Identification and Characterization of New DNA G-Quadruplex

Article pubs.acs.org/jmc

Identification and Characterization of New DNA G‑Quadruplex Binders Selected by a Combination of Ligand and Structure-Based Virtual Screening Approaches† Stefano Alcaro,‡ Caterina Musetti,§ Simona Distinto,*,∥ Margherita Casatti,§ Giuseppe Zagotto,§ Anna Artese,‡ Lucia Parrotta,‡ Federica Moraca,‡ Giosuè Costa,‡ Francesco Ortuso,‡ Elias Maccioni,∥ and Claudia Sissi*,§ ‡

Dipartimento di Scienze della Salute, Università di Catanzaro, Campus “Salvatore Venuta”, Viale Europa, 88100 Catanzaro, Italy Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131, Padova, Italy ∥ Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy §

S Supporting Information *

ABSTRACT: Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand- and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety.

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sequently, cell immortalization.4 A number of studies highlighted the activation of telomerase in several type of cancers. Hence, inhibition of this enzyme can selectively prevent cancer cell growth. Indirectly, G-quadruplex-DNA stabilizers preclude the binding of telomerase and its associated proteins to telomeres and thereby stop the elongation process. Equally important is the stabilization of G-quadruplex in promoter regions or in rDNA and rRNA to achieve selective gene regulation.5−7 Quadruplexes can be formed from one, two or four separate strands of DNA (or RNA) and can show a wide variety of topologies, depending on combinations of strand orientation, loop size, and glycosidic conformation sequence. These different arrangements are dictated not only by the nucleic acid sequence, but, as in the case of the human telomeric DNA, a given sequence can fold into a variety of different

INTRODUCTION G-quadruplex structures are nucleic acid arrangements assumed by guanine-rich sequences and stabilized by the planar pairing of four guanines through eight Hoogsteen hydrogen bonds. These sequences are found in crucial positions of the genome, such as at telomeric ends, ribosomal DNA (rDNA), RNA, or gene promoter regions (for example, c-myc, bcl-2, or c-kit).1 Their composition and location is conserved through evolution and, additionally, several proteins are devoted to recognize or resolve them,2 thus indicating regulatory roles of these structures in multiple biological processes. As a consequence, DNA G-quadruplexes have recently emerged as new molecular targets for therapeutic intervention with a particular focus on anticancer.3 In normal cells, the telomeric DNA is gradually shortened after each replication cycle until a critical limit is reached which leads to cellular senescence and, ultimately, to apoptosis. The enzyme telomerase, a ribonucleoprotein complex with reverse transcriptase activity, adds TTAGGG repeats to telomeres providing their elongation and, sub© 2013 American Chemical Society

Received: September 14, 2012 Published: January 7, 2013 843

dx.doi.org/10.1021/jm3013486 | J. Med. Chem. 2013, 56, 843−855

Journal of Medicinal Chemistry

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conformations according to environmental transitions.8 Indeed, whereas the crystal structure of the DNA G-quadruplex assumed by wild-type Tel22 AG3[T2AG3]3 in K+ is parallel stranded,9 multiple G-quadruplex conformations (Figure 1) have been reported in solution.3,8

stage, compounds with unfavorable ADME (absorption, distribution, metabolism, and excretion) profiles.28 In a second step, LB selected compounds were submitted to ensemble docking simulations on all the four major structurally characterized conformations of the human telomeric sequence.8 The resulting top ranked molecules were considered for clustering analysis and visual inspection. The finally identified compounds were evaluated by biophysical methods to assess their G-quadruplex binding properties.

