela Passeri b, Paolo Filipponi b, Paride Liscio b, Emidio Camaioni a, Roberto Pellicciari a,b, Antimo Gioiello a, Antonio Macchiarulo a,⁎ a b

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Perugia, 06123 Perugia, Italy TES Pharma S.r.l. via Palmiro Togliatti 22bis 06073 Loc. Terrioli, Corciano, Perugia, Italy

a r t i c l e

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Article history: Received 10 April 2014 Received in revised form 14 July 2014 Accepted 15 July 2014 Available online 22 July 2014 Keywords: PARP Polypharmacology Molecular dynamics Fluorescence anisotropy Cancer Neuroprotection

a b s t r a c t The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Poly (ADP-ribose) polymerases (PARPs) are a family of posttranscriptional regulatory enzymes that use nicotinamide adenine dinucleotide (NAD+) as substrate to transfer ADP-ribose moieties to target proteins [1,2]. Although the superfamily of mammalian PARPs is composed of 17 members that have also been named ADP-ribosyltransferases (ARTs) [3], PARP-1 is to date the best characterized enzyme [4]. Its catalytic activity is activated by the recognition of DNA strand breaks; which eventually leads to the recruitment of repairing proteins to damaged sites. As a result, this enzyme promotes important cellular processes such as genome repairing, chromatin remodelling, and cell survival or apoptotic pathways [5–9].

⁎ Corresponding author at: Dipartimento di Scienze Farmaceutiche, Via del Liceo 1 06123 Perugia, Italy. Tel.: +39 075 585 5160; fax: +39 075 585 5161. E-mail address: [email protected] (A. Macchiarulo). 1 Present address: Biochemistry Department, Wintherturerstrasse 190, 8057 Zürich, Switzerland. 2 These authors contributed equally to the work.

http://dx.doi.org/10.1016/j.bbapap.2014.07.012 1570-9639/© 2014 Elsevier B.V. All rights reserved.

Structurally, PARP-1 is composed of six modular domains spanning from the N-terminal DNA-binding region to the C-terminal catalytic region (Fig. 1a). The DNA-binding region comprises three zinc-finger motifs (ZN1-3), with the first and third ones being crucial for DNA binding activity [10–14]. The central region of PARP-1 sequence contains the auto-modification BRCT fold domain, bearing acceptor sites for autoPARsylation of the protein, and the WGR domain, whose function is still poorly understood [15,16]. The C-terminal region includes the catalytic domain of the enzyme (CAT) featuring the helical subdomain (HD, residues 651–785) and the ART subdomain (residues 786–1014), with the latter containing the specific sequence signature (HYYE) involved in NAD+ binding and catalysis [17]. Recent crystallographic studies of a DNA double-strand break in complex with human PARP-1 domains essential for activation (ZN1, ZN3, WGR and CAT) have shown that the enzyme binds to DNA as a monomer, with its domains adopting a collapsed conformation which accounts for the autoPARsylation activity of PARP-1 [18,19]. Efforts to target PARP-1 with small molecule inhibitors have opened novel therapeutic opportunities to treat a range of diseases including stroke, cardiac ischemia, mitochondrial dysfunction, cancer, inflammation and diabetes [20–23]. These studies have so far resulted in a number of compounds advancing to clinical settings as chemosensitizers,

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a

ART

b

HD Zn-1

WGR

Zn-3

Fig. 1. a) Cartoon showing the near full-length PARP-1 structure in complex with a DNA double-strand break (PDB ID: 4DQY). b) Chemical structures of PARP inhibitors.

