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. 2023 Apr 18;3(4):e729. doi: 10.1002/cpz1.729

Binding to the DNA Minor Groove by Heterocyclic Dications: from AT Specific to GC Recognition Compounds

Ananya Paul 1, Rupesh Nanjunda 1,2, W David Wilson 1,✉
PMCID: PMC13528270  PMID: 37071034

Abstract

Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, have found extensive uses in biotechnology, and are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds show that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, compounds can be designed to recognize GC and mixed AT/GC base pair sequences, and stacked dimers can form to recognize specific sequences. © 2023 Wiley Periodicals LLC.

Keywords: AT minor groove binders, DNA complex structures, DNA minor groove, GC base pair specific, heterocyclic amidines, mixed base pair sequence, molecular curvature, molecular dynamic simulations, NMR, stacked cooperative dimer, surface plasmon resonance, transcription factor, X‐ray crystal

Small molecules that bind specifically to the DNA minor groove (MG) have been both influential and experimentally useful in several areas. Many of these compounds, such as DB75, DAPI, cyanines, and Hoechst 33258 in Figure 1A, have intense fluorescence when bound to DNA, and their fluorescence quantum yields can vary significantly depending on which sequence of the minor groove they bind (Hannah et al., 2005; Owens et al., 2019; Karlsson et al., 2003; Stewart et al., 2005; Wheeler et al., 2012; Wilson et al., 1990). This has made some minor groove binders useful cellular DNA stains, as well as sensitive DNA probes for polyacrylamide gel electrophoresis. Sequence‐specific, minor‐groove‐binding compounds have also been developed with affinities rivaling transcription factors, and the compounds have provided a wealth of information on the fundamental principles of DNA recognition (Depauw et al., 2019; Dervan et al., 2005; Lacy et al., 2002; Munde et al., 2014; Tidwell & Boykin, 2004; Wilson et al., 2005, Nguyen et al., 2007). Their effects on transcription factors or other cellular DNA‐targeted proteins have led to the development of MG binders as potential therapeutic agents, and animal studies of some compounds are underway (Cai et al., 2009; Peixoto et al., 2008; Shinohara et al., 2010; Antony‐Debré et al., 2017).

Figure 1.

Figure 1

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Compound structures for the heterocyclic compounds discussed in this paper, as well as some well‐known reference compounds: DAPI, netropsin, Hoechst 33258, berenil, and pentamidine. Synthetic heterocyclic diamidines that are discussed in this protocol are also listed.

Minor groove binding compounds are also used in humans and animals as effective antimicrobial drugs (Cai et al., 2009; Paine et al., 2010; Raskatov et al., 2012; Soeiro et al., 2008; Tidwell & Boykin, 2004; Werbovetz, 2006; Wilson et al., 2005; Wilson et al., 2008). The groove‐binding mode is preferred over intercalative binding for therapeutics due to increased sequence specificity and generally lower toxicity of groove binders. An additional advantage of MG‐binding agents for therapy is that they are not generally mutagenic. A therapeutically successful class of MG binders is based on arylamidines such as DB75, pentamidine, and berenil (Fig. 1A) that have biological activity against parasitic microorganisms (Mukherjee et al., 2006; Paine et al., 2010; Tidwell & Boykin, 2004; Wilson et al., 2005). Pentamidine has been used clinically for over 60 years in humans against a number of microorganisms. It is particularly effective against human African trypanosomiasis (HAT) (Paine et al., 2010; Werbovetz, 2006). Although pentamidine has many successful uses, it has low oral bioavailability and has some limiting side effects. DB75, furamidine (Fig. 1A), was identified as a highly active anti‐trypanosomal drug in animal studies. In a major breakthrough in HAT, an orally available prodrug of DB75, DB289, pafuramidine (Fig. 1A) was synthesized by Boykin and coworkers (Bajic et al., 1996; Boykin et al., 1996; Kumar et al., 1999) and found to be orally active in animals (Mdachi et al., 2009). The prodrug became the first HAT drug to enter a clinical trial pathway that had to comply with current requirements in drug testing. The clinical trials in Africa were highly successful with over 300 patients cured of HAT as the drug reached Phase III clinical trials (Paine et al., 2010). Unfortunately, in a separate and extended Phase I trial, renal toxicity emerged in a small percentage of the treated group following the last treatment with DB289. Although this has halted the HAT trials of the drug, many more active and specific compounds have been discovered, including new compounds that show promising activity against stage II HAT (central nervous system). Because of these successful therapeutic applications of aryl diamidines and the wealth of information they have provided on DNA molecular recognition, binding‐induced conformational changes in DNA, and the energetics of binding and hydration, this protocol will focus on aryl diamidine minor groove binders.

DNA GROOVE SHAPE AND CHEMISTRY

Molecules searching for a groove binding site on DNA find quite different, sequence‐dependent, groove widths and functional groups in the minor and major grooves of DNA (Hamilton & Arya, 2012; Nguyen et al., 2009; Nguyen et al., 2007; Xi et al., 2011). A B‐form DNA structure (Fig. 2A) and both an AT and a GC base pair (Fig. 2B) are shown to illustrate this point. In the minor groove, the purine N3 of A and G, as well as, the C2‐C=O groups of C and T serve as H‐bond acceptors. On the GC base pair, however, G has a C2‐NH2 group, which is not present on A, which H‐bonds with C and forms a steric block to binding of most agents deep into the groove. The groups in the major groove are positioned in a different array. The purine N7 is an H‐bond acceptor while both GC and AT base pairs have C=O ‐ NH2 H‐bonds, but the groups face in opposite directions on the base pairs (Fig. 2B). The C5‐CH3 group on T has both steric and hydrophobic effects on binding of agents in the major groove, especially when several Ts are positioned closely in the sequence. Space‐filling, electron‐density maps at the top of Figure 2B clearly show the strong H‐bond acceptors (in red—negative potential) and donors (blue—positive potential). The schematic illustrations at the bottom of Figure 2B define the atomic positions in the maps. These groups are arrayed along the floor of the minor groove in a sequence‐dependent manner in a DNA double helix (Fig. 3).

Figure 2.

Figure 2

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(A) A B‐form DNA model with the chains colored blue and pink is tilted so that the width of both the minor and major grooves can be seen and compared. (B) Space‐filling models of AT and GC base pairs (top) are shown with electrostatic potential coloring: from blue for positive to red for negative partial charges. The same base pairs are shown as line models (bottom) with the base pairs labeled and lone pair electrons shown on H‐bond accepting groups.

Figure 3.

Figure 3

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The minor groove of a B‐form DNA model with the central sequence 51‐AATT‐31 shown with the DNA as a gray stick model. The connecting distances between the 51‐As and 31‐Ts on opposite chains are shown.

In A‐tract sequences (consecutive As on one strand and Ts on the other), or in some shorter sequences such as the AATT in Figures 2A and 3, the minor groove generally assumes a narrow width that provides for tight binding of the heterocyclic‐amidine system of the dications in Figure 1A, while the groove in GC or mixed base pair sequences is wider (Neidle, 2001; Nguyen et al., 2009). In the narrow AT base pair sequences, single heterocyclic compounds can fit tightly and deeply into the groove such that groups on the inner face of the compounds can interact, typically through H‐bonding and van der Waals interactions, with complementary groups on the edges of the AT base pairs (Neidle, 2001; Wilson, 1996; Wilson et al., 1990; Nguyen et al., 2007). The position of the H‐bond acceptors change with the AT sequence, groove shape, and curvature so that bound agents can distinguish even sequences composed of only AT base pairs.

