Thermodynamics of DNA Minor Groove Binders - Journal of Medicinal

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Thermodynamics of DNA minor groove binders Hasan Y. Alniss J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00233 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Journal of Medicinal Chemistry

Thermodynamics of DNA minor groove binders Hasan Y. Alniss*

Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, P.O. Box 27272, Sharjah, United Arab Emirates.

July 2018 *Address Correspondence to this author at: Department of Medicinal Chemistry College of Pharmacy University of Sharjah P.O. Box 27272, Sharjah United Arab Emirates Tel: +(971)-6-5057427 Email: [email protected]

Keywords: DNA; Minor Groove Binders (MGBs); Thermodynamics; Enthalpy; Entropy; Transcription Factors

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Abstract Understanding the thermodynamic and binding characteristics of DNA minor groove binders (MGBs) is important for the rational design and development of novel MGBs; however, there are contradicting results in the literature regarding the thermodynamic signature of MGBs. The expansion of the thermodynamic database for MGBs in the literature was encouraging to evaluate and critically test the previously reported hypothesis that MGB binding is mainly entropically driven. In this review, the thermodynamic data of a group of MGBs published in the literature were analyzed to better understand the factors that drive minor groove recognition. Analysis of the enthalpic and entropic contributions to the free energy of binding for 20 interactions from a total of 14 different compounds reveals that MGB binding can be driven by enthalpy, entropy or by both and that’s mainly dictated by ligand structural heterogeneity. These findings could be useful in the design of MGBs for therapeutic purposes.

1. Introduction Minor groove binders (MGBs) are a class of small molecules that bind to the minor groove of duplex DNA. Some of these compounds are of natural origins e.g. the polyamides, netropsin and distamycin3 while others are synthetic compounds e.g. thiazotropsin A4 and the hairpin7 structure (Figure 1). These molecules have crescent shapes that match the curvature of DNA in the minor groove and can interact non-covalently in a sequence specific fashion with the targeted DNA base sequence by a combination of hydrogen bonding to the DNA base pairs, van der Waals interactions with the walls of the minor groove and non-specific electrostatic interactions with the backbone of DNA. MGBs have recently found wide applications in research as a tool to


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H Hoechst 33258

NH

HO H N

NH2 NH

O

NH2

Distamycin

N HN

HN

H N

N

N N

O

HN O

N N

H2 N

DB224

NH2

N H

O

N H

O NH2

NH2 H 2N

Netropsin

H N

N H

NH2 HN

H N

O

O

N

N O

DB226

H2N N H

NH2

H

N H

O

NH NH

O

Thiazotropsin A N DB75

H2 N

HN

H N

H N

O

NH2

O

NH2

O

O

S

N H 2N

N

H 3C

NH NH

O

Thiazotropsin B

N NH2

Propamidine

H 2N

NH2

N

O

S

N

O

O

NH2

Berenil

H2N

O

N

NH2

O

N

NH

N NH

N

O

O

H N

H N

H N

H N

H N

O

N

HN

N H

N

Hairpin

N N H

H N

O

NH2 O

HN

H N

N

NH2

N

O

O

N

O

H O O

NH

H

NH Compound 3

Compound 2

HN

H N

N

O

NH NH

H N

O N

N O S

HN

H N

N

N N

O

O

HN O

S O

Figure 1: Structures of commonly studied minor groove binders, including natural and synthetic molecules with diverse structures.



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control gene expression,8 by investigating the effect of turning on/off one or more genes, and as potential therapeutics in anti-cancer and anti-infective therapy.11 These small molecules which intervene at the nucleic acid level are capable of turning on/off gene expression without causing permanent DNA damage which is usually observed with the currently available toxic chemotherapeutics. The significance of MGBs in anti-cancer and anti-infective therapy is therefore growing, for instance, MGBs are currently being developed as transcription factor antagonists for the treatment of prostate cancer12 and as new class of antibacterial agents for the treatment of. Clostridium difficile infections.11, 13 The crescent-shaped MGBs bind to the minor groove as a monomer or a side-by-side antiparallel dimer (Figure 2). The dimeric recognition usually distorts the DNA structure by widening the minor groove and bending the DNA helix toward the major groove resulting in major groove compression.8-9 These structural changes make the transcription factors unable to recognize their target in the major grooves. Such structural perturbations in the DNA helix, especially in relation to the groove dimensions, are believed to be responsible for the disruption of transcription factor-DNA interfaces via allosteric modulation.8 Understanding the molecular basis of ligand–DNA associations particularly the structural and thermodynamic details is of prime importance for the rational design and development of novel drugs. With the increasing availability of advanced isothermal titration calorimeters, these highly sensitive instruments have become widely used for the characterization of biomolecular interactions in vitro. Such studies provide a complete thermodynamic profile for bimolecular interactions in aqueous solutions and that includes the determination of the binding affinity (K), stoichiometry (N), enthalpy (∆H), entropy (∆S) and free energy of binding (∆G) for the interaction. Analysis of the enthalpic and entropic contribution to the free energy of binding can 4 ACS Paragon Plus Environment

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Figure 2: A) X-ray crystal structure of a 1:1 complex of netropsin: DNA (PDB ID: 121D).32 B) X-ray crystal structure of a 2:1 complex of Distamycin: DNA (PDB ID: 378D).34

reveal the molecular forces that drive complex formation. Furthermore, studying the binding thermodynamic characteristics of closely related ligand structures to a specific binding site helps to establish how modifications in the structure influence the binding affinity. Information obtained from the analysis and interpretation of thermodynamic data is useful in revealing the pharmacophore of drug which can then be used in directing the structural optimization of candidate compounds during the stages of drug design and development.15 5 ACS Paragon Plus Environment


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Interpretation of thermodynamic data is sometimes very challenging due to the presence of many variables that might affect the thermodynamics of binding including the environment of interaction, structure of ligand, properties of the binding site, and the molecular forces that drive the interaction. The thermodynamic characteristics of MGB binding have been extensively studied by several groups using isothermal titration calorimetry (ITC).1-2, 4-6, 10, 14, 16 One of the major reviews was conducted by Professor Chaires18 who analyzed the thermodynamic data published in the literature for groove-binding and intercalating ligands and proposed a hypothesis that minor groove binding is entropically driven whereas intercalation is enthalpically driven. However, several studies appeared later in the literature and showed that minor groove binding is primarily enthalpically driven with a small or opposing contribution from entropy.5, 16-17, 19 These contradictory findings for the thermodynamics of minor groove binding were based on a limited number of examples and were unrelated to ligand structure. There is therefore a need to expand the database of thermodynamic data for MGBs to evaluate and critically test Chaires’s hypothesis. Furthermore, this hypothesis supposed that the differences in the thermodynamic profile of groove binding and intercalating ligands arise from differences in reaction mechanisms of the two binding modes, which allows the differentiation between groove binding and interaction via their distinctive thermodynamic signature. However, this assumption ignores other critical factors which are known to affect the thermodynamics of interaction especially the structural diversity of DNA binding agents (e.g. MGBs), and how this might affect the reaction mechanism, the molecular forces that drive the interaction and the overall thermodynamic signature of the binding interaction. Although Chaires’s hypothesis is valid for intercalating ligands which possess similar planar geometry with a limited structural diversity, it’s invalid for MGBs which have extremely diverse structures (Figure 1).11 In this review study, to better 6 ACS Paragon Plus Environment

