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Departamento de Farmacología, Universidad de Alcalá, Madrid, Spain
Received June 13, 2005; accepted September 7, 2005
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) but is now obtained in multigram quantities through chemical modification of microbially produced cyanosafracin B. Trabectedin consists of a polycyclic skeleton composed of three fused tetrahydroisoquinoline rings (Fig. 1A) and binds to the minor groove of DNA by virtue of its reactive 21-carbinolamine group (reviewed in Gago and Hurley, 2002). The binding site covers three base pairs, and the exocyclic amino group of guanine is necessary for the covalent binding of the drug (Pommier et al., 1996). Sequence selectivity has been shown to operate predominantly through a set of well defined hydrogen-bonding rules (Gago and Hurley, 2002) such that the preferred target triplets are 5'-RGC and 5'-YGG, where R and Y stand for purine and pyrimidine, respectively, and the underlined base is the guanine that undergoes alkylation by trabectedin. We have reported previously on the remarkable similarity in the manner in which trabectedin and zinc fingers from transcription factors such as EGR1/Zif268 or Sp1 (Marco et al., 2002) induce DNA structural distortions (Gago and Hurley, 2002), especially when three trabectedin molecules bind head-to-tail to three adjacent optimal binding sites on the same strand (Marco et al., 2002). In this case, trabectedin was shown to stabilize a DNA conformation that was intermediate between A- and B-form DNA (Ng et al., 2000).
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). Indeed, footprinting (Stankusm et al., 1992) and NMR spectroscopic (Gao and Patel, 1990) studies have shown strong, cooperative, and symmetrical binding of chromomycin A3 dimers to adjacent TGG sequences on different strands (i.e., TGGCCA) when embedded in different self-complementary duplexes. The recent resolution by X-ray crystallography of the structure of an Mg2+-chelated dimer in complex with a double-stranded d(TTGGCCAA)2 octanucleotide [Protein Data Bank (PDB) accession code 1VAQ; http://www.rcsb.org/pdb/] has provided additional details about metal ion coordination and specific drug-DNA hydrogen bonds (Hou et al., 2004) In addition, this work has confirmed earlier evidence (Gao and Patel, 1990) that the DNA adopts a conformation that is best described by a combination of both B- and A-DNA structural parameters.
It is interesting that both trabectedin (NSC-648766) and chromomycin A3 (NSC-58514) have been shown to exert at least part of their cytotoxicity by interfering with cell replication and transcription (Bonfanti et al., 1999; Chatterjee et al., 2001). Furthermore, comparison of the activity parameters for these two compounds on the panel of 60 human tumor cell lines of the National Cancer Institute (NCI) Anticancer Drug Screen (http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html) has revealed a very high correlation coefficient (0.96) using the COMPARE algorithm (Paull et al., 1989). Thus, despite a very different mechanism of binding to DNA (i.e., covalent versus noncovalent, carbinolamine activation versus ion-mediated dimerization, etc.), both drugs seem very similar in the way in which they induce apoptosis in different tumor cell types. This might be because the structural distortions they inflict on the DNA molecule are indeed comparable. To test this hypothesis, we have built and simulated, using unrestrained molecular dynamics (MD) simulations in aqueous solution, the structure of the self-complementary dodecanucleotide d(GTATGGCCATAC)2 in a complex with two trabectedin molecules, each covalently bonded to a different strand in a tail-to-tail fashion (Fig. 2). For comparison and completeness, the same sequence has also been simulated under identical conditions in the absence of any bound drug. Thus, we have been able to assess the conformational preferences of the TGG triplet sequence, which is ubiquitously over-represented in human viral and eukaryotic sequences (Burge et al., 1992), including sites at or near replication origins of human mitochondrial DNA (Kang et al., 1997). Moreover, its complementary sequence is an important component of the so-called "CCAAT box" that is present in a high number of promoters (Mantovani, 1998), and the juxtaposition of the two (i.e., TGGCCA) has been dubbed by some X-ray crystallographers as a "natural bending element" (Grzeskowiak et al., 1993).
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) was updated with new DNA parameters (Cheatham et al., 1999) for improved sugar pucker phases and helical repeat (parm98). Bonded parameters and quantum mechanically derived 6-21G* RESP charges for guanine-bonded N12-protonated trabectedin have been described previously (Marco et al., 2002).