This is illustrated in the classical B‐form structure in Figure 3, for example, wherein the terminal base pairs of the central AATT sequence, the N3s of A are separated by over 12 Å, while the keto groups of T are separated by less than 10 Å (Nguyen et al., 2009). Such differences dictate the sequences to which compounds with specific H‐bond donating groups can bind with high affinity. It should be remembered, however, that there is a certain amount of flexibility in the DNA double helix, as well as the unfused heterocycles that are characteristic of most minor groove binders. Induced fit interactions in the minor groove are, thus, quite common (Moretti et al., 2008; Chaires, 2008; Bostock‐Smith et al., 2001; Miao et al., 2005; Tanious et al., 2007).

In addition to the position of functional groups at the floor of the groove and the width, the minor groove has a helical curvature that can also vary slightly with sequence. This helical curvature is easily seen in the DNA duplex models at the top in Figures 2 and 3. Agents that bind to the minor groove must, thus, have a concave shape that complements that of the groove. Molecules that bind tightly and specifically to the groove (Fig. 1) will fit well within the walls of the groove (groove width), form optimum H‐bonds with base edges at the floor of the groove (functional groups), and match the local curvature of the groove (compound shape). Again, induced fit changes in both DNA and compounds with unfused heterocyclic systems give some flexibility to all of these recognition features.

As noted above, molecular curvature is an important feature for minor groove recognition and couples with compound molecular stacking surface, H‐bonding to minor groove bases, and charged groups to determine the energetics of binding. To establish a systematic method to evaluate the molecular curvature of minor groove binders, a graphical approach for the determination of comparative molecule curvature values was developed. To use this method, compounds are energy minimized with a software package such as SPARTAN. Optimization calculations are then performed on the compounds at the DFT/B3LYP theory level. By using PowerPoint for compound visualization, the next step is to define a reference circle that passes through both amidine carbons. The reference circle with a radius to allow it to pass as closely as possible through the center of each molecular unit of the entire molecule and the two amidine carbons is then selected. This is illustrated with the DB75 and DB818 compounds in Figure 4. Two lines are next connected from the amidine carbons to the circle point at the center of the molecule. The angle between two lines defines a relative curvature value for comparison of each molecule (Fig. 4). Analysis of minor groove binding compounds of different structures by this method indicates a value of 140 to 145° is optimal for compounds binding strongly in the DNA minor groove. As can be seen, the curvature of DB818 is in this range while DB75 is too curved to make optimal contacts with the groove, and it binds approximately 10‐times weaker than the thiophene. The two amidines of DB75 contact the groove surface, but the center of DB75 loses direct contact with the minor groove. Due to this lower flexibility and curved structure, the furan and adjacent atoms of DB75 are pushed away from the floor of the groove. This relative comparison thus provides a useful numerical value to use to evaluate new compounds for possible synthesis. It is a useful factor in evaluating components in binding energies (Guo et al., 2021).

Figure 4.

Figure 4

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Molecular curvature for selected minor groove binding compounds.

The difference in groove width in A‐tracts and GC, or mixed sequences, has been noted above. A number of groups have pointed out, however, that there are more minor, but quite significant, variations in local groove width with sequence (Parker & Tullius, 2011; Zhou et al.,. 2013; Bishop et al., 2011). By using sequence‐dependent variations in hydroxyl radical cleavage of DNA, for example, Tullius and coworkers have established a large database of variations in the minor groove with local sequence (Parker & Tullius, 2011; Bishop et al., 2011). The PDB set of experimental DNA structures shows significant variations in minor groove width. The Rohs group has used these results to catalog the sequence‐dependent variation in DNA parameters, including minor groove width. They incorporated the results into web software (http://rohslab.cmb.usc.edu/DNAshape/) that can make reasonable predictions of minor groove width for any input sequence.

Honig, Rohs and coworkers have correlated the variations in groove width with electrostatic potential and the favorable binding affinity of different amino acid side chains, particularly arginine, into the minor groove (Zhou et al., 2009; Bishop et al., 2011). It should be emphasized, however, that the wider groove and different functional groups in GC‐containing sequences of DNA can also be effective binding sites for minor groove agents, particularly if they have H‐bond accepting groups to interact with G‐NH2 functional groups. This feature is discussed in more detail below.

CLASSICAL‐AT‐SPECIFIC MINOR GROOVE BINDERS

The dicationic polyamide, netropsin, in Figure 1A is the paradigm for classical AT‐ specific MG binders. It was isolated in the early 1950s (Finlay et al., 1951) and found to have some activity against microorganisms and cancer cells. Zimmer and coworkers conducted an extensive series of studies on netropsin in the early 1970s and found its AT specificity, as well as, very different effects on DNA as compared to intercalators (Zimmer, 1975). Dickerson and coworkers determined the crystal structure of netropsin bound in the AATT minor groove of the oligomer duplex sequence, d(CGCGAATTCGCG)2 (Kopka et al., 1985) and these results firmly established the minor groove binding mode. The netropsin structure was the first detailed molecular‐level view of a DNA minor groove complex and the same DNA sequence has been used in many other studies of MG binders, including the heterocyclic amidines (Neidle 2001; Ogbonna et al., 2022). The netropsin results established much of the detailed molecular basis of the classical AT‐specific minor groove binding model: the need for a narrow groove, the steric hindrance that would be caused by the G‐NH2 group, the molecular curvature and twist of the bound molecule to match the DNA groove and the advantage of H‐bond donating and positively charged groups. Many studies with other minor groove binders have confirmed the netropsin model.

Starting in the 1990s, our laboratory, along with Boykin and coworkers (Tanious et al., 1994), initiated an extensive series of studies on the minor groove complexes of aromatic diamidines. These studies clearly showed that there are a number of important features of the diamidines that lead to their strong MG binding to DNA, as well as their biological activity (Nguyen et al., 2007, 2009; Tidwell & Boykin, 2004; Wilson et al., 2005; Wilson et al., 2008). The early studies by the Neidle group yielded the DNA complex structures of a large number of diamidines (Neidle, 2001). Important molecular features in DNA MG binders, which have been derived from the initial studies of numerous DNA‐targeted aromatic amidines, include a crescent shape that complements the helical minor groove, positively charged ends to enhance electrostatic interactions and solubility, H bond donor/receptors for sequence recognition, and a somewhat‐flexible structure to allow thermodynamic optimization of the compound‐DNA interactions. These features fit the classical model, but as will be shown below, some of these requirements, especially the requirement for curvature matching and an AT sequence, can be relaxed in certain special cases. As noted above, the narrow width of the AT minor groove and lack of the third H‐bond of GC base pairs is optimum for many MG binders such as those shown in Figure 1A. In AT base pair sequences, aromatic amidines can fit into the groove such that groups on the inner face of the compounds can interact, through H‐bonding and van der Waals interactions, with complementary groups on the edges of the AT base pairs. The torsional flexibility of the aromatic groups of the compounds lets them make optimum surface contacts with the DNA groups that form the walls of the minor groove receptor site. All of these features of the dicationic compounds increase their affinity and specificity for interaction with sequences of four or more AT base pairs (Neidle, 2001; Nguyen et al., 2007; Wilson et al., 2008).