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understand the thermodynamics of DNA minor groove binders, the enthalpic and entropic contributions to the free energy of binding of a group of MGBs were analyzed and linked to the structural diversity of MGBs and the molecular forces that drive the interaction. Furthermore, the principle of ITC experiment and its application in drug discovery was briefly summarized along with an interpretation of the binding thermodynamic data of MGBs with special focus on the chemical structure, rationale of drug design and mechanism of action. 2. Isothermal titration calorimetry Isothermal titration calorimetry (ITC) is a biophysical technique that is used to directly determine the thermodynamics of intermolecular binding at constant temperature. It can provide a complete thermodynamic profile of biochemical equilibrium interactions and has become one of the fastest growing techniques used in biomedical research20-22 since the emergence of the first commercial ITC instrument (Omega model, MicroCal) in 1989.23 The principles and applications of ITC for studying the thermodynamics of biomolecular interactions have been comprehensively described in the literature.24-26 The ITC instrument is composed of two identical cells surrounded by an adiabatic jacket (Figure 3). The sample cell is usually filled with a macromolecule solution (e.g. DNA, protein, etc.) and a reference cell which is filled with buffer or water. Small volumes of ligand are titrated by a computer-controlled stirring syringe into the DNA solution in the sample cell. Alternatively, the ligand solution in the cell can be titrated with the solution of the macromolecule, which is recommended for poorly soluble compounds. If there is a binding interaction between the reactants, heat is either released or absorbed in direct proportion to the amount of binding that occurs. The instrument detects



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temperature differences (∆T) between the reference and sample cell and maintains ∆T zero by increasing or decreasing

Figure 3: Schematic representations of isothermal titration calorimetry (ITC) instruments. (A) An ITC instrument prior to performing a titration. The sample cell and the reference cell are kept at the same temperature, which is typically 25°C. (B) An ITC instrument performing a titration. When an injection is made, heat is released or absorbed in direct proportion to the amount of binding (endothermic or exothermic). In the raw data presented in the inset, each injection is accompanied by an interaction where heat is given out (exothermic). As more ligand is injected, the binding sites in the sample cell are gradually saturated, and the exothermic effect diminishes before new endothermic signals appear as a result of the heat of dilution of the ligand in the buffer.


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the feedback power applied to the sample cell when the reaction is endothermic or exothermic, respectively. When the macromolecule in the cell becomes saturated with added ligand, the heat signal diminishes until only the background heat of dilution is observed. Because the amount of free macromolecule available decreases after each successive injection, the intensity of the raw data peaks becomes progressively smaller until complete saturation is reached (Figure 3). Measurement of this heat change allows for the determination of binding constants and a thermodynamic profile of the reaction that includes the observed molar calorimetric enthalpy (∆Hobs), entropy (∆Sobs), and the change in free energy (∆G) 3. Rationale of the ITC dataset selection In order to establish a reliable comparison for the binding thermodynamics of DNA minor groove binders, thermodynamic data were collected from the literature 1-2, 5-6, 9, 14, 16-17, 19, 27 using the enthalpy values which were calorimetrically determined under similar solution conditions (pH 6.8-7.0, 20-100 mM NaCl, 25 °C). The value of ∆H for a binding reaction is most reliably generated by isothermal titration calorimetry rather than indirectly from van’t Hoff determinations.28-29 However, it’s difficult to obtain a dataset compiled from literature for different compounds analyzed by different research groups under identical experimental conditions. For instance, different salt concentrations and buffers were employed in the studies reported in this review e.g. PIPES,4 ACES,9 cacodylate,1 MES,6 and phosphate buffer.2 The enthalpic and entropic contributions to the free energy of binding is affected by the buffer employed if protons are exchanged upon ligand-DNA association. The buffers mentioned above have different ionization enthalpies, and if protons are released or up taken upon complex formation, there will be a contribution from the buffer to the observed experimental binding 9 ACS Paragon Plus Environment


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Table1: Enthalpy changes for the dissociation of protonated buffers used in this study.30 Buffer

pka

∆H (kJ mol-1)

∆H (kcal mol-1)

Cacodylate

6.14

-1.96 ± 0.02

-0.47 ± 0.005

PIPES

6.71

11.45 ± 0.04

2.73 ± 0.009

ACES

6.75

31.41± 0.05

7.51 ± 0.012

Phosphate

6.81

5.12 ± 0.03

1.22 ± 0.007

MES

6.07

15.53± 0.03

3.71 ± 0.007

enthalpy. The ionization enthalpies of the buffers mentioned above are summarized in Table 1.30 The possible influence of ionization enthalpy of different buffers on the experimental binding enthalpy was previously investigated by conducting parallel experiments under different buffer conditions (e.g. PIPES vs. ACES).9 The results showed that the observed experimental enthalpies in PIPES and ACES buffers were nearly equal. A difference of 1.5-2.0 kcal mol-1 was observed between the experimental enthalpies measured in PIPES and ACES buffers. This difference, which is within experimental error, was not attributed to the buffer effects as the difference between the ionization enthalpy of PIPES and ACES buffer is around 4.8 kcal mol-1 (Table 1).

Another study conducted by Buurma et. al.31 showed that using different concentrations of NaCl & NaBr (20-150 mM) and different buffers have no significant effect on the enthalpy of interaction. Nevertheless, even if we suppose that the observed differences in the measured enthaplies arise from the potential influence of buffer and salt concentration, these slight differences (±10%) are not expected to significantly affect the enthalpic contribution to the free energy of binding or to entirely change the thermodynamic profile from being entropically driven to enthalpically driven or vice versa. Since the current review is qualitative in its nature, the limited potential contributions of buffers and salt concentration to the thermodynamic


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profiles of the different compounds reported in this study do not affect the validity and conclusions of this review. In order to make the analysis feasible, only the binding profiles for the interaction of different compounds with short double-stranded oligodeoxynucleotides that offer a single binding site in the minor groove were selected for this study. Long DNA molecules may contain more than one site, or some overlapping sites, that severely complicates any analyses due to competing effects and the lack of certainty for the stoichiometry of ligand interaction with long DNA molecules. The stoichiometry of the interactions and the exact location of the binding site in the minor groove of DNA for all complexes reported in this study were confirmed by different biophysical techniques including NMR spectroscopy and X-ray crystallography. Based on these considerations, a dataset composed of 20 binding interactions from a total of 14 different compounds was assembled from the literature. The resultant dataset is summarized in Table 2 and divided into three categories based on the contribution of enthalpy and entropy to the free energy of binding. The data were carefully selected to have a representative sample for each category.

