Chapter 1 Predictive Binding Geometry of Ligands to DNA Minor Groove: Isohelicity and Hydrogen-Bonding Pattern Juan C. Stockert Abstract The interaction of drugs and dyes with nucleic acids, particularly when binding to DNA minor groove occurs, has increasing importance in biomedical sciences. This is due to the resulting biological activity and to the possibility of recognizing AT and GC base pairs. In such cases, DNA binding can be predicted if appropriate helical and hydrogen-bonding parameters are deduced from DNA models, and a simplified geometrical rule in the form of a stencil is then applied on computer-drawn molecules of interest. Relevant structure parameter values for minor groove binders are the length (4.6 < L < 5.4 Å) and angle (152 < σ < 156.5°) between three consecutive units, measured at the level of hydrogen donor or acceptor groups. Application of the stencil shows that predictive methods can aid in the design of new compounds, by checking the possible binding of isohelical sequence-specific ligands along the DNA minor groove. Key words Binding mechanisms, DNA ligands, Fluorescent probes, H bonds, Minor groove, Molecular modeling, Predictive binding

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Introduction At present there is an increasing interest in the study of small natural or synthetic DNA ligands (drugs, dyes) and their specific binding mechanisms and biological activities [1–5]. Binding modes and properties of DNA ligands are relevant for molecular biology studies on pharmacological activity of drugs, chromatin structure and function, nuclear labeling, chromosome banding, use of fluorescent probes, etc. [6–10]. Obviously, to analyze DNA-ligand interactions within living organisms, specific quantitative structure-activity relations must be fulfilled for uptake of compounds into living cells [5, 10]. Numerous methods have been used to study the interactions of small molecules with DNA, which correspond to three binding modes (intercalation, groove binding, and nonspecific outside stacking (see ref. [11]). In the case of groove binders, specific drugs, dyes, and proteins can “read” the base sequence by sensing

Juan C. Stockert et al. (eds.), Functional Analysis of DNA and Chromatin, Methods in Molecular Biology, vol. 1094, DOI 10.1007/978-1-62703-706-8_1, © Springer Science+Business Media New York 2014

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Fig. 1 Scheme of the base pairs thymine-adenine (TA) and cytosine-guanine (CG) showing atom numbers, H bonds, electron pairs (black dots), H bond donors (d), and acceptors (a) on minor (m) and major (M) grooves. The C1′ atom of deoxyribose, helix axis (+), and positions of adenine H2 and nearest HL from ligand (circles) are also shown

the pattern of hydrogen (H) bond donors and acceptors on the groove floors. Minor groove DNA binders are cationic compounds containing a curved array of aromatic rings linked by groups or bonds with torsional freedom [6, 12–14]. These ligands can interact with atoms in the minor groove at the level of adenine-thymine (AT) and guanine-cytosine (GC) base pairs (see Fig. 1). As they must be complementary to the DNA curvature along the minor groove (and therefore isohelical) [15–17], molecular modeling studies are very suitable for analysis and prediction of the possible binding of flexible and curved ligands to DNA minor groove [18–22]. It is important to note that these molecules, because of their specific mode of binding, have relevant biological activities (e.g., genotoxic, antiviral, antibacterial, antiprotozoal, trypanocidal, and antitumor activity) [6, 23–31]. Three types of ligands can be expected to interact with the DNA minor groove: (a) symmetric or asymmetric non-H-bonding ligands, (b) symmetric H-bonding ligands, and (c) asymmetric (repetitive) H-bonding ligands. In the first case, bowed drugs

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Fig. 2 (a) Skeletal structure of the DNA segment (dA)12·(dT)12 generated with Dtmm87 showing the H2 helical curve. M and m: major and minor grooves, respectively. (b–d) Simplified views of (a) with the ligand Q-dmPOPOP on the HL curve (b), observed after 90º right rotation (c), and then 45º tilt downwards (d) to show the planar projection and curvatures of the ligand (see refs. 17, 35). (a) DNA axis

and dyes such as SN-18071, NSC-57153, CC-1065, auramines, cyanines, and Q-dmPOPOP seem to present this binding mode [9, 12, 25, 30, 32–38]. In the case of symmetric H-bonding ligands, examples are berenil, pentamidine, propamidine, furamidine, M&B 939, etc. [7, 12, 19, 20, 39–42]. Finally, other ligands show asymmetric units and H-bonding groups, examples being netropsin, distamycin A, and Hoechst dyes such as 33258, DAPI, 2-hydroxystilbamidine, and SN 6999 [6, 13, 14, 30, 43, 44]. In all cases the interacting molecular surfaces have complementary structural features (e.g., shape, H donor and acceptor groups, charge). 1.1 Curvature of DNA and Ligands