Neidle and coworkers used the sequence d(CGCGAATTCGCG)2, as with the netropsin structure described above, to determine the structure of the furan‐diphenyl‐diamidine, DB75 (Fig. 1A; Laughton et al., 1996), and a number of analogs (Neidle, 2001). A derivative structure with a cyclopropyl substituent on each amidine of DB75 is shown as an example in Figure 5 (Laughton et al., 1996). As with DB75 (Laughton et al., 1996; Nguyen et al., 2009), the amidines form H‐bonds with the terminal T‐C2 carbonyl groups on opposite chains of the AATT sequence (Fig. 5). The compound fits snugly between the walls of the minor groove while the cyclopropyl groups fit closer to the top (outside) of the groove and partially overlap with the GC base pairs at the ends of the AATT sequence. The compound excludes water from the groove, particularly from the AATT region, which, as described above, is strongly hydrated in the free DNA structure. It also binds to this sequence slightly better than DB75, which is undoubtedly partially due to the removal of the hydrophobic cyclopropyl groups from the water environment.

Figure 5.

Figure 5

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(A) A space‐filling crystal model of a cyclopropyl substituted diamidine derivative (DB193 in Fig. 1) bound to an AATT sequence is shown with the DNA strands colored blue and red, and the compound shown as a tube model. (B) The complex is shown tilted so that the compound (space‐filling model) can be seen bound in the DNA (tube model) minor groove.

Other alkyl‐amidine derivatives of DB75 (Laughton et al., 1996; Simpson et al., 2000) from ethyl to cyclopropyl (Fig. 5), are essentially isomorphous. They assume the twist of the minor groove and have a curvature to closely match the groove. They fit between the walls of the narrow AATT groove and both unmodified amidine –NH groups form H‐bonds with the edges of the AT base pairs at the floor of the groove, except for the cyclopropyl, which forms a single H‐bond. All of the alkyl groups fit into the minor groove near the top edge as in Figure 5.

A particularly informative study and structure was with the phenyl‐thiophene‐benzimidazole, DB818 (Fig. 1A), analog of DB75 (Mallena et al., 2004; Ogbonna et al., 2022). The phenyl‐furan‐benzimidazole compound, DB293, is similar to DB75, with conversion of one phenyl to benzimidazole, and has a similar binding constant for AATT to DB75 (Wang et al., 2002). DB818 has only a one atom difference from DB293, thiophene to furan, and it was thus a surprise to find that it binds to AATT over 25‐times more strongly than DB293. The increase in binding is entirely due to the binding enthalpy, which is over 4 kcal/Mol more favorable for DB818, while it has a less‐favorable entropy of binding (Mallena et al., 2004).

The crystal structure of DB818, and molecular modeling of DB293 for comparison, provides an explanation for these observations. The larger size of sulfur and the resulting bond angle change from thiophene to phenyl, DB818, relative to furan‐phenyl, DB293, results in an increased radius of curvature for DB818 (Fig. 6). This slightly flattens DB818 relative to DB293 (and other furans), which allows it to fit more optimally against the base pair edges at the floor of the groove. This allows the formation of a better benzimidazole H‐bond in DB818 than in DB293, as well as more‐favorable van der Waals contacts with the floor and walls of the groove (Mallena et al., 2004; Ogbonna et al., 2022). DB818, for a molecule of its size, can be viewed as an optimum classical AT specific‐binding agent and serves as an excellent example for this class of minor groove compounds (Nguyen et al., 2009). The tight and favorable interactions of DB818 account for both its more‐favorable binding enthalpy and less‐favorable entropy. The smaller radius of curvature of DB293 (Fig. 6), due to the furan bond angle, pushes the benzimidazole slightly away from the floor of the minor groove and weakens the benzimidazole‐base H‐bond. The indole analog of DB818, DB1879, was also determined to bind to the d(CGCGAATTCGCG)2 sequence. The indole binds with the DNA bases with similar interactions in the minor groove to the benzimidazole. It does, however, bind slightly weaker than the benzimidazole (K D for DB818, 1.5 nM and 6.5 nM for DB1879. With the free compounds, the three linked aromatic systems in both are essentially planar while the two‐terminal amidines are twisted 30‐40o out of the aromatic plane. The electrostatic potential map shows partial positive charges located on the amidines and the ‐NH groups of the two compounds. With the indole, the remaining charge spreads fairly evenly over the compound parts and is close to zero. With the benzimidazole, there is a partial negative charge on the =N at the outer edge of the compound and a more positive charge at the inner edge of the compound and ‐NH group. Such a distribution is favorable for binding the DNA minor groove and it is thus not surprising that DB818 binds more strongly than DB1879.

Figure 6.

Figure 6

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An overlay tube model of optimized structures for DB293 (gray) and DB818 (brown) is shown with the furan O in red and the thiophene S in yellow. The slight difference in bond angles in the furan and thiophene, due to the size difference of O and S, creates a significant curvature difference between the compounds that gives DB818 a better binding constant for the DNA minor groove. The two arcs clearly show the differences in the radius of curvature for the compounds.

VARIATIONS ON THE CLASSICAL MINOR GROOVE MODEL

Minor Groove Binding and Intercalation of Small Compounds

As described above, the classical model of AT‐specific minor groove complexes, starting with netropsin and continuing with the diamidines, successfully provided criteria for strong MG interactions. This model works quite well for the diamidine DB75 (Laughton et al., 1996; Trent et al., 1996), but we noticed that the compound could also assume a planar shape of the diphenylfuran core that was similar in size to intercalators. We asked the question of what would happen to the binding mode if some or all of the AT base pairs in the binding site were replaced by GC. The answer was quite clear, based on a broad array of biophysical methods, DB75 switches from a groove‐binder to an intercalator in GC‐containing sequences (Wilson et al., 1990; Tanious et al., 1994). The compound in 0.1 M NaCl buffer has a KD of ∼100 nM in AT binding sites (minor groove), and this increases by over a factor of 100 in GC sequences (intercalation). The cost of opening an intercalation site and loss of favorable MG interactions clearly cause this significant reduction in affinity (Wilson et al., 1990; Tanious et al., 1994). Other similar small and relatively planar AT minor groove binders are also able to intercalate into GC sequences. Compounds that are nonplanar, or that are too large to fit well between base pairs, do not form stable intercalation complexes.

Strong Binding of Compounds with Non‐Standard Shapes to the MG

The finding of intercalation of some minor groove binding agents stimulated our interest in another question: how far can we go in the design of nonclassical minor groove binding agents that do not perfectly match the shape of the minor groove? The results described above for DB818 emphasize the importance of shape matching. Understanding the limits of minor groove complex formation by compounds that do not fit the classical model is important for extending our knowledge of the broad aspects of DNA molecular recognition. New types of compounds that target DNA in different ways can also have many fundamental and applied uses. Two isomeric biphenyl‐benzimidazole diamidine derivatives, DB911 and DB921 in Figure 1A, have complementary and very interesting MG binding results. The central meta‐substituted phenyl of DB911 has a classical‐type curvature for MG binding, similar to that of DB75 and related minor groove binding agents. The central para‐substituted phenyl of DB921, however, has a much more linear shape that clearly cannot match the curvature of the DNA minor groove. The biphenyl component of both compounds is expected to be significantly twisted. Based on the dogma established through classical groove binders, DB911 should bind in a similar manner to DB75 but DB921 should bind very weakly to the MG.