4. Thermodynamics of ligands binding to the minor groove of DNA The thermodynamic characteristics of minor groove binding have been the object of studies since the appearance of sensitive titration calorimeters. Understanding the molecular basis of minor groove recognition by small molecules requires not only the determination of the thermodynamic parameters (∆G, ∆H, ∆S, K) of MGB binding, but also linking these parameters to the various possible factors that may influence the interaction, including the structural diversity of the ligand, the heterogeneity in the DNA target sequence, experimental conditions and the intermolecular forces that drive the interaction such as H-bonding, van der Waals forces, and



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electrostatic interactions. If similar experimental conditions are employed for all experiments under comparison as discussed earlier in section 3, the two main factors that affect the thermodynamic profile are the structure of the ligand and the DNA sequence, which will determine the main forces underlying the formation of the ligand-DNA complex. The effects of ligand structural heterogeneity on the thermodynamics of interaction can be assessed by comparing the binding profiles of different ligands to the same binding site whereas the effects of heterogeneity in the DNA target sequence can be determined by comparing the binding profile of one ligand to different DNA binding sites. Analysis of the thermodynamic characteristics of MGB binding data published in the literature for a dataset composed of 20 binding interactions has revealed that minor groove recognition by small molecules can be driven by enthalpy, entropy, or by both (Table 2). The thermodynamic data are shown in Figure 4 as bar graphs, ranked in an increasing order of enthalpy contribution to the Gibbs free energy of binding. These findings disagree with the previously reported hypothesis that minor groove binding is mainly driven by entropy.18 A major drawback in this study was that it was unrelated to ligand structure and based on a limited number of MGBs which possess a similar thermodynamic profile. The thermodynamic signature of MGBs is highly affected by ligand structure. MGBs are typically composed of several (hetero)aromatic rings such as pyrrole, furan, benzene or benzimidazole which are connected by a bond with limited torsional freedom such as an amide or a conjugated biaryl bond (Figure 1). Analysis of ITC data of MGBs has shown that the amide-linked MGBs (netropsin, distamycin and their analogues, e.g. thiazotropsin A) have a completely different thermodynamic profile compared with the conjugated biaryl MGBs (Bis-benzimide derivatives e.g. Hoechst 33258 and the diphenylfuran derivatives e.g. DB224, DB226 etc. ).11 12 ACS Paragon Plus Environment

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Table 2: Thermodynamic data of a group of DNA minor groove binders at 25 ºC. Compound

DNA binding site

Stoichiometry

∆G

∆H

Ligand: DNA

(kcal mol-1)

(kcal mol-1)

(kcal mol )

-T∆S

Reference -1

Entropy-driven interactions Hoechst 33258

d(CGCGAATTCGCG)2

1:1

-11.8

+10

-21.8

1

Hoechst33258

d(CGCAAATTTGCG)2

1:1

-11.7

+4.3

-16

2

Berenil

d(CGCAAATTTGCG)2

1:1

-8

+0.6

-8.6

5

DB226

d(CGCGAATTCGCG)2

1:1

-8.5

-0.5

-8

6

Propamidine

d(CGCAAATTTGCG)2

1:1

-7

-1.1

-5.9

5

DB75

d(CGCGAATTCGCG)2

1:1

-9

-2.2

-6.8

6

DB244

d(CGCGAATTCGCG)2

1:1

-9.9

-2.3

-7.6

6

Enthalpy and entropy-driven interactions* Propamidine

d(CGCGAATTCGCG)2

1:1

-8.2

-3.3

-4.9

5

Thiazotropsin B

d(GCGACTAGTCGC)2

2:1

-7.3

-3.5

-3.8

9

DB293

d(CGCGAATTCGCG)2

1:1

-9.6

-3.6

-6

6, 10

Distamycin 1:1

d(CGCAAATTTGCG)2

1:1

-10.5

-5.8

-4.7

5

Netropsin

d(CGCAAATTTGCG)2

1:1

-8.7

-5.8

-2.9

5

Enthalpy-driven interactions Hairpin

d(CATTGTTAGAC)2

1:1

-9.2

-6.7

-2.5

14

Thiazotropsin B

d(GCGACGCGTCGC)2

2:1

-9.2

-7.4

-1.8

9

Netropsin

d(CGCGAATTCGCG)2

1:1

-8.8

-8.6

-0.2

5

Thiazotropsin A

d(CGCGCTAGAGCG)2

2:1

-9.5

-12.0

2.5

4, 17

Thiazotropsin A

d(GCGACTAGTCGC)2

2:1

-10.4

-12.8

2.4

19

Compound 2

d(GCGACTAGTCGC)2

2:1

-9.1

-14.3

5.2

19

Compound 3

d(GCGACTAGTCGC)2

2:1

-9.5

-15.6

6.1

19

Distamycin 2:1

d(CGCAAATTTGCG)2

2:1

-7.9

-15.7

7.8

5

*Associated with at least 33 % contribution from either ΔH or TΔS to ΔG.



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Figure 4: Thermodynamics of DNA minor groove binders showing three distinctive profiles: I- Enthalpy driven interactions (the top of the graph) II- Entropy driven interactions (the bottom of the graph) III- interactions that are driven by both enthalpy and entropy (the middle of the graph). Binding free energies (∆G) are indicated by the black bars. Binding enthalpy values (∆H) are indicated by the red bars. Entropic contributions to binding (-T∆S) are indicated by the blue bars.

The interaction of amide-linked MGBs with DNA is mainly enthalpically driven with an opposing or slight contribution from entropy to the free energy of binding, whereas the binding of the conjugated biaryl MGBs with DNA is mainly entropically driven with an opposing or slight contribution from enthalpy (Table 2 and Figure 4). For instance, the binding of Hoechst 33258 and netropsin to the same DNA sequence “5’-AATT-3’” generated a favorable entropy 14 ACS Paragon Plus Environment

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value of

-21.8 kcal mol-1 with an enthalpic penalty of +10 kcal mol-1 for Hoechst 33258, while

netropsin binding generated a favorable enthalpy value of -8.6 kcal/mol with a slight contribution from entropy (-0.2 kcal mol-1). These results indicated that the binding of Hoechst 33258 was entropically driven via hydrophobic and electrostatic interactions2,

5

and that there was an

enthalpic penalty associated with the complexation process. However, the interaction of netropsin with the 5’-AATT-3’ was mainly enthalpically driven via hydrogen bonding and/or van der Waals interactions.5 A similar trend is observed when comparing the binding profile of ligands with diverse structures to the same binding site e.g. the interaction of Hoechst 33258 & distamycin

with 5’-AAATTT-3’, DB226 & netropsin with 5’-AATT-3’ and berenil &

distamycin with 5’-AAATTT-3’.

But the MGBs which have similar structural features

generated comparable thermodynamic profiles e.g. the interaction of Hoechst 33258, DB226, DB75 & DB244 with “5’-AATT-3’” is mainly entropically driven, whereas the interaction of thiazotropsin A, compound 2, & compound 3 with 5’-ACTAGT is mainly enthalpically driven. These findings clearly show the role of ligand structural heterogeneity in dictating the thermodynamics of MGBs. Amide-linked minor groove binders can bind to DNA as a monomer (e.g. netropsin)5,

32

or a

dimer (e.g. distamycin,33-34 thizaotropsin A17). A significant breakthrough in the MGB research was achieved with the observation that distamycin could bind in the minor groove as an antiparallel dimer, side-by-side, in a head to tail fashion.35

This opened avenues for the

development of MGBs with base pair selectivity.36-39 Netropsin binds exclusively in a 1:1 complex with DNA,40-41 almost certainly due to the repulsive force which would occur by having two positively charged groups side by side (amidine groups). Distamycin however, whilst binding in a 1:1 complex in a similar way to netropsin, also can bind in a 2:1 complex with 15 ACS Paragon Plus Environment