Particular geometrical parameters must be taken into account when considering binding to the DNA minor groove. For symmetric or asymmetric ligands, prediction of isohelical binding requires knowledge of both the DNA and ligand curvature [15]. A simple method to assess the curvature of the DNA minor groove, and of feasible binders, was described some time ago [17]. This procedure allows comparison of the degree of geometrical correspondence between curvatures of both ligand and biopolymer, and hence permits prediction of binding possibilities. As seen in isolated AT base pairs (Fig. 1), and in the oligomer (dA)12·(dT)12 (Fig. 2a), the H2 atoms from adenines mark the middle point on the convex and electronegative floor of the minor groove. By connecting consecutive H2 positions the helical curve H2 is obtained. When an additional H atom from a ligand (HL) is incorporated (see Fig. 1), a new helical curve HL, expanded by 2.4 Å, is generated (Fig. 2b, c). The planar projection obtained by tilting both curves 45° (Fig. 2d) corresponds to a circular segment

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Fig. 3 Chemical structures of minor groove AT binders. From left to right, Dst distamycin A, D-288/45, Hoechst 33258, DAPI, and antrycide. The curved thick line at left represents the convex floor of DNA minor groove in AT regions. Arrows indicate electron donors for bifurcated H bonds along the “6-atoms-in-a-row” pattern, which is shown as thick bonds. Asterisks show other possible H bonds. W van der Waals contacts of HL with adenine H2

of 11 Å radius (rDNA). As no H atom of the ligand can penetrate within this radius, the corresponding DNA curvature (CDNA) determines the nearest approximation to the minor groove floor. By connecting the exposed H atoms on the concave side of ligands, their curvature can be also defined (CL), which directly leads to a corresponding ligand radius (rL). As an example, the fitting of CDNA with CL from the ligand Q-dmPOPOP [35] is illustrated in Fig. 2b–d. The comparison between CL and CDNA indicates the geometric correspondence, which is expressed by the curvature index, CI = rL/rDNA [17]. Best fitting occurs when CI is 1; if CI is higher or lower, CL will be too small or too large, respectively, to complement CDNA. On the other hand, symmetric H-bonding ligands are generally non-repetitive, and show two H-bonding groups considerably distanced from each other. Therefore, isohelical and repeating H bonds are lacking, and the correspondence of curvatures would be the relevant parameter. 1.2 Isohelical H-Bonding

The relationship between the bowed conformation of asymmetric H-bonding ligands and the curvature of DNA minor groove in AT segments with regularly spaced H acceptors is schematized in Fig. 3. Interestingly, a “six-atoms-in-a-row” pattern appears as a common structural motif in these binders, with the first and last atoms being H donors. Taken into account the inclination (about 45°)

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that ligands should adopt to fit into the minor groove, the 6-atom motif just permits appropriate placing of H donors with respect to the corresponding H acceptors (O2 and N3 from thymine and adenine, respectively). In addition, adenine H2 atoms make van der Waals contacts with H atoms from the ligand [13, 43] (see Figs. 1 and 3). Terms such as “lexitropsins” [45] and “isolexins” [15] have been proposed to define molecules that satisfy the geometrical parameters required for recognition of AT and GC base pairs. Likewise, “combilexins” show DNA-intercalating and sequence-reading capacity [30]. In addition to monomeric minor groove binding, dimer ligands such as antiparallel face-to-face pyrrole-amide antibiotics (netropsin, distamycin) and certain cyanine dyes can also be accommodated in an expanded AT minor groove [46–49]. In a further step to achieve selective minor groove binding to AT and GC base pairs, modified molecules were designed and tested, resulting in ligands (e.g., hairpin polyamides) that can specifically recognize those sequences [30, 50, 51]. Subjecting hypothetical compounds to such geometrical rules has demonstrated that predictive methods could aid in the design of new minor groove ligands [15]. After appropriate helical parameters were deduced from DNA models, a simplified geometrical rule in the form of a stencil could be applied to predict DNA binding [16]. The stencil is therefore a simple and useful instrument for checking the possible binding of isohelical sequence-reading ligands along the minor groove.