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RESULTS AND DISCUSSION Query Selection. With the aim to find new promising scaffolds able to recognize the human telomeric G-quadruplex, we applied computational methods which take into account structural information from known ligands. To build this data set, we analyzed active compounds included in De Cian et al.29 and we enriched the selection with other ligands reported in the literature (Figure S1 and Table S2 in Supporting Information (SI)). From the first analysis, a great diversity of scaffolds appeared, hence we chose multiple-reference searching to run our similarity screening. The queries, RHPS4,30,31 CX-3543 (Quarfloxin),32,33 SYUIQ5,34,35 Triazine-115405,36 Braco 19,37,38 Phen-DC3,39−41 and BMVC42,43 were selected based on their high affinity and selectivity toward DNA G-quadruplex versus duplex, chemical diversity, in vitro activity in tumor cells, and, eventually, preclinical and clinical studies (Figure 2a). Virtual Screening Strategy. For VS, we followed the workflow summarized in Figure 2b. In particular, we applied two LB virtual screening similarity methods on several vendors databases available in the ZINC44 repository to rationally choose the compounds to purchase. The similarity of each molecule in the database was calculated with reference to each query, and the compounds were ranked according to their maximum similarity score. MACCS fingerprint is a 2D similarity method which uses a predefined set of definitions and creates fingerprints based on pattern matching of the structure to the defined “key” set.24 Instead, ROCS recognizes similarity between molecules based on their three-dimensional shape. It quantifies the maximal overlap of the volume of two molecules and a combined score based on ShapeTanimoto similarity and the chemical matching score.27 The top ranked molecules, identified for each query with the applied similarity approaches, were merged obtaining the so-called “data fusion”. This group fusion technique turned out to be more efficient in retrieving hits than searches performed using a single reference compound.45,46 Overall, 3906 compounds were selected with MACCS and 2592 compounds with ROCS. The two methods exhibited very low overlap because only 73 compounds were found in common. After discarding the duplicates, we ended up with 6425 compounds. Scaffold hops are often predicted using ligand-based similarity approaches primarily for proven applicability and speed of software implementation. Both methods chosen in this study were quite fast in analyzing the database of ∼2.7 million compounds and downsizing it. MACCS keys are often more in line with chemical intuition to the loss of versatility encountered by other fingerprints with bigger descriptor sets.24 ROCS was applied with success and showed to have very good enrichment in new47−49 and retrospective studies.46,50,51 The two methods use very different measures for the Tanimoto values so there was no guarantee to obtain similar hits list: one measures the Tanimoto coefficient, while ROCS

Figure 1. Schematic and 3D representation of the (a) antiparallel or basket-type (PDB code 143D),10 (b) parallel or propeller-like (PDB code 1KF1),9 (c) hybrid type-1 (PDB code 2HY9),11 and (d) hybrid type-2 (PDB code 2JPZ)12 DNA G-quadruplex conformations of d(AG3[T2AG3]3) sequence.

Several studies have been devoted to develop novel Gquadruplex binders. They showed that these ligands may be accommodated in multiple sites of the structure: mainly they can stack on the G-tetrads, but they can also be located into the grooves or can interact with the loop bases.13 Although some of them achieved preclinical and clinical phase, none reached the market so far. The major limitations associated with their questionable biological use are the poor selectivity for quadruplex DNA, the propensity to aggregate in aqueous media, the poor water-solubility, and the chemical instability.14 To address this issue, a large synthetic effort have been merged to computational methods. In particular, molecular descriptors able to provide preliminary information to discriminate binding affinities of π-stacking ligands,15 or combination of descriptors in quantitative structure−activity relationship (QSAR) models,16 have been applied. Most of the modeling studies exploit structure information with docking methods to rationalize the activity of the analyzed molecules or to search for new leads.17−21 As far as it concerns 3D-ligand-based approaches, two ligand-based (LB) virtual screening (VS) applications, both successfully applying pharmacophore models, have been reported until now.22,23 In the present study, we selected, as target, the human telomeric sequence. In the attempt to identify novel pharmacophoric units for this G-quadruplex forming sequence, we integrated two LB methods, based on the assumption of a relationship between chemical structure and biological function, with structure-based (SB) approaches. Thus we selected compounds similar to known active drugs and evaluated their complementarities with the receptor. The applied methods were fingerprints Molecular ACCess System (MACCS) MDL public Keys,24 implemented in Pipeline Pilot,25 which allows similarity measures based on binary keysets,26 and Rapid Overlay of Chemical Structures (ROCS), which aligns molecules according to their shape and chemical similarity.27 These similarity filters were combined with a tool able to predict molecular properties in order to discard, at an early 844



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Figure 2. (a) Selected queries for similarity virtual screening and (b) virtual screening workflow.