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DNA, which is only possible because the positively charged tails are well separated (Figure 2). The monomeric association of the amide-linked MGBs is mainly driven by favorable enthalpy and enhanced by positive entropy (e.g. netropsin and distamycin, 1:1 binding mode). This lead to the conclusion that the first monomer of distamycin which is associated with favorable entropy dehydrates the groove, and the second monomer does not release water molecules from the DNA.5, 18 However, the observed dimeric association raises the question ‘do distamycin and its analogues form 2:l complexes with the minor groove even at low [ligand]/[duplex] ratios, or 1:1 complexes are formed at [ligand]/[duplex] ratios up to 1:1, then at higher ratios, 2:l complexes are generated?. The answer to this question was obtained from the NMR titration studies and it seems that the mode of binding relies on the minor groove width of the target DNA sequence. The binding sites which contain GC base pairs or alternating AT base pairs e.g. 5’-ATATA-3’,42 5’-AAGTT-3’,42 & 5’-ACTAGT-3’4 have a wider minor groove compared with the consecutive A/T rich sequences. NMR titration studies have shown that distamycin and its analogues bind to these sequences exclusively in a non-stepwise dimeric 2:1 mode. However, for consecutive A/T rich sequences e.g. 5’-AAAA-3’, 5’-AAATT-3’, and 5’-AAATTT-3’, only 1:1 distamycin complexes are observed at low ratios up to 1: l, & at higher ratios, 2: l [ligand]/[duplex] complexes are formed.43-44 These results reveal that the binding modes of distamycin are dependent on the base sequence of DNA and the width of the minor groove. The effect of binding site heterogeneity on the thermodynamics of interaction can be evaluated by comparing the binding profiles of different DNA sequences with the same ligand. For instance, the interactions of Hoechst 33258 with two dodecamers containing the central 5’AATT-3’ and 5’-AAATTT-3’ sequences were associated with favorable entropy values of -21.8 and -16 kcal mol-1 for 5’-AATT-3’ and 5’-AAATTT-3’ respectively. In addition to the number 16 ACS Paragon Plus Environment

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of AT base-pairs, the dodecamer containing the 5’-AATT-3’ binding site differs from the 5’AAATTT-3’ dodecamer with respect to the number of GC base pairs. The 5’-AATT-3’ dodecamer has more GC base pairs and that makes its minor groove wider than the 5’-AAATTT3’ dodecamer. The binding of Hoechst 33258 to 5’-AATT-3’ showed an entropically driven enhancement in binding free energy and this may reflect the release of more water and/or counter ions from the minor groove upon complexation as the 5’-AATT-3’ dodecamer has a wider minor groove compared to the 5’-AAATTT-3’ dodecamer. In contrast to the binding profile of Hoechst 33258 with the 5’-AATT-3’ sequence, the binding of netropsin to 5’-AATT-3’ showed an enthalpically driven enhancement in binding free energy compared to the 5’-AAATTT-3’ sequence and this may reflect the role of water molecules in assisting the complex formation by bridging between the nucleic acid and the ligand through hydrogen bonding.45-48 These data suggest that the thermodynamics of MGBs is affected by both sequence-dependent minor groove width and the molecular forces that drive complex formation. However, in these two examples different molecular forces were responsible for the complexation and that was dictated by the structures of the ligands “Hoechst 33258” and “netropsin” since the binding site in both cases is identical (5’-AATT-3’). These findings reveal that ligand structural heterogeneity has a more profound effect on the thermodynamics of MGBs than the heterogeneity in the DNA binding site. The order of DNA base-pairs in the binding site affect the width of minor groove and the position of binding regions in the grooves. However, binding sites in the minor groove of DNA can provide all types of non-covalent interactions with a ligand such as hydrogen bond acceptor/donor, van der Waals forces and electrostatic interactions. The binding site therefore plays an important role in dictating the occurrence of an interaction, but if there is an interaction, it’s the ligand structure that will determine the thermodynamics of MGBs by dictating the exact 17 ACS Paragon Plus Environment


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forces underlying the formation of the ligand-DNA complexes. On the contrary to ligand structure, there are no examples in the literature that show significant effects for the heterogeneity in the DNA binding sequence on the thermodynamics of MGBs i.e. reversing the thermodynamics of interaction from being enthalpically driven to entropically driven or vice versa.

For example, the interactions of the distamycin analogue, thiazotropsin A, with

dodecamer oligodeoxyribonucleotides containing central 5’-ATCTAGT -3’, 5’-TCTAGA-3’, 5’GCTAGC-3’, and 5’- CCTAGG-3’ sequences were enthalpically driven via hydrogen bonding and/or van der Waals forces.4 The interaction of the thiazotropsin A with four duplexes showed remarkably similar thermodynamic signatures; however, a relatively weak binding was observed with 5’-CCTAGG-3’ compared with other sequences due to the presence of the guanine NH2, which sterically impedes the ligand from being fully accommodated in the minor groove of DNA. This observation was confirmed by using an equivalent dodecamer containing inosine instead of guanine (5’-CCTAGI-3’), which has no exocyclic amino group protruding from the groove floor. This change was accompanied by a significant increase in binding affinity from a ∆G of -8.5 kcal mol-1 to -11.1 kcal mol-1. These observations support the conclusion that ligand structure is responsible for dictating the exact forces underling the complex formation and thus the thermodynamics of interaction.

5. Interpretation of the thermodynamic data The favorable enthalpy associated with distamycin and its analogues has been explained by the ability of the NH groups of amide links to form hydrogen bonds with the DNA bases on the groove floor in addition to van der Waals interactions between the aromatic rings of the ligand and the minor groove walls of DNA. There are two types of ligand-DNA base hydrogen bonds


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that may be critical to the sequence affinity and specificity exhibited by MGBs: hydrogen bonding between the imidazole/thiazole nitrogen and the 2-amino hydrogen of guanine; and hydrogen bonding between the ligand amide hydrogens and either the O2 oxygen of thymine, the O2 oxygen of cytosine, or the N3 nitrogen of adenine (Figure 2 & 8). Furthermore, the presence of water molecules trapped between the ligand and the binding site could facilitate hydrogen bond formation by enabling the ligand to interact indirectly with DNA base pairs, sugars or phosphates through a connecting water molecule. The favorable entropy associated with the monomeric association with of distamycin and netropsin is likely to be due to the release of counter ions and/or water from the minor grooves upon binding.5 There is also a minor conformational disruption to the DNA helix with monomeric binding, which also has less of an impact on entropy. A similar thermodynamic signature was observed with the hairpin polyamide. Its binding into the minor groove is primarily enthalpically driven (73 %) with a small contribution from entropy. The unfavorable entropy observed with the dimeric binding mode of distamycin and its analogues means that the association induced relatively unfavorable conformational changes in either the ligand or DNA. This conclusion is supported by several NMR

4, 9

and X-ray

8, 34

structural studies which showed ligand-induced widening of the minor groove and bending of the DNA helix toward the major groove resulting in major groove compression. Furthermore, the unfavorable entropy may also mean that the binding interface does not undergo complete desolvation upon binding. Binding into the minor groove as a dimer can potentially trap more water molecules between the ligand and the binding site and facilitate complex formation by bridging through hydrogen bonding. Trapping of these water molecules could also explain the unfavorable entropies. The X-ray crystal structure study conducted by Chenoweth et. al.8 19 ACS Paragon Plus Environment