Biosensor‐SPR experiments with these compounds, and an AATT binding site, provided very surprising results for the binding kinetics and affinities (Fig. 7). DB921 binds much more strongly than DB911 (K A of approximately 2 × 108 M−1 for DB921 and 2 × 107 M−1 for DB911) and has much slower dissociation kinetics (k d of 0.014 sec−1 for DB921 and 0.24 sec−1 for DB911). As expected, DB911 binds very similarly to DB75 but contrary to expectations, DB921 binds more, not less, strongly than these classical compounds. Isothermal titration calorimetry (ITC) of the two compounds showed that the stronger binding of DB921 was due to a more favorable binding enthalpy (Miao et al., 2005; Liu et al., 2011). The ΔH for binding of DB921 to the AATT site is −4.5 kcal/Mol at 25°C, while the value for DB911 is only −2.5 kcal/Mol. The –T∆S contributions to the binding free energy are more similar, −7.0 kcal/mol for DB921 and −7.5 kcal/Mol for DB911. The overall binding, for both compounds, is entropy driven with the AATT site but the favorable binding enthalpy provides a much higher affinity for DB921.

Figure 7.

Figure 7

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In a Biacore T200 biosensor surface plasmon resonance (SPR) instrument, the DNA hairpin shown in the figure was immobilized on a Biacore streptavidin‐coated sensor chip by biotin capture. After cleaning the instrument and chip with standard protocols (Nanjunda et al., 2012), buffer flow was started until a stable baseline was obtained. To monitor the compound‐DNA association reaction, DB921 (A) and DB911 (B) was then injected over the DNA surface (the bracket region in both panels) at concentrations from 1 to 100 nM and binding was monitored by the change in SPR signal (RU) observed in real time. After sample injection, buffer flow was again started and the dissociation reaction was observed. Fitting these curves with a global 1:1 kinetic fit model (black lines through the curves in both panels) provides the association and dissociation rate constants and the equilibrium constants for binding of both compounds (Nanjunda et al., 2012). The binding in the figure is only shown to 10 nM to illustrate the much stronger binding of DB921. The flow rate in the experiment was 100 μL/min at 25°C and 0.01 M MES buffer at pH 6.5 with 0.2 M NaCl.

Insight into the reasons for these binding and thermodynamic differences was proved by a crystal structure from the Neidle group (Miao et al., 2005; Nguyen et al., 2009) and by molecular dynamics calculations (Athri & Wilson, 2009). As expected, DB911 binds to the AATT site in agreement with the dogma from the classical model (see Fig. 5), but DB921 is clearly too linear to bind in this manner. In the crystal structure, the benzimidazole‐amidine end of DB921 is anchored strongly to the floor of the minor groove by amidine and benzimidazole H‐bonds to the base edges at the floor of the groove (Fig. 8). The central phenyl makes van der Waals contacts with the base edges and walls of the groove. The benzimidazole end of DB921 thus binds in a relatively classical manner. The terminal phenyl and especially the amidine attached to it, however, rise off of the floor of the groove due to the linear structure of DB921, as can be seen in Figure 8B. The molecule is “rescued” as a strong MG complex, however, by an interfacial water molecule that bridges the distance between the floor of the groove and the amidine. An amidine–NH H‐bonds to the water O, while an –OH group on the same water forms an H‐bond to an AN3 at the floor of the groove. This water is also H‐bonded to a network of other waters that are in the same local area of the minor groove (Athri & Wilson, 2009; Wilson et al., 2005; Liu et al., 2011). The ternary complex thus forms an optimized set of compound interactions with DNA both directly and through the water, which forms a somewhat flexible linker that allows optimization of the interactions. The DB921‐DNA and water interactions that stabilize the complex so strongly are illustrated in Figure 8. A combination of H‐bonds, van der Waals, and favorable electrostatic interactions with DB921, water and DNA, as well as release of water from the compound and tightly bound water from the minor groove, contribute to the affinity through the binding entropy.

Figure 8.

Figure 8

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The figure is from a crystal structure by Neidle and coworkers (2B0K.pdb). (A) The DNA duplex is shown in a space‐filling model with one strand in blue and one in red. DB921 is shown as large tubes in the AATT minor groove sequence. The phenyl‐amidine that rises off the floor of the groove points towards the reader in this view. The interfacial water that connects this end of the DB921 complex to the floor of the groove can be seen near the floor of the groove with important DNA contact atoms in space filling representation, T‐C=O in red and AN3 in blue. H‐bonds are shown as dashed lines. (B) The contacts that help with the strong affinity of DB921 are highlighted. Starting at the phenyl‐amidine end (bottom of the diagram) there is the interfacial water‐AN3 H‐ bond (this is the first A of AATT) and the water is closely H‐bonded to other water molecules in the minor groove. The phenyl proton that is meta to the amidine points into the groove and makes a close contact with the interfacial water. The central phenyl has a close ‐CH AN3 contact that certainly provides a stabilizing interaction. The benzimidazole ‐NH points into the center of the groove and forms a bifurcated H‐bond with the two middle T C2=O of AATT. The benzimidazole amidine ‐NH that points into the groove forms an H‐bond with the last T C2=O of AATT. These contacts, as well as the van der Waals contacts with the floor and walls of the groove and the amidine positive charges, result in a very high binding for DB921 to the AATT sequence. These features could not be seen without the structural model.

To evaluate how the bound water in the DB921 complex contributes to affinity, DB1055 (Fig. 1A), which has the terminal amidine group of DB921 moved from the para to the meta position, was synthesized. As expected, crystallographic results show that this structural change removes the need for the interfacial water in the complex (Liu et al., 2011). DB1055 binds slightly more weakly than DB921 (loss of 0.5 kcal/Mol in ΔG) with a significant decrease in the binding entropy relative to DB921, but with a more favorable ∆H of binding. The terminal phenylamidine of DB1055 forms a more rigid H‐bonded system with the floor of the minor groove with some‐what better stacking with the groove walls than the water‐linked complex of DB921. This leads to an improved binding enthalpy for DB1055 but a lower entropy with a slight loss in the Gibbs energy. Changes such as that from DB921 to DB1055 have frequently been considered as a way to improve binding energetics through the replacement of linking water with a fixed compound group and a subsequent improvement of binding entropy. The results with DB1055 do show that it is able to replace the interfacial water of DB921, but with slightly less favorable energetics. This result illustrates that a terminal dynamic and flexible linking water molecule can be energetically favorable in a complex. Clearly, incorporation of water, at least near the end of DNA minor groove complexes, can be favorable and is a feature that deserves more attention.

EXTENSION TO GC BASE PAIR RECOGNITION

The Move of Minor Groove Binding from All AT to GC Recognition

The remarkable success of the human genome project not only opened a broad horizon of possibilities to further enhance our fundamental understanding of DNA and its associated biochemical processes but also to explore novel strategies to effectively use DNA as a practical therapeutic target. The decoding of the human genome at the nucleotide level also brought attention to the presence of the GC‐content (almost 40%) interspersed within the AT‐rich genome. GC‐rich sequences are dispersed within many biologically important stretches of DNA and play pivotal, as well as accessory roles in various biological pathways (Gardiner‐Garden & Frommer, 1987; Gruss et al., 1991; Khuu et al., 2007; Kim & Tinoco, 2000; Zerial et al., 1986; Zhang et al., 2004).