Construction and Refinement of the Starting DNA Structures. A model of the free oligonucleotide was built using optimized parameters for B-DNA. The covalent complex between d(GTATGGCCATAC)2 and trabectedin (one covalently modified guanine in the middle of each TGG triplet) was modeled using the same oligonucleotide in which the TGG triplet in each strand had been replaced by a TGG-trabectedin adduct as found in the energy-refined representative structure of the equilibrated (trabectedin)3-d(GTGGCGGCGGCC) x d(GGCCGCCGCCAC) complex (PDB entry 1KML) reported previously (Marco et al., 2002). The target TGGCCA was flanked by a 5'-TA-3' sequence on each side because both TpA and ApT steps are considered to context-independent, and a terminal G:C pair at each end was used to prevent fraying. This initial geometry was then refined by means of 1000 steps of steepest descent energy minimization followed by 2000 steps of conjugate gradient energy minimization of only those atoms belonging to trabectedin and the replaced nucleobases. This procedure allowed readjustment of covalent bonds and van der Waals contacts without changing the overall conformation of the complex.
Molecular Dynamics of Free and Trabectedin-Bonded Oligonucleotides in Water. Each molecular system was neutralized by addition of the appropriate number of sodium ions, placed in positions of negative electrostatic potential and immersed in a rectangular box of 
4600 transferable intermolecular potential three-point model water molecules. Each water box extended 8 Å away from any solute atom, and the cutoff distance for the nonbonded interactions was 9 Å. Periodic boundary conditions were used, and electrostatic interactions were represented using the smooth particle mesh Ewald method with a grid spacing of 1 Å. The SHAKE algorithm was applied to all bonds involving hydrogens, and an integration step of 2 fs was used throughout. The simulation protocol for both the free dodecanucleotide and the covalent adduct was essentially as described previously (Marco et al., 2002) and made use of the SANDER module in AMBER version 6.0 (http://amber.scripps.edu/). In brief, solvent molecules and counterions were relaxed by energy minimization and allowed to equilibrate during 50 ps of MD at 300 K and constant pressure around the atoms of the DNA or the drug-DNA complex, which were restrained to their initial positions with a harmonic restraint of 25 kcal mol-1Å-2. These restraints were gradually reduced in a series of successive minimizations, and the unrestrained system was then heated from 100 to 300 K during 10 ps followed by 0.5 ns of equilibration and 8.0 ns of data collection. System coordinates were saved every 2 ps for further analysis.
Analysis of the Molecular Dynamics Trajectories. Three-dimensional structures and trajectories were visually inspected using the computer graphics program PyMol (http://www.pymol.org). Root-mean-square deviations (rmsd) from the initial or final structures and interatomic distances were monitored using the CARNAL module in AMBER. The conformational and helical parameters of the DNA dodecamers were analyzed by means of program CURVES (Lavery and Sklenar, 1988). The magnitude and directionality of the bending was related to the local helicoidal parameters roll and tilt, and quantified in terms of the angle of axis deflection (
) and its orientation relative to the major groove (
). Graphical display of these quantities for individual base pair steps was performed using polar plots that allow both the magnitude and the direction of the helical bend to be represented in the form of "bending dials" (Young et al., 1995), in which and are the radial and angular coordinates, respectively. Points on the northern hemisphere of the dial reflect positive roll and compression of the major groove, whereas bending into the minor groove is plotted on the southern hemisphere. Each ring on our bending dials indicates a 10° deflection of the helical axis, and runs clockwise from the top.
Solvent-Accessible Surface Area Calculations and Hydrophobic Effect. The reduction in DNA solvent-accessible surface area (SA) brought about by trabectedin binding was estimated as the difference between the total SA of the free DNA and the SA of DNA in the trabectedin-DNA complex using the program NACCESS (http://wolf.bms.umist.ac.uk/naccess/) and a water probe with a radius of 1.4 Å. The buried SA for trabectedin was likewise calculated as the difference in SA between twice the value of one isolated molecule and that of the two trabectedin molecules in the covalent complex. The hydrophobic effect involved was approximated by making use of the conversion factor of 20 cal · Å-2 obtained from a linear correlation between the SA of hydrophobic side chains in proteins and their free energy of transfer from a polar to a nonpolar solvent (Chothia, 1974).