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revealed the presence of four water-mediated hydrogen bonds anchoring the cyclic polyamide to the floor of the DNA minor groove. Moreover, the solvent-exposed surface of the polyamide ligand was hydrated by 22 water molecules which formed a network of hydrogen bonds across the carbonyl oxygens of adjacent amides linking the polyamide heterocyclic rings 8. The conjugated biaryl minor groove binders (e.g. Hoechst 33258, DB226, DB224 and DB75, Figure 1) recognize the DNA as monomers and there are no examples of dimeric association per

one site by this class of compound to date. Their binding into the minor groove of DNA is driven by large favorable entropy changes with small contributions from enthalpy (Table 2, Figure 4). The main forces that drive complex formation of these compounds are the hydrophobic forces, in addition to the electrostatic interactions between the positively charged functional groups of the ligand and the negatively charged sugar-phosphate backbone of the DNA. Favorable entropy is due to the release of water from the minor grooves upon binding, and the release of counterions from the backbone of DNA. The hydrophobic effect removes the ligand from the water phase into the binding site to reduce the unfavourable interactions between water and non-polar atoms. The hydrophobic effect happens mainly because non-polar atoms are unable to form hydrogen bonds with the polar solvent, and it is the inability to form hydrogen bonds with water, rather than the attractive forces (van der Waals) between non-polar groups that is responsible for the hydrophobic effect.49 Non-polar molecules aggregate together to minimize water-exposed accessible surface area, and water is forced to form structured cavities to accommodate these entities, which results in a loss of conformational entropy and an energetic penalty. The hydrophobic effect is an entropy-driven process, which allows reduction of the free energy of a system by minimizing the surface interface between non-polar molecules and aqueous solvent. This process is energetically favorable because the entropic cost of separating water and non20 ACS Paragon Plus Environment

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polar molecules is smaller than the entropic cost of ordering water molecules around large hydrophobic-water interfaces, which can lead to mixed non-polar solutes with water.49 This close association enables van der Waals interactions between the aromatic rings of the ligand and groove walls that make small enthalpic contributions to the overall binding event. Overall, there is a drive to move hydrophobic molecules from an aqueous environment to a less polar binding site in a macromolecule. Endothermic binding events arise also from electrostatic interactions i.e. entropically favourable release of counterions upon binding.2, 5 Electrostatic effects have long been recognised as one of the most important non-covalent attractive forces between charged molecules. Electrostatic interactions between protonated ligands and the negatively charged DNA backbone play an important role in stabilizing ligand-DNA complexes. Detailed analysis of the electrostatic contribution to the free energy of binding has been accomplished for various ligand-DNA,38, 50-51 protein-DNA,52 and protein–protein complexes.53

6. Enthalpy, entropy and MGB drug design Determination of the thermodynamic parameters that drive the binding of small organic molecules into the minor groove of DNA gives invaluable insight into the understanding of ligand–DNA interactions. The enthalpy changes (∆H) relate directly to the heat of interaction between the ligand and its target in the bound and unbound states. This change in enthalpy reflects the total contribution from the formation or removal of non-covalent forces in the system upon binding.54 In ligand-DNA interactions, hydrogen bonding and van der Waals forces are usually associated with an exothermic favorable (negative) enthalpy changes, while the hydrophobic and electrostatic interactions (polyelectrolyte effect), which are manifested



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entropically, are associated with either small positive or negative enthalpy and a favorable (positive) entropy.2, 5 The entropic term represents the change in the order of the system. This incorporates changes in conformational entropy (a gain/decrease in the conformational freedom of certain groups of the ligand or DNA) as well as desolvation entropy (the release of water and counter ions from the minor grooves upon ligand binding). A positive T∆S results in entropically favoured binding- i.e. the system becomes more disordered. Favourable entropy changes are mainly caused by the hydrophobic effect, where an increase in solvent entropy is generated by the removal of nonpolar hydrophobic groups from the aqueous environment and the release of water upon binding. The electrostatic interactions, for example those associated with the release of counterions from the backbone of DNA upon binding, also contribute to the positive T∆S.2,

5

The binding of a

ligand to a macromolecule is usually associated with a decrease in conformational entropy as the degrees of freedom of both the ligand and the macromolecule are restricted upon binding. This decrease in entropy disfavors the interaction between the ligand and its target, but is often partially or completely offset by the release of water molecules from the hydrophobic surfaces of both the ligand and the binding site. Upon ligand binding to the macromolecule, both the ligand and the binding site are completely or partially desolvated. The hydrophobic effect is usually associated with a desolvation of the binding interface, whereas the hydrophilic interactions (i.e. hydrogen bonding), sometimes involve water molecules being trapped between the ligand and the binding site and help complex formation through a bridging effect. 45-48 From the standpoint of rational drug design, the determination of these important thermodynamic parameters (∆H & ∆S) for the binding of a ligand to its target can give a clearer understanding of the important attributes of binding. ITC is widely used nowadays in the field of drug design and 22 ACS Paragon Plus Environment

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optimization to understand the principles of molecular recognition. The establishment of a link between structure and energy can lead to real progress in understanding the mechanisms of molecular associations and help reveal the important structural features that drive the binding event. Introducing a moiety with high enthalpic potential such as one capable of forming hydrogen bonds and van der Waals forces within the binding site is expected to improve binding. However, there are some drawbacks; enthalpy/entropy compensation is an ubiquitous phenomenon, which results from the nature of non-covalent interactions. Enthalpy-entropy compensation analysis (Figure 5) shows that the MGB interactions with high enthalpy (red circles) or high entropy (red stars) contribution to ∆G are associated with entropic or enthalpic penalty respectively. Whereas the interactions which are driven by both enthalpy and entropy (filled squares), both have shown moderate contribution to the free energy of binding. These data illustrate the hurdle which enthalpy/entropy compensation poses to the optimization of binding affinity. This mainly happens because enthalpy and entropy are driven by opposing forces. Mathematically, this is due to the Gibbs free energy equation (∆G = ∆H – T∆S): as the enthalpy changes (∆H) and the entropy changes (∆S) have opposite signs, when both terms are increased, there will be very little change in the free energy of binding (∆G). Increases in bonding are often offset by an entropic penalty, reducing the magnitude of the change in affinity. For instance, the enthalpic gain from a hydrogen bond can be counteracted by conformational entropic losses from immobilization of the constituent groups. Another problem is the flexibility of some binding sites, which can become restricted on binding a ligand and result in a conformational entropic penalty. The balance between enthalpic and entropic contributions to MGB binding has been the subject of extensive research5,

18, 27

and appears to vary with both the MGB structure and the

binding sequence of the DNA. Another major factor is the role of water. X-ray studies of DNA 23 ACS Paragon Plus Environment


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Figure 5: Enthalpy-entropy compensation plot for the MGB binding data. The open circles represent enthalpically driven interactions. The filled squares represent interactions that are driven by both enthalpy and entropy. The filled stars represent entropically driven interactions. The red-colored circles and stars represent interactions associated with entropic and enthalpic penalty respectively. The straight line represents a linear least square fit showing a strong negative correlation between ∆H and -T∆S (R2 = 0.96875). have revealed a highly structured spine of water molecules in the minor groove.55-56 Displacement of these water molecules by MGBs leads to significant entropic gains which are considered a major driving force in binding. However, in certain complexes water molecules play a vital role in facilitating ligand-DNA association by bridging between nucleic acid and


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ligand structure through hydrogen bonding and that’s mainly dependent on the ligand structure which dictate the molecular forces that drive the interaction. Results obtained from determination of the thermodynamic profile of ligand-DNA associations can be used to reveal the molecular forces that drive the interaction, conduct structure-activity relationship studies, and optimize target interactions by improving binding affinity, selectivity, and the physicochemical properties of lead compounds. Isothermal titration calorimetry (ITC) which generates a complete thermodynamic profile for the interaction is an important biophysical tool that can be used as a guide during these stages of drug development and optimization.