calculates ShapeTanimoto combining to the ColorScore.52 This explains the small overlap of the two methods. The consistency of a small number of common hits according to the two selection procedures along our workflow allows assumption that several positive hits could be missed by using a single protocol. As above stated, drug-likeness is a key parameter to take into account starting from the early stages of the drug discovery process. Indeed, considering the observed distribution of some key physico-chemical properties of approved drugs, many Gquadruplex-binders do not show good ADME profiles (SI Table S1 ), and, therefore, the development of some of them has been discontinued. Thus we filtered the hits with a succession of filters created by Pipeline Pilot allowing for the range exhibited by the drugs in the market (SI Table S1). The properties considered in the refinement protocol were: molecular weight, number of rotatable bonds, hydrogen bond donors and acceptors, lipophilicity (by predicting ALogP),

polar, molecular, and solvent accessible surface area, molecular volume, aqueous solubility (Log S), and number of atoms of each molecule.28,53 Only 4005 compounds out of 6425 passed this multiple filter. They comprise 2726 deriving from MACCS selection, 1255 from ROCS, and 24 were overlapping. The selected hits were submitted to ensemble docking experiments considering the four quadruplex folds shown in Figure 1. In fact, the human telomeric sequence has been shown to fold into at least four distinct structures. They derive from remarkably different arrays of guanine pairing which result in distinct strand orientations and loop arrangements. Therefore, an induced fit docking or even a dynamic simulation would not be able to represent all these folds in a reasonable CPU time. However, according to the DNA strand orientation, the G-quadruplex structures could be summarized by four conformations: parallel, antiparallel, and two mixed types with both parallel and antiparallel features.54 Hence, an efficient approach to overcome the polymorphism hitch is to consider 845



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terminal vynil, enol ether, more than one hydroxamic acid, too many heteroatoms, and phosfonamide groups) were discarded.56 By sampling the diversity space of the clustered ligands, according to availability from the vendors, we reduced our data set to 28 compounds: 19 were selected from MACCS hit list, eight from ROCS, and one by both methods (Figure S3 and Tables S3−S4 of SI). In addition, to take into account the reciprocal ligand-target flexibility, the best poses of the 904 compounds generated by the AutoDock Vina57 docking were submitted to full energy minimization for each of the four G-quadruplex folds. The overall induced effect is summarized by the RMS deviation computed onto the receptor conformations optimized in the absence and presence of the ligands. This value, equal to 0.94 ± 0.42 Å, has demonstrated a general low perturbation onto the targets. Single effects on each fold are shown in the SI (Figure S4a). As expected, the major contribution of the mobility can be addressed to the loops, with an average ratio higher than 2.3 with respect to the core (SI Figure S4b). Details about the procedure are reported in the Experimental Section. Biophysical Screening of Selected Compounds. The potential efficiency of the selected compounds in binding Gquadruplex structures was assessed by fluorescence melting studies on a labeled sequence based on the human telomeric one (HTS), an approach extensively validated for screening of G-quadruplex ligand library and already applied to our queries.58 Most of our selected molecules were not able to increment the thermal stability of the telomeric G-quadruplex when used in the 0.5−10 μM concentration range (SI Figure S5). Nevertheless, one compound induced a quite relevant shift of the G-quadruplex melting temperature (Tm ≈ 14 °C at 10 μM ligand concentration). This efficiency was reduced in comparison to those exerted by two queries (Tm ≈ 32 and 30 °C at 10 μM Braco-19 and RHPS4, respectively) in the same experimental conditions (SI Table S5). This was not unexpected due to the selection procedure, thus we considered it worth of further investigation. This positive hit, labeled P1, was selected by MACCS fingerprints based on similarity with the reference query CX3543. This active compound contained a linear furocoumarin moiety also known as psoralen. Thus, all psoralen derivatives identified by the virtual screening were selected (P1−P7). The resulting new small library was further incremented by “in house” available psoralens structurally related to the hit (P8− P13) (Figure 4 and SI Table S6). All of them were tested in a wider concentration range to confirm the hit (Figure 5a). Moreover, a short double-stranded DNA sequence was used as additional target in order to evaluate the ligands selectivity (Figure 5b). As far as the telomeric sequence is concerned, this screening confirmed P1 and P3 as the most effective G-quadruplex stabilizers. Also P2, P6, and P8 were effective, although higher concentrations were required. Interestingly, none of the selected ligands were able to stabilize the double helix. These results suggest phenyl-psoralen as the basic scaffold required to selectively stabilize the tested G-quadruplex. The DNA interactions of the two most effective Gquadruplex binders were further investigated by CD (Figure 5c,d). The two tested psoralen derivatives behaved comparably: when added to the G-quadruplex folded telomeric sequence Tel22, they incremented the intensity of the band located at 290 nm and induced the formation of a negative band at 260 nm. The increased CD intensity upon binding is consistent