All calculations were performed on the R8000 Power Challenge (SGI, Mountain View, CA) at Alcalá University Computer Center, on the SGI R14000 [GenBank] Origin 3800 at Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (Madrid), and locally on SGI R5000 O2 workstations.
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), bonding of trabectedin resulted in increased DNA stabilization as assessed by the smaller rmsd fluctuations in the simulation of the complex relative to the simulation of the free oligonucleotide. An advantage of using a self-complementary DNA sequence is that the extent of sampling for each base-pair step is actually doubled because they are all represented twice. In addition, the similarity of the results for identical steps provides an indication of the consistency of the calculated structural parameters.
Description of the (Trabectedin)2-d(GTATGGCCATAC)2 Complex. The overall structure of the 2:1 complex is one in which each subunit A of trabectedin protrudes perpendicularly off the helix in front of the guanine to which it is bonded (G5 and G17), the B subunits stack over the sugar rings of C20 and C8 in a manner reminiscent of that of typical nonintercalative minor groove binders, and the C subunits expose one flat side to the solvent, whereas the other side makes extensive contacts with the sugar-phosphate backbone of the two nucleosides downstream of each covalently bonded guanine (Fig. 3). In the previous model with three trabectedin molecules bound "head-to-tail" to consecutive DNA triplets in the same strand, no interdrug contacts were apparent; in the present "tail-to-tail" arrangement, however, both B subunits established favorable van der Waals contacts at the junction of both DNA triplets. This may account for the negative roll detected at the G6/C7 junction, and also for the observation that the increase in positive roll at adjacent G/G steps (Table 1) is less than that described in previously studied trabectedin-DNA (Gago and Hurley, 2002; Marco et al., 2002) and EGR1-DNA and Sp1-DNA complexes (Marco et al., 2003).
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Monitoring the relative rotation of successive base pairs about an axis perpendicular to the plane of the base step along the trajectory showed the variation in helical twist angles among the different dinucleotide steps, which was averaged over time (Table 1). The greatest unwinding as a consequence of drug bonding takes place at T4/G5 and C8/A9 (= T16/G17) steps, which also show the largest values of roll angle (relative rotation of the base pairs about the long axis of the base step), in good accord with the known inverse relationship between this parameter and helical twist (Gorin et al., 1995). Both T4/G5 and C8/A9 steps are found in the predominant conformation CA+ (Table 1) that displays relatively small values of twist (30°) and positive roll. This twist-roll relationship is also valid for G6/C7 in that this step displays both negative roll and the largest helical twist within the TGGCCA sequence.
Because roll and tilt components are known to contribute to bending of the double helix (Young et al., 1995), these values were calculated and displayed in the form of "bending dials" (Fig. 4). Bending, which compresses the major groove and arises from the positive roll reported in Table 1, is clearly apparent at T4/G5 and C8/A9 (= T16/G17) steps (dots plotted on the northern hemisphere of the dial) and of comparable magnitude in both despite the opposite tilt preferences. At T4/G5, the tilt component is negative (note the dots on the top left quadrant of the dial), indicative of bending into the phosphate backbone of its complementary Watson-Crick C20/A21 step. At C8/A9, the tilt component is positive, but it would be negative if measured for the complementary T16/G17. Compression toward the minor groove is observed only at the junction between the TGG triplets and is probably a consequence of van der Waals interactions between subunits B of both drug molecules, because this was not apparent in the simulation of the free DNA (see below).