The structures of the anti-infective compounds, netropsin and distamycin have served as the major leads for molecular design of novel anticancer and antibacterial drugs. The major aims for the structural modification strategy of these compounds were to achieve selectivity toward different DNA bases, reduce toxicity and improve the binding affinity & physicochemical properties of lead structures. The rationale of MGBs drug design along with their thermodynamic profiles and mechanism of action in targeting various diseases will be discussed in detail in the following sections. 6.1 Design of transcription factors antagonist Transcription factors are DNA binding proteins that regulate the transcription of specific genes into mRNA which in turn is translated into protein synthesis.57-58 Malfunction of transcription factors leads to a variety of human diseases such as cancer59-62 and inflammation;63-65 they are therefore important therapeutic targets. The activity of several transcription factors can be antagonized by kinase and phosphatase inhibitors targeting a cell signaling pathway involved in 25 ACS Paragon Plus Environment


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the transcription process. However, inhibition of an upstream kinase or phosphatase enzyme is not anticipated to selectively inhibit a specific transcription factor activity and this may lead to non-specific side effects.66 On the contrary, direct antagonism of transcription factors is expected to generate more profound therapeutic outcomes with minimal off-target side effects compared to the indirect approaches.48, 55-56 Direct inhibition of transcription factors by small molecules can be achieved by the disruption of protein–protein associations,67 protein–DNA interactions68 or by allosteric modulation of protein-DNA interfaces11,

69

(Figure 6). However, the disruption of

protein–protein or protein–DNA interactions involved in the transcriptional process is a highly challenging task since the binding interfaces are large and not well defined due to the fact that proteins are not always correctly folded prior to dimerization or DNA binding in protein-protein or protein-DNA interactions respectively.70-71 Moreover, it is difficult for such small molecules to compete effectively on the binding site and overcome the large free energy of binding between protein-protein and protein-DNA interfaces.68,

72

Alternatively, the non-competitive allosteric

modulation of transcription factors by DNA minor groove binders (MGBs) would likely afford a more therapeutically viable approach. These small molecules, which bind selectively to the minor grove of DNA in a sequence-specific fashion, can cause significant structural allosteric perturbations of the DNA helix resulting in major groove compression which makes the transcription factors unable to recognize their target in the major grooves (Figure 7).11, 73 The ligand induced structural perturbation of the DNA helix, was reflected on the thermodynamic signature of amide-linked MGBs, which were associated with a conformational entropic penalty.4,

9, 74

The loss of entropy during ligand-DNA association reflects the conformational

changes in either or both species in order to achieve better hydrogen bond/van der Waals contacts between the ligand and the DNA duplex (induced fit). Large negative unfavorable 26 ACS Paragon Plus Environment

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Figure 6: Possible small-molecule targets for the disruption of transcription factor activity. As an example, the androgen receptor-DNA complex (PDB ID: 1R4I) is shown (red & blue = androgen receptor; orange = DNA). Figure was developed using PyMOL.75

A)

B)

Figure 7: Representation of the NMR refined solution structure (PDB ID: 2MNF) of the complex between AIK18-51 (CPK) and d(CGACTAGTCG)2 (stick and tubes) showing (A) the ligand-induced widening of the minor groove and (B) bending of the DNA helix toward the major groove resulting in major groove compression. Figure was developed using PyMOL.75



entropy changes are often indicative of an “induced fit” interaction.19,

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76-77

Moreover, the

association itself is also expected to constrain the complex structure and to incur an entropic penalty due to the losses in the rotational and translational degrees of freedom when two molecules are bound together to form a complex.15 Such induced fit and allosteric regulation are common phenomena in regulating biological processes.78-79According to the “central dogma of molecular biology”, nucleic acids alone can specify the sequence of protein products, while proteins cannot specify a particular nucleic acid or protein sequence.80 Interference at the DNA level is therefore the highest level of target control and can provide efficient ways of switching on/off the synthesis of protein products of interest. The number of genes present in a cell is far fewer than the corresponding mRNAs or protein products, and therefore theoretically requires much smaller quantities of compound to target the DNA. For these reasons, DNA is considered a primary target for binding or chemical modification by several classes of molecules,68, 81 which can be isolated from natural sources or synthetically prepared. The structural simplicity of such molecules, combined with facile synthesis, has accelerated efforts directed toward understanding the relationships between molecular structure and DNA recognition.22, 81 The design of sequencespecific DNA-binding molecules was inspired by the natural products netropsin and distamycin A, whose structures served as the major template for molecular design of novel MGBs as transcription factor antagonists. The main aim of the structural modification strategy of these leads was to achieve selectivity toward different DNA bases by allowing the ligand to discriminate one Watson-Crick base pair from the other three combinations. Selectivity for G:C82-83 base pair over A:T was achieved by introducing N-methylimidazole/or thiazole as a heterocyclic monomer; this removes the steric clash with the N-2 of guanine and replaces it with a new hydrogen bond to the proton on N-2 of guanine (Figure 8A). Distamycin showed a 28 ACS Paragon Plus Environment

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Figure 8: A) Illustration of Imidazole binding a G of a G:C base pair, and the steric clash which results with pyrrole B) Specific recognition of different DNA bases in the minor groove by a hairpin structure via hydrogen bonding. Circles with dots: lone pairs of N(3) and O(2) of pyrimidines, circles containing a H: the 2-amino group of guanine, dashed lines: hydrogen bonds, Py: N-methylpyrrole, Im: N-methylimidzole, and Hp : 3-hydroxy-N-methylpyrrole.

significant property of binding in a 2:1 complex with DNA. Each molecule binds to a single strand of DNA (Figure 2), this allows potentially for more subtle sequence discrimination which could distinguish G:C from C:G and A:T from T:A base pairs. This work largely performed by Dervan et al, led to a set of pairing rules for minor groove recognition (summarized in Figure 8B).37,

67, 72, 84

The N-methylimidazole/N-methylpyrrole pairs distinguish G:C base pair from

C:G whereas the selectivity for T:A base pair over A:T was achieved by introducing a new heterocycle, 3-hydroxy-N-methylpyrrole, which forms a specific hydrogen bond between the 3OH group and the O-2 of thymine. 29 ACS Paragon Plus Environment


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The N-methylpyrrole / N-methylpyrrole pair is degenerate and binds to either an T:A or A:T base pair.81,

85

The concept was exploited further by Dervan et. al. who developed hairpin

linked86 and cyclic dimers8 (Figure 9 ). These compounds benefit from the reduced number of possible conformations compared to the unlinked distamycin dimer and this led to a significant improvement in binding affinity owing to a high increase in the binding enthalpy.14 This new class of polyamides were designed by covalently linking the terminal amine and carboxylic acid groups with γ-aminobutyric acid (γ-turn) to generate antiparallel side-by-side hairpin linked dimers. Incorporation of γ-turn is important for keeping the heterocyclic units unambiguously paired and avoiding the “slipped” binding modes, observed with unlinked dimers, when targeting a specific binding site.59, 87-88 Furthermore, the γ-turn recognizes A:T/T:A base pairs and helps to maintain the active confirmation of hairpin linked dimers, which markedly enhances binding affinity and sequence specificity. Incorporation of β-alanine between two imidazole rings in place of one of the heterocyclic units was found to be useful in resetting the curvature of hairpin structure to better match the helical shape of DNA minor grooves, which significantly improves the binding affinity specially when the number of aromatic rings in the hairpin dimer is increased (Figure 9).59, 75, 87, 89 It seems that the flexibility afforded by the β-alanine (β) moiety allows a proper orientation for the two flanking imidazole rings (Im-β-Im) to form hydrogen bonds with the exocyclic amine of G, whereas for the Im-β-Py subunit, only one Im-G hydrogen bond is formed, thus the flexibility generated by β-alanine in this subunit is not crucial.90 These findings lead to the development of new hairpin polyamide scaffold in which the antiparallel pairing of imidazole opposite to βalanine recognizes a G:C base pair, and β-alanine /imidazole pair recognizes C:G, whereas 30 ACS Paragon Plus Environment