multiple rigid receptor conformations. With ensemble docking, a single ligand library is docked to each target conformation. To properly compare the score energy of the complexes generated by the docking experiments, the receptors are required to have the same sequence. Consequently, the two additional nucleotides at the head and tail caps of the hybrid receptors were removed.55 We picked up the best compounds according to the highest docking consensus score (240 nm was observed (SI Figure S6). This suggests the presence of multiple photoprocesses occurring in solution involving structural modifications in the chromophore moiety. Interestingly, a different kinetic in the photoreactivity of P1 and P2 emerged; indeed, the variation of their spectroscopic 847



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Figure 5. Variation of DNA melting temperature induced on (a) HTS or on (b) double-stranded DNA by selected psoralens. Representative CD spectra of (c) Tel22 and (d) ctDNA recorded in the presence (dotted line) or absence (solid line) of 10 μM P1.

G-quadruplex stabilization observed for some of them would be the result of UV conversion into the inactive form. Finally, we recorded the CD spectrum of Tel22 irradiated at 308 nm for 25 min in the presence of 2 equiv of psoralen. The resulting CD spectrum, compared to the one belonging to the free oligonucleotide, looked similar in shape but slightly less intense. This can reflect dissociation from the nucleic acid of the psoralen photoproduct due to its low affinity. Nevertheless, loss of DNA ordered structure cannot be excluded. Psoralens Photoreactivity on Distinct DNA Folds. Because of the chemical properties of psoralens, cross-reaction on DNA can occur. Thus, the reactivity of P1 and P3 promoted by UV was monitored in the presence of DNA sequences folded into different structures, in particular G-quadruplex, single-stranded, and double-stranded DNA. Each compound was added to DNA, and the whole solution was irradiated at 308 nm and loaded on a sequencing polyacrylamide gel electrophoresis (PAGE) (Figure 7). By monitoring the process after increasing irradiation time, we observed some DNA modifications occurring within 20 min. As described above, further increments in the irradiation time actually led to ligand degradation, thus we can assume that they do not contribute in defining psoralen-mediated DNA damage. On all the tested DNA templates, we identified the formation of DNA adducts characterized by a low electrophoretic

The chromatogram of P3 showed one peak at RT 17.2 min associated to a m/z value of 459.26 which identified the monocharged free compound (MW = 458.23); upon UV irradiation, it rapidly evidenced the appearance of an additional peak at RT 16.6 min with m/z value of 463.23, corresponding to the 6-benzoyl-7-hydroxycoumarin derivative. However, in this case, the coexistence of these two species was conserved even after 100 min of irradiation. DNA Interaction Properties of Photoproducts. To assess if photoproducts share the DNA recognition properties of the starting compounds, the G-quadruplex stabilization properties of P1 and P3 were compared to those of the irradiated derivatives. Fluorescence melting data summarized in Figure 6c clearly showed that the efficiency of G-quadruplex stabilization was largely impaired after UV irradiation. In line, the UV irradiated psoralens did not induce any change in the CD spectrum of Tel22 (Figure 6d). Because we applied conditions which are not causing psoralens degradation but their conversion into the 6-benzoyl-7-hydroxycoumarin derivatives, these data indicated that these photoproducts are not able to recognize the G-quadruplex template. In this context, it is important to underline that all tested psoralen derivatives showed comparable absorption profiles and photoreactivity with respect to P1 and P3. Thus we can exclude that the lack of 848



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Figure 6. (a) Normalized absorbance of P1 (full symbols) and P3 (empty symbols), recorded at 305 and 308 nm, respectively, as a function of UVirradiation time. (b) Scheme of photoconversion of tested psoralens into the corresponding 6-benzoyl-7-hydroxycoumarin derivatives. Effect of 25 min of irradiation at 308 nm on psoralen on (c) the thermal stabilization of HTS and on (d) the CD spectrum of Tel22 (4 μM Tel 22 alone, solid line; upon addition of 8 μM P1 not irradiated, dashed line; or of 8 μM P1 previously irradiated, dotted line). In (d), the spectrum of the irradiated Tel22−P1 mixture is also included (gray line) for comparison.