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The conformational features of the DNA in the (trabectedin)2-d(GTATGGCCATAC)2 complex are reminiscent of those previously found in the (trabectedin)3-d(GTGGCGGCGGCC)·d(GGCCGCCGCCAC) complex and are best described as intermediate between those of A- and B-form DNA (Table 2). Average values for slide (relative translation of the base pairs about the long axis of the base step), twist, and minor groove width are closer to those of regular A-DNA. The negative sign of the X-displacement in both types of complex means that the base pairs have been displaced from the helical axis in the direction of the minor groove to create a deeper major groove, as found in A-DNA. With respect to sugar puckering and backbone conformation, three different situations are observed: 1) deoxyriboses with a C3'-endo conformation, such as those attached to the cytosines (C8 and C20) that are complementary to the trabectedin-bonded guanines (G5 and G17); 2) phase angles that oscillate during the trajectory from C3'-endo to O1'-endo conformations, as in C7, C19, T4 and T16, thus differing from the behavior expected for a regular B-DNA in which values are distributed between C2'-endo and C1'-exo; and 3) deoxyriboses in C2'-endo with low standard deviation in their phase angles, as seen in those linked to the trabectedin-bonded guanines and the purines in the complementary strand (Table 3). These differences in phase angles seem to be correlated with distinct values in the glycosyl torsional angle, 
, such that nucleosides with high standard deviations in sugar puckering have average values midway between those corresponding to A-DNA ( = -154°) and B-DNA ( = -90°), whereas typical B-DNA values are found in nucleosides with low standard deviation in their phase angles. This effectively means that B-type nucleotides in the triplet region harboring the drug are paired with A/B-type nucleotides in the complementary strand. These altered sugar parameters correlate with the observed changes in slide and roll that are known to be necessary for the B
A transition (Ng et al., 2000; Vargason et al., 2001).
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Interactions between DNA and the Two Trabectedin Molecules Bonded in Opposite Strands. In the equilibrated complex of d(GTATGGCCATAC)·d(GTATGGCCATAC) with two trabectedin molecules covalently bonded to G4 and G17 (underlined), each drug is held in position by an intermolecular hydrogen bonding arrangement coincident with that originally proposed based on NMR experiments (reviewed in Gago and Hurley, 2002) and essentially identical to that found in other trabectedin-DNA complexes (Gago and Hurley, 2002; Marco et al., 2002): the protonated N12 of trabectedin is engaged in a hydrogen bond with the N3 acceptor atom of A21 in the first triplet and A9 in the second triplet; the methylendioxy oxygen facing the minor groove is involved in a hydrogen bond with the amino N2 of G5 and G17 (the bases after the guanines that are covalently modified by each trabectedin molecule), and the OH on subunit C is hydrogen-bonded to the O1P of the phosphate linking C7–C8 and C19–C20 (Table 4).
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Upon formation of the 2:1 trabectedin-DNA complex, the buried solvent-accessible surface areas of DNA and both trabectedin molecules are 590 and 770 Å2, respectively, of which 185 and 340 Å2 correspond to nonpolar atoms. Thus, a substantial hydrophobic effect can be expected that would translate into an important energetic contribution (10.5 kcal/mol) to the stabilization of the double helix.
Comparison of d(GTATGGCCATAC)2 in the Free State and with One Trabectedin Molecule Bonded to Each Strand. The positioning of the bulky drug in the central region of the dodecanucleotide results in a notably wider minor groove at the TGGCCA sequence relative to the free DNA (Table 5). Similar results were found for the AGC-, TGG- and CGG-containing oligonucleotides reported previously (Gago and Hurley, 2002; Marco et al., 2002).
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By comparing the average twist values for the different base steps in the trabectedin-DNA complex and in the free dodecanucleotide (Table 1), it can be noted that ApT and TpA steps retain their natural tendency to be underwound and overwound, respectively (Gorin et al., 1995), in both simulations. The most marked changes brought about by bonding of trabectedin, on the other hand, affect T4/G5, C7/C8, C8/A9, and A9/T10 steps, which not only are all unwound relative to the free DNA but also display much less fluctuation about their average values. This finding is in consonance with the enhanced DNA structural stabilization brought about by drug bonding discussed above (Supplemental Fig. S1).
The reported differences in twist translate into similar differences in roll so that T4/G5 and C8/A9 steps appear bent toward the major groove (Fig. 4). Even though T4/G5 and C8/A9 are shown to be intrinsically bent, greater bending is clearly observed upon trabectedin bonding, as demonstrated previously for an isolated triplet site (Gago and Hurley, 2002). Despite the relatively small increase in positive roll at G5/G6 and C7/C8 (= G17/G18), these steps show the largest negative slide (Table 1) that is associated with having trabectedin covalently bonded to G5 and G17, and this correlates with the observed changes in minor groove width and glycosyl torsion angles (Tables 3 and 5).