When GC‐base pairs are inserted into an AT‐rich DNA sequence, the physical and chemical characteristics of the sequence are significantly altered. For example, GC‐containing sequences have higher thermal stability because of their increased base‐stacking potential, and partly due to the additional H‐bond within the GC base pair. As described above, GC‐containing sequences also significantly alter the groove dimensions (both major and minor) of DNA. In contrast to the narrow and deep minor groove characteristics of AT‐containing sequences, the grooves of GC‐ sequences are wider, with decreased electronegative potential, and the third base pair in the minor groove affects a number of properties. The fact that the G‐NH2 group and GC base pair constitute a negative recognition element for binding of many small molecules in the minor groove of DNA has now been demonstrated using modified DNA bases in which the amino group has been either deleted from guanines and/or added to adenines (Bailly & Waring, 1995; Waring & Bailly, 1994). Given the G‐amino group in the minor groove and its hydrogen bonding properties, the introduction of a H‐bond acceptor heteroatom in the pyrrole rings of netropsin should allow the drug to bind to GC‐containing sequences (Edwards et al., 2011; Dervan et al., 2005; Goodsell & Dickerson, 1986; Lee et al., 1988, 1989; Kawamoto, et al., 2018; Shinohara et al., 2010). The wider minor groove of GC sequences also favors the formation of stacked heterocyclic complexes within the groove to target both strands of DNA. These concepts have been extensively exploited and developed into a series of polyamides that have increased selectivity for GC‐containing sequences (Walker et al., 1997; Kielkopf et al., 1998; Satam et al., 2015; Reddy et al., 1999). Despite the elegant design strategy for GC‐specific sequence recognition using polyamides, the biological activity of this class of minor groove binders has not yet led to clinically useful drugs (Nozeret et al., 2018; Hargrove et al., 2012; Nishijima et al.; 2010).

Small molecules that have the appropriate features to recognize AT stretches, as well as GC motifs, provide understanding to develop selective ligands with increased therapeutic potential. In an effort to expand the repertoire of heterocyclic diamidines for duplex DNA recognition beyond the AT sequences, several new modules were rationally designed and developed with acceptor groups for the G‐NH2 group. Systematic modifications were performed on the heterocyclic ring systems of AT binding compounds to give modules that were designed to specifically recognize the potential H‐bond donors and acceptors of GC base pairs. The synthetic accessibility and the excellent cell uptake of this class of minor groove binders provide alternate ways to better understand and establish new DNA recognition principles and also to develop the next generation of ligands with better therapeutic potential.

Heterocyclic Amidines Can be Modified to Recognize GC Base Pairs

The initial breakthrough in recognizing a GC‐containing sequence with a heterocyclic diamidine was provided by substitution of one of the phenyl moieties of DB75 with a benzimidazole, DB293 (Fig. 1A). DB293 has a strong footprint with a mixed DNA sequence containing a single central GC step, 51‐ATGA‐31 (Bailly et al., 2001; Wang et al., 2000; Wang et al., 2001). The benzimidazole ring of DB293 decreases the curvature when compared to DB75, while increasing the stacking surface. The benzimidazole unprotonated nitrogen, or the furan‐oxygen atom, can also participate in H‐bonding with the G‐NH2 group. Thermal melting studies of DB293 with an oligomer containing the ‐ATGA‐ motif resulted in an increase in the melting temperature (T m ) of the DNA by almost 30°C. Unlike most heterocyclic diamidines, the T m values do not saturate until a compound to DNA ratio of 2:1 is reached. The intermediate ratio of 1:1 exhibits an unusual biphasic transition in the melting curve with the two transition values coinciding with the T m values of free DNA and the 2:1 complex. The T m results thus indicate that the recognition of GC‐containing DNA by DB293 is characterized by the formation of a dimer complex. SPR results with the ‐ATGA‐ oligomer further showed the formation of a strong, cooperative DB293 dimer with the second compound having a binding affinity approximately 25‐fold stronger than the first (K 1 = 2.8 × 106 M−1; K 2 = 7.3 × 107 M−1) (Tanious et al., 2003). NMR analysis of DB293 with an ATGA‐containing oligomer confirmed the highly cooperative dimer formation (Fig. 9). The COSY spectra at the intermediate ratio of 1:1, for example, contained peaks from the 2:1 complex and the free DNA clearly supporting positive cooperativity in complex formation.

Figure 9.

Figure 9

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Two‐dimensional COSY spectra of the ‐ATGA‐ sequence and with DB293 complex showing the T‐CH3 and T‐H6 correlations at different ratios of DB293. The DNA sequence contains six thymine residues and all the six methyl‐aromatic proton scalar couplings are observed in the absence of DB293 (0:1, top). At the intermediate ratio (1:1, middle), a doubling in the number of signals is observed, suggesting the presence of two distinct species. One set of these peaks corresponds to the free DNA (indicated with solid lines between 0:1 and 1:1). The other set of peaks corresponds to the 2:1 complex based on the spectra at 2:1 ratio (bottom, indicated with broken lines between 1:1 and 2:1). The absence of any free DNA cross peaks at 2:1 highly suggests the complete saturation of free DNA at 2 Molar equivalents of DB293. The presence of two distinct sets of signals at the intermediate ratio of 1:1 for the free DNA and the 2:1 species clearly shows the formation of a strong and highly cooperative stacked complex by DB293 with the ‐ATGA‐ sequence as also observed in SPR studies.

High‐resolution 2D‐NMR studies of DB293 with the ‐ATGA‐ sequence further showed that the unsymmetrical phenyl‐furan‐benzimidazole units stacked in an antiparallel orientation in the minor groove of ‐ATGA‐. Strong cross peaks were also observed between the two stacked molecules of DB293 and both DNA strands, suggesting the sequence‐specific recognition of both strands. The discovery of DB293 forming a highly cooperative, antiparallel dimer with a GC‐ containing mixed DNA was the first instance where a heterocyclic diamidine was shown to recognize a non‐AT DNA sequence. Other dications, such as netropsin, have not been discovered to form stacked dimers with any DNA sequences. The dimer with a local charge of +4 is an unusual result (Wang et al., 2000, 2001). The stacked‐dimer recognition mode of these ligands is completely different from the traditional DNA recognition exhibited by polyamides, and offers ideas for developing new agents to recognize diverse motifs.

Design of Broad‐Based Sequence‐Specific Minor Groove Binding Agents

The design of sequence‐specific minor‐groove‐binding agents which have the potential for biotechnology applications and clinical activity is an important area of current research in nucleic acid interactions. The compounds in Figure 1A are AT‐specific binders but, as noted above, important new uses of these compounds will require broader sequence recognition. Until recently, recognizing mixed base‐pair sequences in the minor groove of DNA has followed the original ideas with pyrrole and imidazole groups in polyamides. Such compounds have an advantage of design simplicity, but have been plagued by solution and synthetic difficulties (Nozeret et al., 2018; Hargrove et al., 2012; Nishijima et al.; 2010). Heterocyclic amidine minor groove binding derivatives, on the other hand, are attractive for the development of GC‐specific agents since they have good solution properties and cell uptake, for example, Furamidine, DB75), berenil, DAPI, and pentamidine (Fig. 1A) The overall compound design goal is to form a set of innovative minor groove modules with extended AT and GC base pair sequence‐specific DNA interactions. None of the new compounds will contain a pyrrole or imidazole group or an amide linkage as in the polyamide minor groove binders that have had no clinical success. Three groups of compounds that provide important new DNA‐targeted reagents have been discovered through designed synthesis and screening. These compounds are exciting new therapeutic candidates with favorable development potential. The designed compounds have structures that contain three quite different mixed‐sequence DNA minor groove binding modules. The three basic structural units for GC base pair recognition are azabenzimidazole, pyridine, and thiophene‐N‐methyl benzimidazole (Fig. 1C). The new compounds replace the pyrrole‐amide units of polyamides with newly designed H‐bond‐accepting units (Fig. 1C). These modules can recognize GC base pairs and H‐bond with the guanine NH2 group that protrudes into the minor groove. Compounds with these units flanked by AT base pair recognizing modules with H‐bond donating groups, such as amidines, benzimidazole derivatives, etc., were designed and prepared. They provide novel ways to recognize mixed sequences of DNA by modifying AT selective heterocyclics as well as by using entirely new GC bp recognition units. Surprisingly simple changes in substituents can frequently convert a heterocyclic unit from AT to GC‐specific recognition, for example, a switch from benzimidazole to an azabenzimidazole function.