Table 1. Summary of LC-MS Data Obtained before and after UV Irradiation of P1 and P3 P1 irradiation time

P3

RT (min)

calcd MS

obsd MS

0

17.2

451.15

451.16

3 min

16.1 17.2

455.15 451.15

25 min

16.1 17.2

455.15 451.15

100 min

several peaks

irradiation time

RT (min)

calcd MS

obsd MS

0

17.0

459.23

459.26

455.16 451.16

3 min

16.6 17.0

463.23 459.23

463.24 459.24

455.16 451.16

25 min

16.6 17.0

463.23 459.23

463.25 459.25

multiple subproducts

100 min

16.6 17.0

463.23 459.23

463.24 459.25

based virtual screening approaches, selecting only the compounds with favorable predicted ADME properties. At the end of our virtual screening workflow, we selected 28 hits. Most of them presented a planar scaffold, often decorated with flexible side chains which are predicted to interact with DNA grooves and/or loops (SI Figure S8). Experimental evaluation of the ability to bind and stabilize the telomeric G-quadruplex structure by fluorescence and CD techniques allowed us to identify one psoralen as novel a G-quadruplex binder. This positive hit, P1, was selected by MACCS fingerprints with reference to the query CX3543. A retrospective analysis of ROCS selection results showed that the Comboscore for this molecule was 0.8, while the threshold for selection was set to values >1.2. This suggests that a less restrictive range score should provide better consensus results.

mobility; in addition, DNA degradation was clearly promoted. This was extremely remarkable in the presence of P3. From our data, the occurrence of radical-mediated reactivity did not allow a targeted reactivity on the G-quadruplex structure, although it appears less prominent for duplex DNA. The comparable reactivity observed for G-quadruplex and single-stranded DNA suggests that the residues in the loops (which can be structurally compared to a single stranded portion) could represent the target of our psoralens derivatives. This is also consistent with the presence of thymines in these regions, which represent the most favorable nucleobases for psoralenadduct formation.

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CONCLUSION In summary we have screened ∼2.7 million compounds from the ZINC database with a combination of ligand and structure849



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Figure 7. Sequencing gel of the reaction products occurring upon irradiation of (a) single-stranded, (b) G-quadruplex Tel22, or (c) double-stranded DNA in the presence of P1 and P3, and (d) percentage of unmodified DNA detected after 20 min of irradiation in the presence of 20 or 50 μM psoralens.

photoconversion of P1 and P3 into the corresponding 6benzoyl-7-hydroxycoumarin derivatives is detrimental for Gquadruplex recognition because the system loses its planarity, a key parameter to provide stability to the complex. By analyzing the energy minimized docking poses of the best compound P1, these considerations were sustained (Figure 8). In particular, the positively charged substituent was found to interact with the negatively charged phosphate backbone of the G-quadruplex within the grooves/loops, whereas the aromatic portion was alternatively involved in stacking with the bases. It is worth underlining that these considerations summarize different binding modes detected accordingly to the Gquadruplex folds. Nevertheless, in each case, no substantial variations in the G-quadruplex folds were found after docking experiments and energy minimization simulations. This is in line with the generally conserved CD spectral features of the telomeric sequence observed in the presence and absence of the active compounds which suggest poor G-quadruplex structural rearrangement upon binding. A good agreement between theoretical calculation and experimental data was obtained also by comparing the docking results of compound P1 with reference compounds Braco-19 and RHPS4. In this instance, we observed that all of them stack on the G-tetrad when the receptor assumes a parallel conformation (1KF1). However the flexibility of the side chains in Braco-19 allows them to better accommodate into the respective grooves of the G-quadruplex folds, whereas RHPS4 forms extensive π−π interactions between its huge aromatic portion and the G-quartets in three of the four analyzed