i.e. enhancers of the action of chemotherapeutic drugs [24,25]. More recently, embracing the concept of synthetic lethality in cancer drug development, PARP-1 inhibitors are also being investigated as single agent therapy in cancer cells endowed with genetic defects of DNA repairing pathways, such as BRCA1 or BRCA2 deficient cells [26–29]. Although PARP-1 inhibitors hold great promises for the development of innovative and personalized anticancer drugs [30,31], the lack of selectivity and/or biological data annotations of available compounds demands further investigations to grasp the polypharmacology of PARP inhibitors and avoid misinterpretations of results from biological and clinical studies [32,33]. While we have reported that some of the current drug candidates such as Rucaparib, Olaparib, and Veliparib show inhibitor activity towards additional members of PARP superfamily [34], Murai and coworkers have elegantly demonstrated that the interaction of some of these drug candidates with PARP-1 and PARP-2 is able to induce the formation of cytotoxic complexes by trapping the enzymes on damaged sites of DNA [35]. In particular, they showed that this activity of PARP inhibitors is independent from the potency of the compound to inhibit the catalytic activity of the enzyme, with Olaparib having a greater cytotoxic poisoning effect than Veliparib. The authors hypothesized that an allosteric reverse signal couples the interaction of the inhibitors within the C-terminal CAT domain to the N-terminal DNA-binding domain, contributing to the enhancement of DNA binding. This idea is further supported by the finding that when the DNA-binding domain of PARP-1 is bound to DNA, it allosterically activates the CAT domain [16,18,19]. Although the poisoning of PARP-1 and PARP-2 on DNA is a very effective strategy in contributing to the inhibition of the repairing functions of the enzymes, the molecular principles underlying the allosteric reverse signalling are still elusive. Grounding on the crystallographic structure of PARP-1 domains bound to a DNA double-strand break [18] and as a continuation of our efforts in the field of design and synthesis of PARP-1 inhibitors [36–39], in this work we have employed microsecond molecular dynamics (MD) to investigate the ligand-induced conformational changes of PARP-1 that may underlie the allosteric reverse signalling promoted by Olaparib (1, Fig. 1b) and Veliparib (2). The results gathered from MD simulations are then used to study the allosteric reverse signalling of HYDAMTIQ (3), a potent PARP inhibitor endowed with neuroprotective properties as well as ability to reduce chronic lung inflammation in asthmatic patients [37,40,41].

2. Materials and methods 2.1. Chemicals Olaparib and Veliparib were purchased from Selleck (USA), [http:// www.selleckchem.com]. HYDAMTIQ was synthesized by a multistep flow synthesis consisting of Suzuki coupling reaction, intramolecular thermal cyclization, Mannich-type reaction and final hydrolysis (Fig. S1 of supporting information) [42]. 2.2. MD simulations The structure of human PARP-1 in complex with a DNA doublestrand break was taken from the PDB (PDB ID: 4DQY) and prepared using the Protein Preparation Wizard protocol of Schrödinger software (Schrödinger 9.3.5, LLC, New York, NY). Two missing loops were added to the structure, copying and pasting the atomic coordinates of the first loop (Leu575–Trp584) from the crystal structure of the WGR domain of PARP-1 (PDB ID: 2CR9) after structure superimposition, while modelling the second loop (Asp644–Lys662) with the loop completion script of Modeller 9.11 [43]. These modelled loops occupy a region on the opposite side of the catalytic cleft of the enzyme. To reproduce physiological pH conditions, side chains of aspartate and glutamate residues were negatively charged, those of lysine and arginine residues were positively charged. The bioactive conformations of Olaparib, Veliparib and HYDAMTIQ were extracted from the relative crystal structures of the conserved ART subdomain of TNKS-2 (PDB ID: 3U9Y) [43], PARP-2 (PDB ID: 3KJD) [42], and PARP-1 [37]. The structures of these compounds were processed with the LigPrep protocol of Schrödinger software. Compounds 2–4 were docked into the active site of the near-full length PARP-1 (4DQY), using Glide 5.8 with the XP scoring function [44]. Solutions with the lowest root mean square deviation (RMSD) from the atomic coordinates of the relative experimental bioactive conformation of ligands were used as input for MD simulations. MD simulations were carried out on: (i) PARP-1/DNA double-strand break complex bound to Olaparib (two independent simulations); (ii) PARP-1/DNA double-strand break complex bound to Veliparib (one simulation); (iii) ligand-unbound PARP-1 DNA double-strand break complex (one simulation); (iv) PARP-1/DNA double-strand break complex bound to HYDAMTIQ (one simulation). Each of the four systems was solvated in a cubic box of TIP3P water molecules,