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Materials Modeling programs currently available for teaching and research are appropriate for computer-assisted drawing of chemical structures, as well as for precise viewing and comparison [52]. Software packages Dtmm87, MDL ISIS Draw 2.5, ACD-ChemSketch 10, Avogadro 1.1.0, ChemDraw Ultra 8.0, and HyperChem 8 can be used for molecular modeling (see Note 1).

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Methods

3.1 Modeling of Chemical Structures

1. The chemical structures of compounds are generated either with modeling software or by appropriate drawing. Whenever modeling programs are not available, simple graphical procedures can generate adequate chemical structures, e.g., using plastic templates or direct drawing, taking into account appropriate chemical parameters (see Note 2). Whichever procedure is adopted, great care must be taken to avoid errors in the chemical structures (see Note 3).

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Fig. 4 Possible minor groove binders with three consecutive asymmetric units, showing bond length (L) and angle (σ) between H donors (N, black circles) or H acceptors (O, empty circles). Note the bowed shape and aromatic rings (pyrrole, indole, benzimidazole, benzoxazole) linked by bonds with rotational freedom. Only single bonds and not all hydrogens are drawn. PK pyrrole-ketone, VP vinyl-pyrrole, AP azo-pyrrole, I indole, BI benzimidazole, BIA benzimidazoleamine, BOA benzoxazole-amine, BIK benzimidazole-ketone. In the three last molecules, a new “8-atoms-in-a-row” pattern is present

2. In the case of computer drawing, the commands “energyminimization routine,” “clean structure,” or “energy optimization” should be used to optimize the geometry of molecules. A rapid geometry optimization is achieved by using molecular mechanics force field (MM+ and MM2) (see Note 4). 3. After geometry optimization, the length (L) between H bond donors and the angle (σ) between repeating subunits are directly recorded from the molecule. The use of a rule and of an angle protractor will be required for measuring simple drawings. 4. At least three adjacent units are necessary to measure the angle σ between them. Although the angle τ was early suggested [16], in the present case the angle σ was easier to measure using the direct value (180° − τ = σ). Several types of molecules showing different L and σ parameters are illustrated in Fig. 4. 3.2

Stencil Modeling

1. According to Zasedatelev’s parameters [16], length and angle between units must satisfy a range of values, and therefore, the use of a stencil with minimum and maximum limits is the best strategy for comparing real and allowed values.

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Fig. 5 (a) Stencil representation for checking three consecutive positions of H donors and acceptors, showing the permitted L and σ values for minor groove binding. (b) Corresponding comparison of a given ligand (BOA) with the stencil. A close fitting occurs between stencil limits and ligand H donor atoms. ψ torsion angle between units

2. The stencil is easily modeled or drawn using the following parameters: 4.6 < L < 5.4 Å and 152 < σ < 156.5° (Fig. 5a) (see Note 5). 3. The stencil image can be photocopied on a transparent sheet, which is then applied on the structure of interest, or the stencil is simply merged with the molecule on the computer screen. An example ligand, benzoxazole-amine (BOA), with the stencil placed on its H donor positions, is shown in Fig. 5b ( see Notes 6–8). 3.3

Example Results

1. Some example results illustrate modeling approaches for isohelical binding and selective H-bonding. Oligomer duplexes (dA·dT)8, (dA)8·(dT)8, and (dG)8·(dC)8 were generated with Dtmm87 and HyperChem software, using standard bond lengths and angles. To form the bifurcated H-bonding pattern, the positions of equidistant H donors from ligands for thymine O2 and adenine N3 of (dA·dT) and (dA)·(dT) oligomers were calculated. Taking into account the usual H bond length (2.6– 3.0 Å, see ref. [16]), an average value (2.8 Å) was employed. Likewise, the positions of ligand H donors for cytosine and ligand H acceptors for guanine were also calculated in the oligomer (dG)8·(dC)8, placing H donor and H acceptor sites at a distance of 2.8 Å from cytosine O2 and guanine N2, respectively. Three adjacent donors or acceptors in the different duplexes