A weakness of 3D methods is related to the complexity in finding a proper conformation for the screened compounds. OMEGA, with default settings, generates 200 conformations which were confirmed to possibly comprise the bioactive conformation.66,67 Concerning P1, only 56 conformations were retrieved. By increasing the energetic windows from 10 up to 20 kcal/mol above the global minimum value, the number of generated conformations rose up to 123, but the 0.8 Comboscore was maintained. Thus, OMEGA parametrization should not be responsible for the missed identification of P1. Then we can postulate that the scaffold of P1 is too different from the query to be identified by ROCS. Nevertheless, it is worth reminding of the absence of a unique binding mode for the G-quadruplex ligands. Although this can explain the diversity of scaffolds shown by the active compounds, it can turn the selection of ligands interacting by computational methods into a very challenging process. By using a small psoralen scaffold focused library we found that by increasing the distance between the aromatic core and the amide group a reduction in activity occurred, P1 and P3 (which have one methylene linker) being the most active compounds. Furthermore, a strong influence of the side chain was highlighted: it should be not too rigid as in P5 and P7, and a small (P1, P2, P3) or a flexible chain (P6 and P8) is preferred. The role of the aromatic ring at position 3 of the psoralen core is relevant: its presence was confirmed to be essential, as P7 and P9−P13 are inactive, and the bulkiness/ electronic properties of the substituents are able to modulate the activity. Accordingly, the opening of furan ring and the 850



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Figure 8. Optimized docked poses (orange) compared to original docking pose (blue), of complexes P1 (in green)-G-quadruplex conformations of d(AG3[T2AG3]3) sequence. (a) Antiparallel PDB code 143D), (c) parallel (PDB 1KF1), (e) hybrid type-1 (PDB 2HY9), and (f) hybrid type-2 (PDB 2JPZ). Compound interactions with each receptor were analyzed by means of LigandScout software. 2D depictions show hydrogen bond (HB) donors (green arrows), HB acceptors (red arrows), aromatic interactions (blue circles), and ionic interactions (blue rays). ZINC44 repository associated with nine vendor companies (Vitas, Specs, Ibs, Enamine, NCI, Asinex, Aurora, Chemblock, Maybridge). Seven active compounds, reported in Figure 2, were used as queries. The database structures were “washed” by removing all inorganic components and by adjusting ionization states to pH = 7.4. Furthermore, the compounds were energy minimized using the MMFF (Merck Molecular Force Field) force field. The 2D similarity score for the lead compounds was calculated by means of Tanimoto coefficient (Tc) and using MACCS fingerprints24 as implemented by Pipeline Pilot.25 Only compounds with Tc ≥ 0.7 were selected, for a total of 3906 compounds. The same database was screened with ROCS shape-based methodology.27 A conformer library was generated using the program OMEGA,70 with default parameters. The 2592 compounds were selected by considering ComboScore (>1.2) values. This score takes into account two distinct aspects: the shape similarity (“ShapeTanimoto”) and the chemical pattern (“ColorScore”) similarity. Both components obtain values between 1 and 0 and are summed up for the

conformations (SI Figure S9−S10). These indications, which are in line with X-ray and NMR structural data available for these compounds, justify the better G-quadruplex stabilization they exert in comparison to P1.68,69 This suggests that, by increasing the aromatic interaction of our compounds or reinforcing the contacts of the lateral side chain with the grooves, we could expect to improve the efficiency of our lead as G-quadruplex binder. In conclusion, the workflow herein applied was successful in retrieving a new promising drug-like scaffold different with respect to known binders. Thus, this represents a highly valuable starting point for an optimization procedure concerning this new class of G-quadruplex binders.

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EXPERIMENTAL SECTION

Similarity Methods. The similarity search was conducted by virtually screening ∼2.7 million of compounds publicly available in the 851