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3.1. Binding modes of Olaparib, Veliparib and HYDAMTIQ to PARP-1 The analysis of RMSD values of Olaparib (RMSD = 1.77 ± 0.85 Å), Veliparib (RMSD = 1.42 ± 0.10 Å) and HYDAMTIQ (RMSD = 0.93 ± 0.35 Å) along the MD trajectories shows that all the inhibitors are stably anchored into the nicotinamide binding pocket of the ART subdomain, even though a large conformational rearrangement is observed for Olaparib at ~600 ns of simulation time (Fig. 2). All compounds feature specific conserved interactions which include hydrogen bonding with Gly863 and Ser904 as well as a π-stacking interaction with the side chain of Tyr907. In particular, the pyridazinone ring of Olaparib forms two hydrogen bonds with the backbone atoms of Gly863 (occupancy 88.3% and 77.8%), and one hydrogen bond with the side chain of Ser904 (occupancy 43.8%). During the first half of the simulation (~620 ns), one hydrogen bond is formed between the cyclopropyl-carbonyl tail of Olaparib and Arg865 (occupancy 32.8%). Thereafter, this interaction breaks due to a conformational rearrangement in which the piperazine ring and cyclopropyl tail extend towards the HD subdomain (see movie_1.pps of supporting information), resulting in the formation of a hydrogen bond with Met890 (occupancy 14.2%, Fig. 3a). This conformational rearrangement is not observed in a second 1 μs MD simulation of Olaparib-bound PARP1 (supporting information, movie_1b.pps and Figs. S2–S3), albeit steric bumps produced by Olaparib on the WGR domain are still present as evidenced by the higher fluctuations of this region of the enzyme (see discussion part and supporting information, Fig. S4). Conversely, Veliparib and HYDAMTIQ are not involved in any significant conformational rearrangement during the simulation (see movie_2.pps, movie_3.pps of supporting information). Veliparib is stabilized through three hydrogen bonds with Gly863 and Ser904 (occupancy 83.4%, 76.1%, 54.9%), a specific salt bridge with Glu763 (occupancy 39.7%), and a water-mediated hydrogen bond with Glu988 (occupancy 95.1%, Fig. 3b). HYDAMTIQ shows a pattern of interactions similar to Veliparib (Fig. 3c). It forms two hydrogen bonds with Gly863 and one with Ser904 (occupancy 52.8%, 36.9% and 4

3.5

3

2.5

2

1.5

1

0.5

2.4. Cytotoxicity assays

1000

750

Time (ns) Olaparib

Cell viability was evaluated by measuring ATP levels using Cell Titer Glo (Promega), according to the manufacturer's instructions. Tamoxifen (Sigma) was used as positive control for the goodness of the assay.

500

0 250

The fluorescence anisotropy experiment was performed following the protocol reported by Murai and coworkers [35]. The assay was carried out using 30 bp DNA duplex labelled with Alexa Fluor488 (5′Alexa Fluor488 ACCCTGCTGTGGGCdUGGAGAACAAGGTGAT, Integrated DNA Technology). The labelled DNA was annealed to its complement in buffer containing 50 mM potassium acetate, 20 mM tris acetate, 10 mM magnesium acetate, 1 mM DTT pH 7.9 and then incubated for 2 h at 37 °C with APEI and UDG (New England Biolabs). To evaluate DNA/ protein binding, 250 nM of recombinant PARP-1 active protein (Active Motif) was incubated for 30 min with 1 nM DNA and increasing concentrations of a PARP inhibitor were added in an assay buffer containing 50 mM Tris–HCl pH8, 100 mM NaCl, 4 mM MgCl2 and 10 ng/ml BSA. The enzymatic reaction was started with 5 mM NAD+, and fluorescence anisotropy was measured 30 min after adding the cofactor NAD+ using the Envision 2103 Multilabel Reader. The normalization of FA activity was calculated as percentage relative to the maximum signal of Olaparib (FA = 100%) and its background signal (FA = 0%).

3. Results

0

2.3. Fluorescence anisotropy DNA-binding assay

Briefly, 2500 HeLa cells were stimulated with the indicated concentrations of compounds in a white 96-well microplate for 72 h at 37 °C. Luminescence signals were read with a Victor Light multiplate reader (PerkinElmer Life and Analytical Sciences).

RMSD (Å)