Comparison with the (Chromomycin A3)2-d(TTGGCCAA)2 Complex. Although two independent complexes in different packing environments are present in the asymmetric unit of the crystal lattice (Hou et al., 2004), they were found to be virtually superimposable (rmsd of 1.24 Å over 983 atoms). Remarkably, this drug-DNA complex and the (trabectedin)2-DNA complex that we have studied have several features in common such as 1) an unwound DNA and a considerably widened minor groove in the target TGGCCA region; 2) a similar pattern of roll values (increased positive roll at TpG steps and small positive roll at GpG steps, together with negative roll at the central GpC step, which is more marked in the trabectedin-complex); and 3) negative slide at both GpG steps, in good agreement with the finding of values closer to those of A-DNA.
All of these characteristics describe a DNA conformation (Ng et al., 2000; Vargason et al., 2001) that is intermediate between those of A-DNA and B-DNA (Table 2). On the other hand, both drugs span a common triplet DNA site, and the O8 atoms of chromomycin A3 are involved in similar hydrogen bonding interactions with the exocyclic amino group (N2) of G4 and G12 as those observed between the methylendioxy oxygens of trabectedin and the N2 of G5 and G19. The nonbonded equivalent of the covalent bond between the C21 of trabectedin and the N2 of G5 and G17 is the hydrogen bond that is established between the E-ring oxygen of chromomycin A3 and the N2 of G3 and G11 (Fig. 3).
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) or three tandemly arranged triplet sites in the same DNA strand (Marco et al., 2002) have already been published, no structural information is available about the effects brought about by the drug when more than one molecule bind to closely spaced target sites on different strands. We now show that it is indeed feasible for two trabectedin molecules to bind in a tail-to-tail arrangement to two adjacent TGG sites placed on opposite strands.
Previous NMR-based MD simulations in aqueous solution of the covalent complexes between trabectedin and two non–self-complementary DNA nonanucleotides containing a central AGC or CGG triplet revealed that adduct formation brings about widening of the minor groove and restriction of some of the bending motions of free DNA (Gago and Hurley, 2002). These structural changes were shown to translate into significant bending of the DNA duplexes toward the major groove (PDB entries 1EZH and 1EZ5), mostly because of an increase in positive roll at the base pair step involved in covalent bond formation. Both the direction and magnitude of these bends were found to be in very good agreement with the macroscopic curvature detected in earlier electrophoretic gel migration and circularization experiments (Zewail-Foote and Hurley, 1999). It is noteworthy that, compared with their respective free DNA molecules, the CGG triplet seemed to accommodate the drug with less distortion than AGC because of a slightly wider minor groove and both a decreased twist angle and a positive roll angle at the CpG step. This result suggested that the intrinsic bendability of a particular DNA sequence, and its overall preorganization could facilitate specific recognition by trabectedin in much the same way as DNA sequence specificity for the minor groove covalent binders anthramycin and tomaymycin has been shown to correlate with the degree of bending and reaction kinetics (Kizu et al., 1993).
Model building based on this early work also suggested to us that tandem binding of several trabectedin molecules to suitable adjacent DNA sites was sterically and energetically feasible. Indeed, in agreement with the fact that both TGG and CGG triplets represent optimal binding sites for trabectedin (reviewed in Gago and Hurley, 2002), we were able to assess the stability of a (trabectedin)3-d(GTGGCGGCGGCC)·d(GGCCGCCGCCAC) complex (PDB entry 1KML), demonstrating that the expected intermolecular hydrogen bonding scheme between each trabectedin molecule (covalently bonded to the underlined guanine) and each Y-G-G site was maintained along a nanosecond MD trajectory (Marco et al., 2002).