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Fig. 6 Correlation between length (L) and angle (σ) values for H-bonding pattern to DNA minor groove. Square and rhombus symbols correspond to an ideal DNA duplex (L = 5 Å, σ = 154°) and their limit values (4.6 < L < 5.4 Å and 152 < σ < 156.5°), according to Zasedatelev [16]. Note that here the σ angle corresponds to the τ angle of this author (σ = 180° − τ). Circles represent L and σ values for the indicated duplexes

have characteristic values for bond length (L) and angle (σ), which limit the possibility for minor groove binding (Fig. 6). 2. In the case of the oligomer (dA·dT), consecutive 2.8 Å-expanded H donor positions from O2 and N3 atoms allow the representation of the Hd curve in a base pair model (Fig. 7a, b). To illustrate the adequate fitting of a given molecule, the ligand BOA was modeled and matched with the Hd curve (see Fig. 7c), both Hd and ligand appearing in a planar projection. 3. The interaction between the oligomer (dG·dC)8 and the alternating BOA-BIK ligand is represented in Figs. 7d and 8 (see Note 8). The antiparallel face-to-face BOA-BIK dimer fulfills the requirements of both isohelicity and H acceptor/donor pattern in GC sequences of DNA minor groove very well. Obviously, as occurs with AT-binding dimers [46, 47], the minor groove width at GC sequences becomes wider after binding of the BOA-BIK dimer (from 8.3 to 9.5 Å, as measured between C4′ atoms of d-ribose across the minor groove).

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Notes 1. This list is not prescriptive, and previous or later versions of the indicated chemical drawing software may be used, as well as alternative software packages.

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Fig. 7 Front (a) and side view (b, after 90º rotation), of a space-filling base pair model of (dA·dT)8. The helical Hd curve corresponds to consecutive H donor sites for ligand binding. C: cyan, N: blue, O: red, H: white, C1′: pink. a: DNA axis. (c) The structure shown in (b) after 45° tilt downwards, showing the planar projection and Hd fitting of BOA. (d) Stereopair of the sequence (dG·dC)8 with an antiparallel face-to-face (BOA-BIK)2 dimer modeled with Dtmm87 software. Observe the close fitting of the dimer into the expanded minor groove. C atoms in BOA and BIK are green and violet, respectively. C: white, N: blue, O: red, P: yellow, H atoms involved in H bonds: cyan. Vertical line: DNA axis. 3D viewing can be achieved by relaxing eyes, focusing at infinity, and image fusion (see ref. 52)

2. Although only approximate, the following parameters can be used for simplified handmade drawing of chemical structures on paper: bond length equivalence (1 Å = 1 cm [1.4 Å = 14 mm for aromatic C–C bonds]) and 120° and 108° for bond angles in hexagonal and pentagonal rings, respectively. 3. In particular, inadequate chemical drawing and modeling procedures, wrong bond lengths or angles, omission of hydrogen atoms, etc. can result in implausible molecular structures and misleading interactions (see ref. 53).

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Fig. 8 Schematic view of interactions of the BOA-BIK dimeric motif showing the H-bonding pattern and recognition possibilities for alternating GC base pairs in the DNA minor groove

4. When more precise and better optimized molecular structures are desired, the semiempirical energy-minimization PM3 method can be applied (e.g., using ChemDraw or HyperChem software). 5. Note that L values from ligand and stencil must use the same linear scale (e.g., Å, or mm). 6. In practice, this procedure is greatly simplified by using planar (2D) chemical structures. However, when 3D molecular structures are used, a variable torsion angle (ψ, 20° on average) between repetitive units should be introduced, to fit them into the helical ramp of the minor groove [16]. However, the value of ψ does not significantly influence L and σ values. 7. Some flexibility in the conformation of molecules (e.g., bond and torsion angles) is also permitted, allowing a more precise final fitting of ligands into the DNA minor groove [14]. Likewise, the DNA duplex is capable of small conformational changes (bending, unwinding) following ligand binding. 8. The compounds BOA and BIK correspond to a new structural motif, as suggested by Sazedatelev [16].

Acknowledgements I thank A. Blázquez-Castro, J. Espada, and R.W. Horobin for valuable collaboration. This work was supported by a grant (CTQ201020870-C03-03) from the Ministerio de Ciencia e Innovación, Spain.

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