with a minimum distance of 5 Å between the boundary and any atom of the protein. Each system was neutralized by adding sodium and chlorine ions at a concentration of 0.15 M. Periodic boundary conditions were applied to avoid finite-size effects. The VMD 1.9.1 program was used to set up the systems [45]. MD simulations were performed using ACEMD software [46,47], with CHARMM36 force field [48], and a graphical processing unit (GPU) NVIDIA GeForce GTX680. Compounds 2–4 were parameterized with ParamChem [49,50], and CGenFF forcefield [51]. The particle-mesh Ewald methodology was used for long-range electrostatics [52]. The van der Waals interactions were truncated at a cut-off of 9 Å with a switch function starting at 7.5 Å. All simulations were carried out at a constant temperature of 298 K with the Langevin thermostat. Covalent bonds between heavy atoms and hydrogen atoms were fixed with default algorithm [53,54], and hydrogen mass was multiplied by 4 [55], for the use of a time step of 4 fs. Some harmonic constraints were applied on different atoms during the whole simulation to avoid undesired moves, such as the exit of zinc atoms from zinc finger domains and denaturation of DNA. The two zinc atoms were restrained with a force of 25 kcal/mol/Å [56]. The sulphur and nitrogen atoms of side chains of residues coordinating zinc atoms (Cys21, Cys24, Cys56, Cys295, Cys298, Cys311–Cys321 and His53) were restrained with a force of 5 kcal/mol/Å. The 15 nucleotide bases of the DNA tail being not in contact with the protein, were constrained with a force of 1 kcal/mol/Å. For each simulation, a minimization run of 1000 steps was performed with all heavy atoms of protein, DNA and ligand restrained with a force of 1 kcal/mol/Å. These constraints were kept for a first equilibration run of 1 ns and were released for a second equilibration run of 1 ns. Equilibration steps were performed in the NPT ensemble with a Berendsen barostat at 1.01325 bar for a total equilibration time of 2 ns. The production runs were performed in NVT ensemble for 1 μs. The atomic coordinates of each system were saved every 100 ps, achieving a total of 10,000 frames. Before analysing the resulting MD trajectories, water molecules were removed from each frame of the system. The root mean square deviation (RMSD) values of the different domains (ZN1, ZN3, WGR, CAT and DNA bound complex) were calculated from the input structure, using the atomic coordinates of backbone atoms after the alignment on the corresponding domain. GROMACS analysis tools have been used for the analysis of the occupancy of hydrogen bonds and salt bridges.

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Veliparib

HYDAMTIQ

Fig. 2. RMSD plot of Olaparib, Veliparib and HYDAMTIQ along the MD trajectories. Trajectory frames were aligned using the atomic coordinates of backbone atoms.

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a

b

c

Olaparib Veliparib

HYDAMTIQ

Fig. 3. Binding mode of Olaparib (a), veliparib (b) and HYDAMTIQ (c) at the beginning of the simulation (time 0, cyan colour-coded) and at the end of the simulation (time 1 μs, yellow colour-coded). Hydrogen bond interactions are displayed with black dashed lines.

28.1%), weak interactions with Glu763 (occupancy 3.4%), and an additional hydrogen bond with Glu988 (occupancy 43.9%). 3.2. Ligand-induced conformational changes of PARP-1/DNA complexes The RMSD value inspection of backbone atoms of the near fulllength PARP-1/DNA complexes and single domains along the trajectories shows significant differences at the level of WGR and HD domains (Fig. 4), suggesting the presence of specific ligand-induced conformational changes of the enzyme. Specifically, the RMSD value of the WGR domain (residues 532–649) is significantly higher in the Olaparibbound PARP-1 simulation with respect to the other simulations, with an average value of 4.82 ± 1.11 Å. This is congruent with higher drifts observed in the HD subdomain of Olaparib-bound PARP-1 along the trajectory (4.83 ± 0.11 Å) with respect to Veliparib-bound enzyme (3.67 ± 0.78 Å), HYDAMTIQ-bound enzyme (4.04 ± 0.84 Å) and the apo enzyme (RMSD = 4.21 ± 0.62 Å). The presence of higher drifts in WGR and HD domains is confirmed in the second MD simulation of Olaparib-bound PARP-1, with RMSD values of 4.39 ± 1.14 Å and 4.74 ± 0.22 Å, respectively. Beside these movements, different patterns of salt-bridge interactions are observed in the simulations of PARP-1/DNA complexes. Table 1 lists residues involved in stable salt bridge interactions with hydrogen bonding occupancy higher than 25% in at least one simulation.