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ComboScore. Hence, the values range from 0 to 2, where 2 stands for the best possible overlap and 0 for no similarity. Drug-likeness Filter. A Pipeline protocol was created using “property prediction” components to analyze the drug-likeness of the compounds in order to evaluate if the fulfillment of ADME descriptors was assessed. The ranges of drugs28,53 were taken into account as indicated in Table S1 in SI. After applying this series of filters, only 4005 compounds were selected and submitted to docking experiments. Docking. The ensemble docking simulations were carried out using the software program AutoDock Vina.57 Finally 904 compounds were selected. Detailed protocol of docking is described in the SI. Clustering Pose Analysis. The poses analysis of compounds docked into G-quadruplex receptor has been developed by means of the angle descriptor defined by three dummy atoms. Two of them, respectively centered within the external bottom and the intermediate G-quartet planes, are DU1 and DU2. DU3 is defined by the ligands’ centroids computed by the Maestro GUI in best poses. Basing on the DU1−DU2−DU3 angle, the classification of the cluster analysis is defined as follows: cluster bottom (angle within 0−60°), cluster lateral (angle within 60−120°), and cluster top (angle within 120−180°) (Figure 3). The molecules were clustered for diversity by means of Pipeline Pilot’s method using ECFP6 fingerprints.25 The analysis of clusters, scores, and the visual inspection helped the selection of compounds. Considering their availability, 28 compounds were purchased and tested. Complex Energy Optimization. Following our previously reported computational approach,55 the best poses obtained in the docking of 904 selected compounds were submitted to full energy minimization. DNA experimental models and all docking generated complexes were fully optimized using 10000 steps of the Polak Ribiere conjugate gradient71 algorithm and energy evaluated with the united atoms notation of the AMBER* force field72 as implemented in MacroModel version 9.8.73,74 Solvent effects were taken into account by means of the GB/SA water implicit solvation model.75 A convergence criterion equal to 0.05 kJ·mol−1·Å was adopted. The ligand induced DNA structural perturbation was explored by computing, onto the nucleic acid not hydrogen atoms, the root means square deviation (RMSd) among the optimized experimental model and the 904 corresponding complexes. Root-mean-square data values were reported in Å. Taking into account as “core” the atomic coordinates of the guanine tetrads and as “loops” the rest of the target model, the mobility ratio was computed dividing the average RMS deviation of the loops for that of the core. The figures representations of receptor folds and docking poses were made by means of Pymol76 and LigandScout software.77 Photoproducts Formation. Solutions of selected ligands (30 μM) in 10 mM Tris-HCl, 50 mM KCl at pH 7.5 were irradiated at 308 nm (0.022 J/min) for increasing time at 25 °C, and the corresponding absorption spectra in the wavelength range 350−250 nm were recorded with a Perkin-Elmer Lambda 20 apparatus. Absorbance values at 305 nm (P1) or 308 nm (P3) were plotted as a function of UV irradiation time. Mass Spectrometry. Mass spectra of ligands before and after UV irradiation were obtained using an Applied Biosystems Mariner mass spectrometer connected with an Agilent Technology 1290 Infinity UPLC apparatus. The column used was a ZORBAX Eclipse Plus C18 column, whose dimensions are 2.1 mm × 50 mm with a diameter of particles of 1.8 μm. Chromatographic separations were performed at a flow rate of 0.02 mL/min by a linear water/acetonitrile gradient containing 0.1% of formic acid from 4% to 80% in 20 min. The eluted product was detected and the identity was confirmed by ESI-MS analysis. The ESI source was set in positive ion mode with an electrospray voltage of 4.5 kV. Spectra were acquired every 8 s over the m/z range of 100−2500. Fluorescence Melting Studies. Melting experiments were performed in a Roche LightCycler, using an excitation source at 488 nm and recording the fluorescence emission at 520 nm. Target DNA was the human telomeric sequence (HTS) 5′-AGGGTTAGGGT-