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γ-turn β-alanine

O N

O

NH

N

O

N

N H N

N N

O O

N H

O

N

H N

N

O N

O

H N

O

O N

O

N

H N

N

N

H N

N

PA11

O

O

HN

H N

H N

N

N

KA1007

NH

H N

NH3 N H

N

H N

N

N H H N

N

H N

O O

NH

O

O N O

N O

Recognizes 5’-WGGCWW-3’

HN

Recognizes 5’-WGCGCW-3’

O NH O

O

N

NH

O N N

N H

O N Cyclic PA

N H

O

N

O

N

O N

ARE1

O

H N H N

O

O N O

O N O

NH

Recognizes 5’-WGWWCW-3’

N

N

O

N

O

H N

H N

N

O O

HN

N H

H N N

N H

O

N

N

H3N

N

NH3 N H

O

IPA

N

H N

NH

O

H N

H N

H N

N

N H

NH3 N H

O

N

O

(androgen response element)

Recognizes 5’-WGWWCW-3’

HN

O O N

N

NH

O N

N H

O N

H N

N H N

O O

N N

O

NH

O HN

Compd. 1

H N

O

O

H N H N

N O

O N O

N O

N

O

O

H N

H N

N

N H N

N

N H N

N H

O O N

NH3 N H

O

H N

H N

N

N H

N

N

N H

O N

ERE1

NH

O NH3

O N O

NH O

Recognizes 5’-WGGWCW-3’ (estrogen response element)

HN

Recognizes 5’-WGGWWW-3’

(NF-κB response element)

KA1039 NH

H N

O

N

O

N

N H

N

HN

H N

H N O

N

O

N

O

N

O

N

NH

NH

N

O

N

H N O

Recognizes 5’-WGGCW-3’

Figure 9: Structure of some minor groove binders developed as potential transcription factor antagonists. (filled circles = Im; unfilled circles = Py; diamond = β-alanine; half-circle with a plus = the dimethylaminopropylamide tail; semicircle = the achiral γ-aminobutyric acid turn; semicircle linked to a half-circle with a plus = the chiral γ-aminobutyric acid turn). 31 ACS Paragon Plus Environment


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pyrrole/ β- alanine, β-alanine /pyrrole and β-alanine/ β-alanine combinations are degenerate and recognize either a T:A or A:T base pairs.88, 91 These β-alanine /aromatic ring pairings facilitated the design of novel hairpin molecules capable of targeting large, diverse and challenging binding sites, such as the 5‘-GCGC-3‘sequence (e.g. PA11, Figure 9).39 However, although the incorporation of β-alanine in the hairpin structure relaxes its curvature and allows for optimal hydrogen bond formation with the DNA bases, some studies reported a significant decrease in the binding affinity when a β-alanine moiety replaces a pyrrole or an imidazole ring, specially when the substitution is on the N-terminal side of the γ-aminobutyric acid (GABA) turn 92-93 (e.g. KA1007,94 Figure 9). These observations clearly indicate that the influence of β-alanine substitution on the binding affinity is largely affected by the substitution placement. Although the flexibility of β-alanine residues might allow the hairpin dimer to form better contacts with the minor groove of DNA, it might also force the ligand to adopt multiple inactive conformations, leading to a large decrease in binding affinity and sequence specificity, however, that is mainly dependent on the position of β-substitution and the target DNA sequence.

Addition of amino group on the α or β position of γ-turn has shown to change the orientation of hairpin dimer in the minor groove of DNA with respect to 5′-3′ ends. There are two possible orientations; either the N-C terminus of the ligand is aligned in the 5′-3′ or 3′-5′ direction of the DNA (Figure 10). Hairpin dimers with α-amino substitution on γ-turn and flexible β residues between the heterocyclic units showed preference for the 5′-3′ orientation (forward), in which the N-terminus of imidazole is lying toward the 5′ end of the binding site,90 while β-containing flexible hairpin dimers with β-amino substitution on γ-turn prefer a 3′-5′ orientation (reverse),


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Figure 10: Representation of the binding orientations adopted by the conformationally flexible, β- containing hairpin dimers. A) Hairpin dimers with α-amino substitution on the γ-turn prefer the forward orientation; the ligand N-C terminus aligned with 5′-3′ and recognizes the GCGC sequence B) Hairpin dimers with β-amino substitution on the γ-turn prefer the reverse orientation; the ligand N-C terminus aligned with 3′-5′ and recognizes the CGCG sequence. (filled circles = Im; unfilled circles = Py; diamond = β-alanine; half-circle with a plus = the dimethylaminopropylamide tail; semicircle linked to a half-circle with a plus = the chiral amino substituted γ-aminobutyric acid turn).

in which the N-terminus of imidazole points toward the 3′ end of the binding site.93 On the contrary to the structurally flexible β-containing hairpin dimers, the relatively rigid fully ringpaired dimers prefer a 5′-3′ orientation (forward) regardless of the amino group position on the γ-turn.92 The incorporation of isophthalic acid (IPA) (Figure 9) at the C-terminus of hairpin dimers improved their biological activity and cell permeability.88 Thorough investigations on the effect of C-terminus modification confirmed the role of the IPA group in enhancing the cellular uptake of hairpin dimers without compromising the binding affinity and sequence selectivity.59, 91



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Isophthalic acid substitution on the C-terminus exhibited a biological effect comparable to that observed for the fluorescein-labeled hairpin dimers in cervical (HeLa) and glioma (U251) cancer cells.88 These findings indicate the importance of the carboxylic acid group on the phenyl ring, which resembles the carboxylic acid and alcohol groups of fluorescein, in enhancing cellular uptake and nuclear localization. Hairpin dimers have been shown to exhibit high binding affinities similar to those of endogenous DNA-binding transcription factors.37,

74, 95-96

Comparison of the enthalpy and entropy

contribution to the free energy of binding shows that the hairpin-DNA association is dominated by favourable binding enthalpy,14,

94

(e.g KA1039,94 Figure 9) which mainly stems from the

formation of hydrogen bonds between the ligand amide groups and the DNA bases, in addition to van der Waals interactions between the heterocycles of the ligand and the minor groove walls of DNA. Replacing an amide group of MGB binding ligand with an alkene isostere was found to be detrimental to the biological as this modification eliminates a potential hydrogen bonding group, which is in part responsible for sequence recognition and binding affinity. These findings are consistent with the thermodynamic signature of the amide-linked distamycin analogues.