Two groups of salt bridges can be identified: (i) those that are stable and present in all of the simulations with similar occupancies, likely contributing to protein stability and/or DNA binding; (ii) those that are preferentially observed in one simulation, possibly mediating inhibitor-induced conformational effects. Among the first group, salt bridges a and h occur within the ZN1 and WGR domains, respectively. In particular, Lys571 contributes to the formation of salt bridge h, interacting with both Asp534 and Asp644 within the WGR domain of the Olaparib-bound PARP-1. Conversely, Lys571 specifically interacts with Asp534 in the HYDAMTIQ-bound PARP-1 simulation, and Asp644 in Veliparib-bound enzyme as well as in the apo protein. The second group of salt bridges can be further differentiated on the basis of the simulation in which they are observed or not. Salt bridges b, d, f and g specifically occur in zinc domains of Olaparib-bound enzyme with high occupancy of hydrogen bonding. Salt bridges l, m and o accidentally occur within the HD and ART subdomains of Olaparib-bound enzyme, being not observed as stable interactions in the second MD simulation of the complex. The side chains of Lys703 and Glu772 (i) are involved in an intra-domain salt bridge interaction in both Veliparib (3)-bound PARP-1 and HYDAMTIQ-bound enzyme, stabilizing Table 1 Salt bridge interactions and occupancies (%) of hydrogen bonding. Salt bridges that are consistently and specifically stable in Olaparib-bound PARP-1/DNA complex simulations are highlighted in bold and italics (see also Fig. 7).

7

# 6

RMSD (Å)

5

4

3

2

1

0 PARP-1

ZN-1 Apo

ZN-3

WGR

Veliparib

Olaparib

HD

ART

HYDAMTIQ

Fig. 4. Histogram plot of RMSD of near full-length PARP-1 and its functional domains for each of the four simulations.

Acceptor

Donor

Olaparib

50.7% (58.3%)a 62.5% (40%) 33.4% (2.9%) 62.6% (90.3%) d Arg340(ZN-3) 79.6% (92.3%) e Arg282(ZN-3) 90.9% (29.7%) Arg330(ZN-3) 23.4% (0%) Asp354(ZN-3) 8.8% (87.2%) f Asp307(ZN-3) Lys324(ZN-3) 44.1% (25.3%) g Asp314(ZN-3) Lys320(ZN-3) 45.6% (70.9%) h Asp534(WGR) Lys571(WGR) 39.8% (36.2%) Asp644(WGR) 23.1% (0%) i Glu772(HD) Lys703(HD) 6.9% (20.3%) l Asp743(HD) Lys 695(HD) 55.8% (4.4%) m Asp783(HD) Lys796(ART) 39.2% (11.7%) n Asp766(HD) Lys878(ART) 68.5% (82.1%) o Asp800(ART) Lys802(ART) 29.0% (22.7%) p Glu923(ART) Lys852(ART) 32.4% (9.6%) Asp965(ART) 4.4% (31.9%) q Asp981(ART) Lys933(ART) 30.4% (32.0%) r Asp988(ART) Lys903(ART) 10.3% (19.9%) a b c

Asp31(ZN-1) Glu70(ZN-1) Asp81(ZN-1) Asp250(ZN-3) Glu263(ZN-3) Asp285(ZN-3)

Lys30(ZN-1) Arg65(ZN-1) Arg78(ZN-1)

Veliparib Apo

HYDAMTIQ

65.3% 9.7% 0.0% 89.2% 13.7% 0.0% 52.2% 74.6% 3.4% 19.9% 0.0% 64.9% 59.9% 3.2% 12.3% 98.6% 7.9% 6.5% 26.6% 0.4% 5.7%

65.8% 0.6% 0% 1.5% 0.5% 0.2% 4.9% 0% 20.3% 19.9% 72.6% 0% 40.9% 0% 0.2% 0.1% 20.5% 3.6% 32.2% 30.5% 7.7%

54.2% 0.5% 9.2% 94.3% 25.9% 87.0% 43.3% 7.1% 4.4% 16.0% 0.6% 34.6% 0.2% 0% 7.2% 88.1% 12.6% 5.3% 13.0% 24.5% 63.5%

a In brackets the occupancy of hydrogen bonding observed in the second simulation of Olaparib-bound PARP-1 complex.

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100

80

% Vitality

the HD subdomain. In contrast, the interactions between Lys933 and Asp981 (q) as well as Lys903 and Glu988 (r) are missing in Veliparibbound protein simulation, with the latter salt bridge being also absent in the Olaparib- and HYDAMTIQ-bound enzyme. Collectively, Olaparib-bound PARP-1 simulations count a higher number of stable salt bridges (15 salt bridges in the first simulation, 12 salt bridges in the second simulation) than Veliparib-bound PARP1 (7 salt bridges), HYDAMTIQ-bound PARP-1 (5 salt bridges) and the unbound enzyme (7 salt bridges), suggesting a different stabilization of PARP-1/DNA complexes. While these results suggest that HYDAMTIQ induces similar behaviour of PARP-1 as Veliparib and not Olaparib, they point out this inhibitor as having a low poisoning activity, comparable to the one of Veliparib.