TAGGGTTAGGGT-3′, labeled with Dabcyl at the 5′ end and Fluorescin at the 3′ end and a 18 bp double-stranded DNA (5′GTGAGATACCGACAGAAG). Mixtures (20 μL) contained 0.25 μM of target DNA and variable concentrations of P1 and P3, before and after UV irradiation, in 10 mM LiOH, 50 mM KCl, pH 7.4, with H3PO4. They were first denatured by heating to 95 °C for 5 min and then cooled to 30 °C at a rate of 0.5 °C min−1. Then temperature was slowly increased (0.2 °C/min) up to 90 °C and again lowered at the same rate to 30 °C. Recordings were taken during both these melting and annealing reactions to check for hysteresis. Tm values were determined from the first derivatives of the melting profiles using the Roche LightCycler software. Each curve was repeated at least three times and errors were ±0.4 °C. Circular Dichroism Measurements. Circular dichroism spectra were recorded from 230 to 350 nm using a 10 mm path length cell on a Jasco J 810 spectropolarimeter equipped with a Peltier temperature controller. As DNA substrates, we used calf thymus DNA (ctDNA) and Tel22 (5′-AGGGTTAGGGTTAGGGTTAGGG). DNA solutions (88 μM in residues) in 10 mM Tris-HCl, 50 mM KCl, pH 7.5, were added of P1 and P3 (8 μM), before and after 30 min UV irradiation. The reported spectrum of each sample represents the average of three scans recorded with 1-nm step resolution. Observed ellipticities were converted to mean residue ellipticity [θ] = deg × cm2 × dmol−1 (Mol. Ellip.). Photoreactivity on Distinct DNA Folds. G-quadruplex Tel22 or single-stranded DNA (5′-GGATGTGAGTGTGAGTGTGAGG-3′) were 5′-labeled with 32P and T4 polynucleotide kinase by incubating the reaction mixture at 37 °C for 30 min. The kinase was then inactivated by heating the reaction mixture at 85 °C for 5 min, followed by two extractions with one volume of phenol/CHCl3 (50:50). Experiments were performed using 1 μM DNA in 10 mM Tris, 50 mM KCl at pH 7.5 in three different conditions: (1) DNA solutions were added of increasing concentrations of tested compounds and subsequently irradiated at 308 nm for variable time, (2) DNA solutions were added of increasing concentrations of compound previously UV-irradiated for 30 min, (3) DNA solutions were added of increasing concentrations of nonirradiated compound. Reaction products were resolved by gel electrophoresis (20% polyacrylamide gel with 7 M urea) in 1× TBE (89 mM Tris base, 89 mM boric acid, 2 mM Na2EDTA) and finally visualized and quantified on a PhosphorImager (Amersham).

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ASSOCIATED CONTENT

S Supporting Information *

Chemical−physical property filters and comparison with Gquadruplex binders and hits; docking protocol and details; structure and chemical−physical properties of G-quadruplex binders; summary of full energy minimization of best posed 904 selected compounds on each G-quadruplex fold; ligand concentrations required to produce 50% of the maximal Gquadruplex thermal stabilization; structure and chemical− physical properties of the purchased hits; virtual screening scores; chemical−physical properties of the tested psoralen derivatives; screening of the selected compounds by fluorescence melting experiments; UV spectra of P1 and P3 after UV irradiation; LC-ESI MS of P1and P3; docking poses of selected hits, docking poses of reference compounds, 1H NMR. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*For C.S.: phone, +39-049-8275711; E-mail, claudia.sissi@ unipd.it. For S.D.: phone, +39-070-6758550; E-mail, s. [email protected]. 852



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Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the Italian Ministry of Education (Funding for Investments of Base Research) for the years 2009−2014 (code FIRB-IDEAS RBID082ATK), the PRIN 2009 (code 2009MFRKZ8), by AIRC (Associazione Italiana per la Ricerca sul Cancro) and by University of Padova (grant no. CPDA114388). F.M. is grateful to “Commissione Europea, Fondo Sociale Europeo e della Regione Calabria” for her Ph.D. grant. We thank Openeye Scientific Software, Inc. for providing the academic license for the Openeye package, Accelrys Software, Inc. for the free academic license of Pipeline Pilot Student Edition, and Odra Pinato for technical support. We acknowledge Prof. S. Neidle and Prof. R. Griffin for providing them the tested queries.

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DEDICATION This work is dedicated to the memory of Prof. Sergio Caffieri.

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ABBREVIATIONS USED DNA, deoxyribonucleic acid; rRNA, ribosomial ribonucleic acid; Tel22, wild-type telomeric sequence 22 bases long; UV, ultraviolet; CD, circular dichroism spectroscopy; QSAR, quantitative structure−activity relationship; VS, virtual screening; MACCS, Molecular ACCess System; ROCS, Rapid Overlay of Chemical Structures; ADME, absorption distribution metabolism and excretion; SI, Supporting Information; ESI-MS, electrospray ionization mass spectrometry; MW, molecular weight; RT, room temperature; LC-MS, liquid chromatography−mass spectrometry; MMFF, Merck Molecular Force Field; Tc, Tanimoto coefficient; PDB, Protein Data Bank

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Identification and Characterization of New DNA G-Quadruplex

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