The present results now show for the first time that binding of two trabectedin molecules to target sites placed contiguously but on opposite strands is also feasible and leads to distinct structural distortions in the DNA oligonucleotide. Furthermore, the availability of the high-resolution (2.15 Å) structure of a Mg2+-mediated chromomycin A3 dimer bound to d(TTGGCCAA)2 has allowed us to realize the striking similarity of the DNA structural parameters in both complexes (Table 2 and Fig. 4). The rationale for performing such a comparison stemmed from the observation that trabectedin and chromomycin A3 induce highly correlated cytotoxic responses in the COMPARE analysis (r = 0.96). It is remarkable that the overall structure that we find for the TGGCCA sequence in the complex of d(GTATGGCCATAC)2 with two trabectedin molecules is virtually superimposable to the same sequence as found in a DNA octanucleotide containing two chromomycin A3 molecules aligned in a head-to-tail orientation in the minor groove (rmsd of only 2.1 Å over phosphate backbone atoms and 3.1 Å over all nonhydrogen atoms of the common TGGCCA sequence). This structural similarity is presumably accompanied by increased duplex stabilization through interactions of each bound drug not only with both strands but also with the neighboring drug molecule. Given the amount of nonpolar surface area that is buried upon complex formation (525 Å2), we would expect an important hydrophobic contribution not only to the free energy of trabectedin-DNA association but also to the ensuing stabilization of the double helix in the aqueous medium. For a complex of the type reported here it seems safe to assume that the increase in the temperature of DNA thermal denaturation will be larger than that obtained for a DNA oligonucleotide containing a single trabectedin adduct, which has been shown to be of 19° for a 5'-AGC site (Zewail-Foote and Hurley, 2001a). Therefore, it is not unreasonable to think that the known ability of trabectedin to block the activity of exonuclease III (ExoIII) (Dziegielewska et al., 2004) or the helicase activities of both the simian virus 40 (SV40) large tumor antigen (T-antigen) (Zewail-Foote and Hurley, 2001a) and the UvrA2B complex (Zewail-Foote and Hurley, 2001b) derives from this substantial stabilization, which is likely to be amplified if two or more suitable triplets are tandemly or separately arranged in the oligonucleotide sequence.
To distinguish between drug-induced DNA distortions and sequence-dependent intrinsic propensities, the dynamic behavior of the free d(GTATGGCCATAC)2 oligonucleotide was simulated under identical conditions. Our results strongly suggest that this dodecamer shows two distinct bends produced by rolling at each TpG step (Fig. 4), whereas no bending is apparent at the junction between TGG and CCA. These findings are in very good agreement with results from a two-dimensional NMR spectroscopy study reporting that GGC in the related self-complementary decamer d(CATGGCCATG)2 forms a tight stack with parallel bases, and that high positive roll is present at both TpG steps (Dornberger et al., 1998). Nonetheless, they are at odds with earlier X-ray crystallography observations that stacked B-DNA double helices of general sequence C-C-A-x-x-x-x-T-G-G exhibited the same 23° bend across the -T-G-G C-C-A- nonbonded junction in the crystal lattice (Grzeskowiak et al., 1993) that was encountered in the middle of another decamer helix of sequence C-A-T-G-G-C-C-A-T-G (Goodsell et al., 1993). Because of this, the central DNA stretch studied in the present investigation, d(TGGCCA)2, which can be simultaneously targeted by two molecules of either trabectedin or chromomycin A3, was dubbed a "natural bending element", despite the fact that this curvature originated in crystal packing effects.
As reported earlier for complexes containing trabectedin bonded to a central guanine in AGC, CGG, or TGG triplets (Gago and Hurley, 2002; Marco et al., 2002), the minor groove in the present complex is notably wider than in the free oligonucleotide (Table 5), resulting in a compressed and deeper major groove in the central region where the drug is bound. This major groove compression is favored by the distinct behavior of pyrimidine/purine steps compared with purine/purine steps, which makes the former particularly susceptible to roll bending, as detected in experimental DNA structures (Gorin et al., 1995). The preference of TpG/CpA for positive roll leads to decreased stacking interactions, and this could offer an advantage for the binding of both proteins and drugs that induce or require this structural distortion. In fact, this dinucleotide step is an important component of the CCAAT box, which is the core of the binding site for the evolutionary conserved transcription factor NF-Y (Mantovani, 1998). NF-Y makes important contacts with the minor groove and induces large directed bends, a capacity that is thought to be important for its promoter-organizing activities. It is noteworthy that trabectedin, which can bind to the TGG triplet present in the complementary strand and also bends DNA (Zewail-Foote