60

40

Olaparib

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3.3. HYDAMTIQ does not trap PARP-1/DNA complex

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Veliparib HYDAMTIQ -8

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-3

Log [Ligand] M To evaluate the poisoning effect of HYDAMTIQ, a PARP-1/DNA binding assay based on fluorescence anisotropy was performed as described by Murai and coworkers [35]. The trapping potencies of PARP-1 DNA complexes by Olaparib, Veliparib and HYDAMYIQ were thus assessed in vitro. All PARP-1 inhibitors were able to enhance the fluorescence anisotropy signal (Fig. 5), indicating the stabilization of PARP-1/DNA complexes. In agreement with the results of Murai and coworkers [35], Olaparib (2) showed approximately 10-fold more potency than Veliparib in the biochemical trapping of PARP-1. Congruent with indications from MD simulations, HYDAMTIQ was less effective to trap PARP-1 on DNA than Olaparib, and showed a similar fluorescence anisotropy signal to Veliparib. Next, we tested the cytotoxicity of Olaparib, Veliparib and HYDAMTIQ in HeLa cells. As expected, HYDAMTIQ was less cytotoxic than Olaparib and slightly more than Veliparib with cell vitality IC50 values of 98 μM, 20 μM and 130 μM, respectively (Fig. 6). 4. Discussion The binding of the N-terminal region of PARP to damaged sites of DNA triggers the event of allosteric signal leading to the catalytic activation of the C-terminal domain [2,9,16]. Murai and coworkers demonstrated that bulky PARP inhibitors such as Olaparib were able to trap PARP-1/DNA complex, supporting the hypothesis of an allosteric reverse signalling from the C-terminal CAT domain to the N-terminal 150

Olaparib Veliparib HYDAMTIQ

% FA

100

50

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-12 -11 -10 -9

-8

-7

-6

-5

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Log [Ligand] M Fig. 5. Differential biochemical trapping of PARP-1 by Olaparib, Veliparib and HYDAMTIQ as resulting from the fluorescence anisotropy (FA)-binding assay.

Fig. 6. Survival curves of HeLa cells treated with Olaparib, Veliparib and HYDAMTIQ for 72 h. The rate of vitality (%) was calculated with respect to Tamoxifen used as s positive control.

DNA binding domain [35]. We used microsecond MD simulations and the near full-length PARP-1/DNA crystallographic complex [18] in this work to investigate the molecular basis of the allosteric reverse signalling of PARP inhibitors 1–3. These compounds were selected on the basis of the following considerations: (i) a crystallographic ligand bound complex of the ART subdomain was available [37,57,58]; (ii) Olaparib is a potent PARP-1 inhibitor (IC50 = 0.005 μM) [59] exhibiting a poisoning effect; (iii) Veliparib is still a potent PARP-1 inhibitor (IC50 = 0.008 μM) [60] but devoid of poisoning effect; (iv) HYDAMTIQ is a potent PARP-1 inhibitor (IC50 = 0.029 μM) [37] with unknown poisoning effect, thereby a suitable test compound. A caveat of the study was the use of double-stranded DNA in MD simulations that is at odds with recent reports suggesting that PARP-1 preferentially binds to 5′-deoxyribose phosphate (5′-dRP) DNA repair intermediate [61], and that PARP-1/DNA complexes are not induced by topoisomerase-I inhibitor mediated lesion bearing 5′-phosphate groups [62]. Still, its use was motivated by the fact that doublestranded DNA is the only structure so far solved in the crystallographic complex of the nearly full length PARP-1 [18]. The use of such experimental complex thus avoided the uncertainty associated to predict the correct binding geometry of PARP-1 domains to 5′-dRP DNA. Furthermore, we assumed that this approximation could only marginally affect the MD study of the allosteric reverse signalling of PARP inhibitors, since this latter originates from the CAT domain and propagates to the N-terminal DNA binding domain of the enzyme. Conversely, no attempts were made to calculate the binding energy of PARP-1 to double-stranded DNA, since the above approximation would have affected more the correlation between such obtained value and the experimental data of PARP-1 binding to 5′-dRP DNA. The analysis of MD trajectories showed that inhibitors 1–3 establish patterns of conserved and non-conserved interactions with the ART subdomain (Fig. 3a–c). In particular, conserved interactions include hydrogen bonds with Gly863 and Ser904 as well as a π-stacking interaction with Tyr907. Ligand-specific interactions of Veliparib and HYDAMTIQ comprise direct and water-mediated hydrogen bonds to Glu763 and Glu988. Conversely, Olaparib engages with Arg865 a specific hydrogen bond persistent in the first half of the simulation. This interaction is then broken as a consequence of a significant conformational drift of the ligand (~620 ns) that extends its cyclopropyl tail towards the HD subdomain (see movie_1.pps of supporting information), forming a new hydrogen bond with the backbone of Met890 (Figs. 2 and 3a). Although such conformational rearrangement was not observed in a second independent MD simulation of Olaparib-bound PARP-1 (supporting information, movie_1b.pps and Figs. S2–S3), this observation may suggest the