The lead compounds have strong binding and excellent mixed sequence binding specificity for a GC base pair flanked by AT base pair DNA sequences (Paul et al., 2019, Wilson & Paul 2022). The goal of the design of these new minor groove binding agents is the recognition of biologically important DNA sequences. Recognition agents of this type have much broader sequence binding abilities than what has been possible previously. The compounds have good solution and cell uptake abilities with a range of structural and chemical properties that gives them advantages in targeting multiple cell types. The concepts for the development of the three lead compounds, will be presented as examples (Fig. 1C). The concept for this design with a thiophene‐N‐methylbenzimidazole (BI) module was that the thiophene sulfur and the N‐methylbenzimidazole unprotonated nitrogen could form an electron‐rich hole that would pre‐organize the compound shape appropriately for recognition of the minor groove. This shape also orients the unprotonated nitrogen of N‐alkylBI to form a hydrogen bond with the G–NH2 group in the minor groove. The thiophene sulfur and imidazole N in the molecular structure of DB2429 and other similar compounds form a sigma hole interaction that pre‐organizes the system for favorable complex formation with the DNA minor groove. The thiophene‐N‐methylbenzimidazole module in DB2429 can then specifically bind to a mixed sequence DNA with a single GC bp and flanking AT sequences. The next step in the design of this system involved adding groups larger than methyl to the N‐alkyl position. Microstructural variations in minor groove width then allowed the tuning of compound sequence specificity and affinity for a new generation of designed agents (Guo et al., 2016; 2018). With the appropriate compound modifications, these microstructural variations can allow us to selectively target a single GC bp binding site DNA sequence. For example, DB2714, with the incorporation of a bulky cyclopentyl group at the N‐alkyl‐BI position not only increases the single GC binding affinities but also this compound shows very high sequence selectivity. In our experimental conditions, this compound shows weak binding affinities towards pure AT (AAATTT) and two GC bps (AAAGCTTT) sequences (Fig. 10).

Figure 10.

Figure 10

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Representative SPR sensorgrams for DB2714 in the presence of AAATTT, AAAGTTT, and AAAGCTTT hairpin DNAs. With AAATTT and AAAGCTTT sequences, the concentrations of DB2714 are 2 to 500 nM of each compound from bottom to top. With the AAAGTTT sequence, the concentrations of DB2714 from bottom to top are 15, 20, 30, 50, and 100 nM; The solid black lines are best‐fit values for the global kinetic fitting of the results with a single site function.

The azabenzimidazole module has also provided new mixed sequence‐specific binding compounds. Modifying the benzimidazole group to an azabenzimidazole gave improved GC recognition capability, but not at a satisfactory level for cell use. Adding the ‐O‐CH2‐ to the azabenzimidazole significantly improved the GC recognition and gave DB2277 (Fig. 1C) (Paul et al., 2015). NMR analysis of DB2277 with the AAGATA DNA binding sequence and its complementary strand revealed key details of the mixed sequence recognition by this new set of compounds. The azabenzimidazole–benzamidine end of the complex fits tightly into the minor groove with excellent van der Waals contacts, H‐bonds, and solvent interactions (Fig. 11). The experimental structure models from 2D NMR and MD results (Fig. 11) show that the G5‐NH2 in the minor groove of AAGATA is able to form an excellent H‐bond with the aza‐N of the aza‐BI group, as expected from the compound design. The desired DB2277−GC bp interaction is also stabilized by the unexpected formation of an H‐bond between the aza‐BI‐NH that points into the minor groove and the C=O group that H‐bonds with the G (Fig. 2). This is an exciting finding that shows the usefulness of the azabenzimidazole unit and reinforces the rationale for this group in GC base pair recognition (Harika et al., 2016; 2018). The flanking AT sequences also strongly interact with DB2277 and help provide excellent affinity for the compound with the mixed bp sequence. The benzamidine attached at the imidazole side of the aza‐BI group forms a direct amidine H‐bond in the minor groove to an AT bp. The amidine−DNA interaction is also stabilized by an ensemble of dynamic water H‐bonds between minor groove base pairs and the amidines of DB2277 (Fig. 11). This and the other sequence‐specific compounds are also stabilized by phenyl C–H protons that are near the floor of the minor groove and can interact strongly with polar groups on the base pair edges. The aromatic C‐H hydrogens carry a small positive charge and can form stabilizing interactions with DNA A‐N3 and T–C=O groups. The DB2277−DNA interactions coupled to the dynamic stabilizing hydration network and van der Waals interactions provide a very favorable complex and specific DNA recognition for the aza‐BI benzamidine module.

Figure 11.

Figure 11

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(A) Snapshot of NMR‐based MD simulations of DB2277‐d[(5′‐CCAAGATAG‐3′) (5′‐CTATCTTGG‐3′)] complex. The ball and stick model in green– white–blue–red (C–H–N–O) represents DB2277. The DNA bases are represented in tan–white–red–blue–orange (C–H–O–N–P). (B) The important interactions between different sections of the DB2277–DNA complex are illustrated. DB2277 forms four direct H‐bonds (black dashed lines) and one interfacial water‐mediated interaction (purple, ball and stick).

The terminal benzamidine of the complex that is connected by a flexible –OCH2– linker to the aza‐benzimidazole system rises away from the floor of the minor groove such that it cannot form direct interactions with the AT bp at the floor of the groove. Instead, there is an unusual interfacial water molecule that links the inner facing amidine‐NH to T16‐ O2 (Fig. 11) at the floor of the groove (–NH⋯O–H⋯O=T). This water molecule, unlike the other waters that are well‐known to externally stabilize minor groove complexes, is an integral part of a ternary DB2277−DNA−water complex. This is an unexpected ternary minor groove complex that is rare in the AT‐specific compounds of Figure 1A. Such interfacial water molecules show the importance of the design of different types of minor groove binding modules.

Both amidines of the aza‐compound are also stabilized by a dynamic, external and extended water network in the groove that is frequently seen in minor groove complexes. The unusual feature of the DB2277−water interactions is the interfacial water interaction that is required for strong and specific DNA recognition. These results have shown how the flexible –OCH2– linker in DB2277 provides freedom for the benzamidine to project away from the floor of the minor groove of DNA. The flexibility of –OCH2– phenyl helps DB2277 track along the minor groove curvature by forming dynamic water‐mediated H‐bond contacts of the amidine with the bases at the floor of the minor groove. The flexibility and water‐mediated contact allow the phenyl group to rotate several times per microsecond such that the group rotation in a compound bound in the minor groove can be observed in MD simulations. Rapid minor groove expansion motions must dynamically occur in the DNA that result in transient widening of the minor groove to allow the ring to flip. (Harika et al., 2016; 2018) A similar phenyl rotation was observed in the Hoechst33258–AATT complex (Searle & Embrey, 1990).