and Hurley, 1999), is able to abrogate the transcriptional activation of a number of genes that possess a CCAAT box in their regulatory regions, including MDR1,c-Jun, and COL1A1 (Mantovani, 1998). Nevertheless, the effects of trabectedin are not exclusively related to NF-Y binding sites; this drug can also inhibit the trichostatin A-mediated transcriptional activation of the gene encoding p21Cip1 (Friedman et al., 2002). Interestingly, the CCAAT-less p21Cip1 promoter is regulated by major-groove binding of the zinc-finger-containing protein Sp1 to two or more of its G/C-rich motifs ("GC-boxes"), which also provide suitable triplet sites for trabectedin bonding in the minor groove (Gago and Hurley, 2002; Marco et al., 2002). In this regard, the recent finding that mithramycin A (also known as plicamycin), a close analog of chromomycin A3, inhibits the transcriptional activation by Sp1 and p53 of the p21Cip1 and PUMA (p53–up-regulated mediator of apoptosis) gene promoters induced by 5-fluorouracil (Koutsodontis and Kardassis, 2004) can also be indicative of possible similarities in the mechanism of action of these two types of drugs. In addition, we note that transcription factors also bind to auxiliary sequences adjacent to replication origins, where they recruit chromatin remodeling factors that create either nucleosome-free regions or regions of specifically spaced nucleosomes (Melendy and Li, 2001). It is remarkable that a very recent report has revealed strong inhibition, by nanomolar concentrations of trabectedin, of SV40 DNA replication in BSC-1 green monkey kidney cells and HCT116 human colorectal carcinoma cells (Dziegielewska et al., 2004), together with accumulation of unusual DNA structures, which probably suggests the existence of collapsed replication forks. In light of the results shown here and further work in progress (V. Garcia, E. Marco, F. Gago, and A. Domingo, manuscript in preparation), it will be interesting to see to what extent binding of trabectedin can hamper assembly of the prereplication complex and/or the helicase activity of its associated minichromosome maintenance proteins (Lei and Tye, 2001). The extremely low concentrations of trabectedin and analogs that are necessary to cause cell cycle arrest and cell death (Rinehart, 2000) are suggestive of a trans-acting mechanism that probably acts through one or more cellular DNA damage response pathways or checkpoints (Cox et al., 2000). This action would then be reminiscent of that evoked by adozelesin, another minor groove alkylator that, in contrast to trabectedin, binds selectively to AT-rich regions (Liu et al., 2000). In this respect, it is notable that cell sensitivity to trabectedin is somehow dependent on the presence of a proficient transcription-coupled pathway of nucleotide excision repair (Damia et al., 2001; Erba et al., 2001; Takebayashi et al., 2001).
In conclusion, the similarity in DNA structure between the (trabectedin)2-DNA complex studied herein and the recently reported (chromomycin A3)2-DNA crystal complex can be summarized by stating that both of them adopt characteristics associated with A-DNA or DNA-RNA hybrids. The minor groove in TGG sequences provides a well defined arrangement of hydrogen bonding donor and acceptor atoms that can interact with suitable functional groups present in these DNA-binding drugs, but the intrinsic bendability of this sequence can also facilitate an indirect readout. Reaction of trabectedin with guanines embedded in suitable G/C-rich sequences (Friedman et al., 2002) could stabilize or prevent, depending on sequence context, the binding to DNA of transcription factors such as those belonging to the Sp1 family, which can behave as activators or repressors in the regulation of gene transcription. In this regard, it is of interest that inhibition of Sp1 DNA binding in vivo has recently been demonstrated for the mithramycin A (Koutsodontis and Kardassis, 2000), a close analog of chromomycin A3. In addition, the very high Pearson correlation coefficient (r = 0.96) obtained in the COMPARE analysis for chromomycin A3 and trabectedin can be interpreted to indicate that both types of compounds may possess a similar mode of action (Paull et al., 1989). Hence, the structural similarities reported here provide a common foundation that can be of help in the elucidation of the molecular mechanism(s) involved. Among the logical experiments suggested by the present findings would be to compare the effects of these two drugs on gene transcription and to evaluate the possible dependence of chromomycin A3 cytotoxicity on the integrity of the NER system.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: PDB, Protein Data Bank; NSC-648766, trabectedin; NSC-58514, chromomycin A3; MD, molecular dynamics; rmsd, root-mean-square deviation; SA, surface area; SV40, simian virus 40; NF-Y, nuclear transcription factor Y.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Federico Gago, Departamento de Farmacología, Universidad de Alcalá, E-28871 Alcalá de Henares, Madrid, Spain. E-mail: federico.gago{at}uah.es
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