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presence of an alternative extended conformation for Olaparib that originates as a consequence of steric bumps produced by the ligand on the HD subdomain. It should also be mentioned that different reasons may explain why the extended conformation of Olaparib is not observed in the crystal structure. Specifically, the folded conformation of Olaparib for MD simulations was extracted from a truncated form of the conserved ART subdomain of TNKS-2 (PDB ID: 3U9Y) [43]. While we cannot rule out that crystallization conditions and/or crystal packing may favour the stability of the folded conformation, it is also likely that the use of a different PARP isoform and/or truncated form of the enzyme lacking the HD subdomain and WGR domain for crystallization studies may hamper the stabilization of the extended conformation, favouring the folded one. Pushing down the helix composed of residues 755–778, ligand generated steric bumps may be responsible of the significant drifts of the WGR domain observed at ~660 ns of the Olaparib (2)-bound PARP-1/ DNA trajectory (Fig. 7), whose presence is also observed in the second MD simulation of this complex at ~ 400 ns (supporting information, Fig. S4). According to the crystal structure, the WGR domain contributes to the formation of the DNA-recognition module of PARP-1 [18]. It has been proposed to play a crucial role in the activation of the enzyme, triggering distortions of PARP-1 HD subdomain [19]. Hence, it is not surprising that the wider movements observed for the WGR domain may have a role in the Olaparib induced trapping of PARP-1/DNA complex. Interestingly, the ligand-induced drifts of the WGR domain occur in association with the formation of several salt bridge interactions in PARP-1/DNA complex (Table 1). While constituting pivotal contacts for protein stability [63], salt bridge interactions play an important role in regulating the binding of proteins to DNA [64]. The crystal structure of the near full-length PARP-1 bound to a DNA double-strand break contains 95 acidic residues and 103 basic residues. Some of these residues are involved in the formation of 35 salt bridge interactions, with the one made by Asp45 and Arg591 being proposed as key contact between the ZN-1 domain and the WGR domain [18]. The side chain of Arg591 also interacts with the carbonyl group of the backbone of

Lys747 which is located in the HD subdomain, thereby connecting the WGR domain with the CAT and ZN-1 domains. In all MD simulations, Arg591 is in equilibrium between the salt bridge interaction with Asp45 and the hydrogen bond with the backbone of Lys747. Of note, MD simulations of Olaparib-bound PARP-1/DNA complex consistently show a higher number of stable salt bridge interactions (occupancy N 25%, Table 1) that almost double the number of stable salt bridges observed in the remaining simulations. Among these interactions, four salt bridges are exclusively observed in the simulations of Olaparib-bound complex (salt bridges b, d, f and g, Figs. 8 and S4), being located on the DNA binding domain. Three of them (salt bridges b, d, f) increase the occupancy values after the major drifts of the WGR domain (first simulation at t N 660 ns; second simulation at t N 400 ns). This finding supports the notion of an increased stability in the DNA binding domain of Olaparib-bound PARP-1/ DNA complex as a consequence of the steric bumps of the inhibitor to the HD subdomain and ensuing drifts of the WGR domain, strengthening the binding of the enzyme to the DNA. Collectively, these observations support the seminal hypothesis that trapping of PARP-1/DNA complex by Olaparib can be ascribed to a specific allosteric reverse signalling [35]. Moreover, they suggest that the molecular basis of the mechanism starts with steric bumps of the bulky compound on the HD subdomain, propagates through the WGR domain, and eventually stabilizes PARP-1/DNA complex by locking salt bridge interactions in the DNA binding domain. Although these results may suffer from the approximation of using a truncated PARP-1 structure which lacks the ZN-2 and BRCT domains [18], they further implicate the WGR domain as a structural key element, extending its functional role beyond PARP activation to mediator of PARP poisoning. If the steric bumps of Olaparib on the HD subdomain are the triggering event of the allosteric reverse signalling, then we may explain why bulky PARP inhibitors are likely endowed with such a mechanism: they are able to prime WGR drifts and lock key salt bridge interactions. Sustaining this scenario, a very recent study has reported the high trapping activity of BMN-673 at PARP–DNA complexes [65]. Analysing the molecular weight of BMN-673 (MW = 380 Da) as molecular descriptor of its bulkiness, it is worth noting that it is similar to Olaparib (MW = 434 Da) and higher than Veliparib (MW = 244 Da). The structure of