The third lead module is based on pyridine as an acceptor for a G‐NH2 H‐bond. A critical breakthrough in developing strong and selective GC base pair recognition with pyridine modules was with pyridyl‐phenyl‐diamidine compounds, DB2447 (Fig. 1C). To obtain compound curvature to match the minor groove, two terminal phenyl‐amidine groups with −CH2O− likers to a central pyridine are essential. Changes in DNA thermal melting temperature provide an initial ranking of compounds for binding affinity and relative sequence specificity with different DNA sequences. DB2447 resulted in a pronounced increase in the thermal stability of the single GC containing DNA sequences, AAGTT (ΔT m = 10°C) and AAAGTTT (ΔT m = 14°C). DB2447 also showed significant sequence selectivity with very weak binding to the pure AT sequences (AATT ΔT m = <1°C). The ΔT m values of DB2447 with ATGAT and ATAGTAT sequences are 6 and 10°C, while AAAGTTT and AATGAAT sequences are 14 and 8°C. These results suggested that DB2447 has more favorable interactions within the narrow minor grooves of pure A‐tract DNAs compared to the wider groove in alternating AT sequence DNAs. As expected from the previous results and compound design approach, DB2447 did not show any enhancement in thermal stabilities for two GC base pair containing sequences (Paul et al., 2022).

To quantitatively evaluate the interactions of DB2447 with DNA, SPR experiments were conducted with the three DNA binding sites used in T m studies. As expected from the T m results, DB2447 binds most strongly with AAAGTTT and global kinetics fitting generated a single binding site with a K A= 5.5 × 108 M‐1 (K D = 1.8 × 10‐9 M) in 0.1 M NaCl. The strong binding constant of DB2447 is the result of the rapid association (k a = 4.6 × 107 M‐1s‐1) and comparatively slow dissociation rate (k d = 7.7 × 10‐2 s‐1) constants. DB2447 binds to AAATTT as a monomer complex with a 200‐fold lower affinity compared to AAAGTTT. This result indicates high sequence selectivity for the single G•C bp sequence. The sensorgram of AAATTT shows an off‐rate that is much faster, and complete dissociation from the complex occurs within the first few seconds of the dissociation phase (Fig. 12). With the wider minor groove sequence AAAGCTTT, DB2447 shows 30‐fold weaker binding affinity than AAAGTTT under the same experimental conditions (Paul et al., 2022).

Figure 12.

Figure 12

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Representative SPR sensorgrams for DB2447 in the presence of AAAGTTT and AAATTT DNA sequences. The solid black lines for DB2447‐AAAGTTT complex are best‐fit values for global kinetic fitting of the results with a single site function; The steady‐state binding plot for DB2447 with AAATTT are fitted to a steady‐state binding function using a 1:1 model to determine equilibrium binding constant. The listed binding affinities are an average of two independent experiments carried out with two different sensor chips, and the values are reproducible within 10% experimental error.

Heterocyclic Amidines that Recognize DNA Sequences with More Than One GC Base Pair

The next GC‐specific ligands should be able to target sequences with more than one GC base pair—a commonly observed theme in biologically important genomic regions. As noted above, the minor groove characteristics of GC‐containing sequences differ from AT‐rich DNA. The design of compounds that are selective for GC motifs requires reevaluating the design strategies. Heterocyclic diamidines that have unusual shapes and geometry that do not match the curvature of the minor groove, surprisingly, have been shown to target AT‐rich sequences with excellent binding affinities (see DB921 description above). Such compounds can be used as paradigms to test the hypothesis of linear‐shaped ligands for multiple GC recognition. Designing molecules to recognize GC‐rich sequences can be achieved by incorporating H‐bonding partners at appropriate positions.

Using this shape‐based design strategy, a series of linear heterocyclic diamidines that did not match the curvature of the DNA minor groove were rationally designed, and nitrogen atoms were strategically placed at different positions that were proposed to be useful in recognizing important minor groove elements of GC‐rich motifs (Munde et al., 2007). One compound that provided the necessary breakthrough in recognizing multiple GC base pairs using this design strategy was a linear, unfused, tricyclic diamidine, DB1242 (Fig. 1A). The amidine‐phenyl‐pyrimidine‐ phenyl‐amidine units of DB1242 were substituted to give the molecule a very linear shape with zero radius of curvature. DNase I footprinting studies showed the compound exhibited a strong footprint for a unique GC‐rich sequence, 51‐ GCTCG‐31 (Munde et al., 2007). Interestingly, no detectable footprint was seen for DB1242 with any of the all AT sequences even up to high ligand concentrations (5 μM). This was very promising since this was the first instance where a non‐polyamide was shown to target a GC‐rich motif with very high selectivity over AT‐containing sequences. This also suggested that DB1242 has a unique interaction mode with the ‐GCTCG‐ sequence considering the linear shape of the ligand.

Surface plasmon resonance studies of DB1242 with the cognate sequence showed a surprising highly cooperative dimer formation (Munde et al., 2007). In this case, the binding affinity for the second DB1242 dimer was much stronger than the first (K 1 = 2.0 × 104 M−1; K 2 = 9.1 × 106 M−1). In agreement with the footprinting results, the binding affinity of DB1242 for an AATT sequence was very weak (K = 3.1 × 105 M−1) as compared to the affinity of DB293 with the AATT sequence (K = 2.1 × 107 M−1). Detailed molecular modeling studies of DB1242 with the ‐GCTCG‐ sequence revealed very important interactions between the two dimer ligands and the DNA that favored the formation of a uniquely stacked dimer in the minor groove in a sequence‐dependent manner (see Fig. 7 in Munde et al., 2007). The two molecules stack in an antiparallel orientation with the terminal amidines of the two ligands extending to the edges of the binding site and being located deep in the minor groove. Several inter‐dimer and DNA‐dimer H‐bonds are observed in the models that contribute to complex stability. The amidine units of the dimer make strong H‐bonds with the ‐C=O groups of cytosines throughout the binding site while the inner‐facing nitrogens of both pyrimidine rings make H‐bonds with the ‐NH2 group of guanines in the minor groove (Fig. 13). The overall stacked geometry of the two linear compounds of the dimer match the curvature of the minor groove. The overall stabilization of the complex is guided by H‐bond interactions and explains the largely enthalpy‐driven dimer complex formation observed in calorimetry (Munde et al., 2007).

Figure 13.

Figure 13

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A docked model for the stacked complex of DB1242 (Fig. 1A) with the ‐GCTCG‐ sequence is shown. (A) The DNA helix is shown in a space‐filling model with one chain in blue and one in red. The two DB1242 molecules are shown as tube models. Hydrogens are not shown for clarity. (B) Several of the major interactions that help in the stabilization of the complex are highlighted. The amidine of the top molecule (bottom of the figure) makes strong contact with the outer nitrogen of the pyrimidine of the bottom molecule. The inner nitrogen of the same pyrimidine makes strong contacts with the G‐NH2 in the minor groove with the 5'‐G of ‐GCTCG‐ (red‐colored DNA strand). Similar interactions are observed with the amidine of the bottom molecule to the pyrimidine nitrogens of the top molecule and to the G‐NH2 of the first G of the CGAGC sequence of the complementary strand (blue‐colored DNA strand). The other amidine of the top molecule (top of the figure) makes a strong interaction with the carbonyl group in the minor groove of the first C in ‐CGAGC‐ (blue strand). All the DNA‐ligand and the inter‐ligand interactions help in the formation of a very strong and highly cooperative dimer in the wider minor groove of this DNA sequence.

Designed Compounds that Recognize Two AT Sites Separated by One or More Intervening Base Pairs

While DB293, DB2277, DB2477, DB2429, and DB1242 provided the breakthrough in designing compounds that can target single and multiple GC units as noncovalent motifs, covalently linking such molecules using simple linker systems established new DNA recognition principles (Liu et al., 2012). Such covalently linked monomer structures can recognize long DNA sequences with multiple binding sites. The idea behind such a design strategy is that even ligands with relatively weak affinity for a single site can synergistically bind to multiple target sites on a stretch of DNA and induce conformational changes that would be sufficient to elicit a desired biological response.