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Fig. 7. a) RMSD plot of ligand and WGR domain along the first MD trajectory of the Olaparib-bound PARP-1/DNA complex. Trajectory frames where the conformational rearrangements of Olaparib and higher fluctuations of WGR occur are indicated with dashed lines (a: time 620 ns; b: time 660 ns). b) Superimposition of backbone atoms between frames at time 620 ns (black cartoon) and time 0 ns (grey cartoon) of Olaparib-bound PARP-1/DNA complex. The conformation of Olaparib at time 620 ns is depicted showing that it is extending the piperazine ring and cyclopropyl tail, providing steric bumps on the HD subdomain.

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Fig. 8. a) Specific salt bridge interactions with high occupancy (N25%) formed in Olaparib-bound PARP-1/DNA complex during the first simulation. b) Time dependence of salt bridges in the first simulation of Olaparib-bound PARP-1/DNA complex.

HYDAMTIQ (MW = 274 Da), a potent PARP inhibitor, is less bulky than Olaparib and similar to Veliparib. Compliant to the above mentioned scenario, the results of MD simulations for HYDAMTIQ show a binding mode and ligand-induced conformational changes of PARP-1/DNA complex that are similar to those of Veliparib. In line with the expectation, fluorescence anisotropy assays provide the first experimental evidence that HYDAMTIQ is indeed less potent than Olaparib to trap PARP-1/DNA complex, and shows a PARP-1 poisoning activity similar to Veliparib. Furthermore, this is also sustained by the lower cytotoxic activity of HYDAMTIQ (IC50 = 98 μM) with respect to Olaparib (IC50 = 20 μM) in HeLa cells. In conclusion, our results show that the binding of Olaparib to the CAT domain of PARP-1/DNA complex is able to induce significant conformational rearrangement of the WGR domain as well as favours the formation of salt bridge interactions that may account for its poisoning effect. The triggering event could be the steric bumps that Olaparib generates during the simulation on the HD subdomain. We also report that HYDAMTIQ is almost devoid of PARP-1 cytotoxic poisoning activity, supporting our mechanistic hypothesis of allosteric reverse signalling as well as the development of HYDAMTIQ as neuroprotective agent and/or anti-inflammatory agent in asthmatic patients rather than an anticancer compound. Last but not the least; they suggest potential chemical modifications to PARP inhibitors that, increasing bulkiness, may favour the PARP-1 cytotoxic poisoning activity. Supporting Information. Movies of Olaparib, Veliparib and HYDAMTIQ trajectories are shown in movie_1.pps, movie-1b.pps, movie_2.pps and movie_3.pps, respectively. Fig. S1 reports the scheme of multistep flow synthesis of HYDAMTIQ. Fig. S2 shows conformational change of torsional angle α of Olaparib along the trajectories of two independent MD simulations. Fig. S3 shows conformational change of torsional angle β of Olaparib along the trajectories of two independent MD simulations. Fig. S4 reports the RMSD plot of ligand and WGR domain along the second MD trajectory of the Olaparib-bound PARP-1/ DNA complex. Trajectory frames where higher fluctuations of WGR occur are indicated with dashed lines (b: time 400 ns). Fig. S5 shows time dependence of salt bridges in the second simulation of Olaparibbound PARP-1/DNA complex. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbapap.2014.07.012. References [1] B.A. Gibson, W.L. Kraus, New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs, Nat. Rev. Mol. Cell Biol. 13 (2012) 411–424.

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