As shown above, the amidine‐benzimidazole‐ phenyl (ABP) moieties are very effective in recognizing AT‐rich sites and, therefore, were incorporated into the design strategy as the primary recognition units. A series of linked, symmetrical ABP units were rationally designed with varying linker lengths [‐O‐ (CH2)n‐O‐, n = 3, 4, 5; Liu et al., 2012]. Surface plasmon resonance and mass spectrometry results quite interestingly show that the number of intervening base pairs can significantly influence the interaction mode (Liu et al., 2012). In order to recognize long AT sequences, two AT recognition modules connected by a flexible linker were used. As examples, DB2115 and DB2313 are shown in Figure 1B. The flexible linkers allow the compounds to conform to the local shape of the minor groove and form strong AT H‐bonds with the AT recognition modules. The PU.1 transcription factor is involved development of acute myeloid leukemia (AML) and the PU.1 DNA recognition sequence has a 5' AT sequence. We found that both DB2115 and DB2113 bind strongly to that AT sequence and are strong inhibitors of PU.1 binding. Furthermore, cell studies with AML revealed that both compounds are also strong inhibitors of AML in cells (Antony‐Debré et al., 2017).

The symmetric compounds with two ABP units exhibit a large variation in the binding mode and affinities with the different sequences (Liu et al., 2012). However, no significant differences in the affinities among the compounds are observed as the linker length is varied; therefore, only the simplest molecule, DB2232 with an ‐O‐(CH2)3‐O‐ linker, is discussed here. The different modes of binding observed with DB2232 can be extrapolated to the other compounds with varying linker lengths. Interestingly, DB2232 binds very weakly to the single AATT sequence because of the limited conformational flexibility imposed on DB2232 by the narrow minor groove of the AATT sequence. With the two‐site sequence separated by a single GC base pair, however, DB2232 can form a very strong 1:1 complex. Molecular modeling studies show that DB2232 can bind to the AATTGAATT sequence with the two APB units stacking favorably in the two AATT sites, while the linker spans across the single GC site (Liu et al., 2012). As the number of GC base pairs is increased SPR and mass spectral studies show the formation of a very strong 2:1 complex with the AATTGCAATT sequence with the binding affinity of the second molecule much higher than the first ligand (K 1 = 1.1 × 106 M−1; K 2 = 9.4 × 108 M−1). Detailed molecular modeling studies provided a satisfactory explanation for the unusual dimer formation by DB2232 with this sequence (Fig. 14). The presence of the two GC base pairs between the AATT sites creates a wider groove that is energetically favorable to accommodate a stacked complex. The two molecules of DB2232 bind in an offset manner with the maximum overlap region of the two molecules located in the widest part of the minor groove, with the two GC base pairs (Fig. 14B) and the non‐overlapped ABP moieties of DB2232 favorably located in the narrower AATT site. The offset orientation also puts the terminal amidines of the two molecules away from each other and decreases the electrostatic repulsion while forming optimum cation‐π interactions with the inner phenyl rings of the stacked ligand. The complex is, thus, able to form a significant number of favorable interactions with base edges at the floor of the minor groove, as well as with the groove walls.

Figure 14.

Figure 14

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A model for the stacked complex of DB2232 docked into the AATTGCAATT sequence is shown. (A) The DNA helix is shown as a tube model with one chain in blue and one in red. DB2232 is shown in space filled with carbons in light blue, nitrogens in dark blue, and oxygens in red. Hydrogens are not shown for clarity. (B) The two central GC base pairs are shown in space filling representation with the same atom colors as in (A). The two stacked amidine‐benzimidazole‐phenyl modules are shown as purple tube models. The other DNA base pairs and backbone atoms are shown as thin red or blue tubes. The close interactions of the compounds with the GC base pairs and widened minor groove to accommodate the stacked modules are easily seen in this view. In the AATT sequences only single modules fit snugly into the narrow A‐tract type groove structure. This model clearly provides the rationale for the 2:1 model of DB2232 at AATTGCAATT.

CONCLUSION

The model of AT‐specific minor groove binders was developed from the discovery of netropsin in the early 1950s and the beginning studies with DNA by a number of pioneer investigators. Extensive synthetic efforts have provided a variety of heterocyclic diamidines that have provided clinically useful agents, extensive structural models for minor groove complexes, and a greatly expanded understanding of DNA molecular recognition. The synthetic efforts also produced new types of derivatives that helped to show that the original model for AT‐specific minor groove binders was far too limited. The shape recognition requirement was shown to be too limited by very strong minor groove binding compounds that have a linear structure. It is highly likely, based on these results, that an entirely new class of minor groove binders can be developed that incorporate a bound water molecule into their DNA minor groove complex and form a very strong ternary complex. The requirement for an all AT base pair binding site has also been shown to be too limiting. Cooperative stacked dimers of a heterocyclic diamidine that actually have a total charge of +4 can bind into GC‐ containing sequences better than they bind to pure AT sequences. Developments in this area are at the beginning stages and will certainly continue to produce new ways to recognize the minor groove. Developments of the compounds as transcription factor inhibitors and activators are also at an early stage and offer new agents and approaches in chemical biology. Finally, the heterocyclic diamidines have a record of clinical usefulness and they should continue to produce exciting new results in this critical area.

AUTHOR CONTRIBUTIONS

Ananya Paul: Data curation, formal analysis, investigation, methodology, software, validation, visualization, writing ‐ original draft, and writing ‐ review & editing Rupesh Nanjunda: Conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, validation, visualization, writing ‐ original draft, and writing ‐ review & editing; W. David Wilson: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing ‐ original draft, and writing ‐ review & editing.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

Our research in the area of minor groove binding agents for therapeutics and chemical biology has been generously supported by NIH for a number of years and is currently supported by NIH grant GM111749 (W.D.W. and D.W.B.). A number of collaborators have made key contributions without which the research described in this overview would not have been possible. Although their names are listed in the references, we would like to call special attention to a few key scientists. Professor David Boykin and his coworkers have been equally responsible for all of the results and developments described in this paper. Without their compound design and synthesis efforts, these studies would not have been possible. The structures provided by Professor Stephen Neidle and his coworkers have frequently provided the missing link to help us understand puzzling results that continue to take our understanding of minor groove interactions to a new level. Professors Richard Tidwell, Moses Lee, and James K. Bashkin have also provided exciting new minor groove binders over many years that have helped to expand our understanding of minor groove interactions. Dr. Gregory M. K. Poon and Dr. Ulrich Steidl have provided important protein‐DNA models and cellular systems for testing our designed minor groove binding compounds. Dr. Markus Germann has been an important collaborator in the NMR structural experiments described in this review. The footprinting and biological studies of Drs. Christian Bailly and Marie‐He´le`ne David‐Cordonnier have directed our attention to new minor groove recognition possibilities and provided the key information for GC base pair recognition. Not only are these collaborators outstanding scientists, but they have also all become good friends, and have certainly made our research much more enjoyable for many years.

Paul, A. , Nanjunda, R. , & Wilson, W. D. (2023). Binding to the DNA minor groove by heterocyclic dications: from AT specific to GC recognition compounds. Current Protocols, 3, e729. doi: 10.1002/cpz1.729

Published in the Nucleic Acid Chemistry section

DATA AVAILABILITY STATEMENT

No new data were created in this study.

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