Based on these insights obtained from thorough structure-activity-relationship studies, many sequence-specific DNA-binding molecules were designed to disrupt protein-DNA interactions and regulate gene expression. This strategy has been extensively used by Professor Dervan whose work has generated many compounds with potential application as transcription factor antagonists e.g. inhibition of the NF-κB transcription factors57 by targeting 5’-WGGWWW-3’ (W is A or T), inhibition of hypoxia-inducible factor91 (HIF-1) by targeting 5’-WWWCGW-3’, inhibition of estrogen receptor58 by targeting 5’-WGGWCW-3’ and inhibition of androgen 34 ACS Paragon Plus Environment

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receptor transcription59 by targeting 5’-WGWWCW-3’. The later plays a key role in prostate cancer progression. For example, ARE1 (Figure 9) is a pyrrole-imidazole polyamide designed to selectively target the consensus androgen response element (ARE) half-site (5’-WGWWCW3’).12 Malfunction of androgen receptor (AR) can lead to dysregulated gene expression and might cause prostate cancer. Prostate specific antigen (PSA) gene which is commonly used as a marker for the diagnosis of prostate cancer is regulated through the interaction of AR with the androgen response element (ARE) of the PSA gene.97 The AR-ARE interaction can be disrupted via the hairpin and cyclic dimers targeting the androgen response element (ARE) [e.g. ARE112 and Cyclic PA,8, 98 (Figure 9)]. Recent data from Dervan’s laboratory have shown that the eightrings hairpin dimer (ARE1), which binds to the ARE, is able to regulate the expression of AR target genes in cell culture studies.12 This compound is currently being developed by Gene Sciences, Inc. for the treatment enzalutamide-resistant prostate cancer.12 Another example is the inhibition of the NF-κB transcription factors.57 NF-κB is a heterodimeric transcription factor which plays a crucial role in cell proliferation and inflammatory response in conditions such as inflammatory bowel disease, asthma and arthritis.99. NF-κB exists in the cytoplasm in its inactive form bound to an inhibitory protein of the IκB family. NF-κB is activated by IκB phosphorylation, the complex is then degraded to liberate the active NF-κB which subsequently translocates to the nuclei and binds to the regulatory regions of target genes in the major groove (termed κB sites).100 The work of Dervan's research group has involved the synthesis of hairpin dimers which bind to a predetermined DNA sequence which overlap parts of the NF-κB binding site 5′- GGGRNYYYCC-3′, (where R: purine, N : any base, Y: pyrimidine). The results of this work have shown that hairpin dimer (Compd. 1, Figure 9) which



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bind the 5’-WGGWWW-3’ (where W is A or T) portion of the binding site inhibit NF-κB binding in the major groove.57

6.2 Design of minor groove binding alkylating agents Alkylating agents are the oldest class of drugs that have been used for the treatment of cancer. Chemically, these compounds contain electrophilic moieties that can easily react with the nucleophilic sites of DNA or proteins resulting in the formation of permanent covalent bonds, which cause mutations, DNA damage and cell death.101-102 The chemotherapeutic agents, nitrogen mustards and cis-platin are examples of DNA alkylating molecules, (Figure 11) which bind covalently to DNA primarily in the major grooves. However, although these compounds are toxic and have many adverse side effects due to their lack of specificity, they still play a major role in the treatment of certain types of refractory cancers. Tremendous efforts have therefore been made to enhance the specificity and safety of DNA alkylating agents. One strategy that has been attempted to enhance specificity is to attach an alkylating functional group (e.g. nitrogen mustard) to the N-terminus of the MGB structure (Figure 11). Attaching these nitrogen mustards to the MGB structure has been shown to give improved specificity.60-61 One such example is tallimustine which contains a β-chloroethylamine mustard alkylating group.62 Nitrogen mustards are not the only class of alkylating groups which have been incorporated into MGBs, further studies have shown that brostallicin, which contains a latent alkylating bromoamide group, is activated by glutathione within the target cell and benefited from reduced toxicity.63 Brostallicin has reached phase 2 clinical trials for the treatment of soft tissue sarcoma and further development is continuing.64-65


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Nitrogen mustards

Platinum NH3

Cl

Cl

N

Cl

N

Pt NH3

Cl

Cl

Cl Br

H2N

Cl

O Cl

N

NH 2

H 2N

NH2

NH

NH

HN Tallimustine H N

HN

H N

O N

O

O N H

KR12 H N

HN N H

HN

O

N H

O

H N

N

N

O

N

HO

O

O

N

N H

N

HN

NH

O

O

N N H

N

O

Brostallicin

N

O

N

O

O

N

O

N

HN

N

O

N

HN

N

N H

N H N

H N N

O

N

N

O

O

N O

NH H N

NH

N

O

N

O

O

Cl SJG-136 N

O

Duocarmycin

O

O

N

O

O N

O

N

O

O

O O

O

N

Val-Ala dipeptide linker h1F6

O O S

O

N

HN

N O

N H

H N O

N

O

O

O

O

O

O N H

N H

O

N N O

PBD-dimer

O

Figure 11: Structure of some minor groove binding alkylating agents developed as potential anticancer agents.



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Many derivatives of pyrrole-imidazole polyamide (hairpin) with potent anti-tumor activity have also been developed by adding alkylating groups to the C-terminus of the polyamide, an example is the KR12103 (Figure 11). The hairpin dimer (KR12), which is coupled with a seco-CBI alkylating group, was designed to selectively target the oncogenic codon 12 KRAS mutations which are considered the most common mutations in metastatic colon cancer.104 KR12 form covalent bonds with the N3 of adenine bases, resulting in DNA strand cleavage, suppression of colon cancer growth and apoptosis.103

There are several other minor groove binders with alkylating groups which do not contain the pyrrole-polyamide scaffold such as the natural product duocarmycin (Figure 11) and its derivatives.105 The duocarmycins possess a spirocyclopropylquinone which is enclosed in a heterocyclic unit, and has a crescent shape suitable for DNA minor groove binding. The key feature responsible for its biological activity is the cyclopropane ring, which is opened via a nucleophilic attack of the N-3 of adenine or the amino group of guanine to give the alkylated complex, in a reaction that is thermodynamically driven by the aromatization of the diene-one cycle to generate a phenolic ring.106 Additional examples of these minor groove binding alkylating are the pyrrolobenzodiazepines, (PBD) e.g. anthramycin and its derivatives. For example, SJG136 (Figure 11) is a dimer of the pyrrolobenzodiazepine designed to cross link the DNA target through its electrophilic imine group of the diazepine ring, which reacts with the exocyclic amino group (C2-NH2) of guanine


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bases, the reaction therefore happens mainly at the GC tracts of DNA.65 SJG136 has reached phase 1 clinical trials for the treatment of solid tumours.66 Furthermore, recently a PBD dimer has been conjugated to a monoclonal antibody, anti-CD70 h1F6 mAb, by a valine-alanine dipeptide linker. This linker was used as it is known to be stable in blood circulation, but can be easily cleaved in the target cells via protease enzymes. The humanized monoclonal antibody (h1F6 mAb) is specific for targeting the CD70 cancer antigen.107 CD70 protein is highly expressed in both non-Hodgkin lymphoma and renal cell carcinoma,108 however, its expression in normal cells is limited.109 The strategy of coupling the monoclonal antibody h1F6 to the cytotoxic DNA cross-linking PBD dimer aims therefore to provide more potent antitumor effects by enhancing drug specificity for CD70-positive nonHodgkin lymphoma and renal cell carcinoma. In vitro assay results showed that the h1F6 -PBD conjugate was effective and immunologically selective against non-Hodgkin lymphoma and renal cell carcinoma cell lines.110 The alkylation of the guanine amino groups with different PBD compounds was found to proceed in a slow rate.111-112 For PBD compounds, minor groove recognition via non-covalent interactions takes place prior to the relatively slow alkylation reaction. In order to accurately determine the non-covalent thermodynamic profile of PBDs, and to separate the effects of covalent bonding with the guanine amino groups, equivalent DNA duplexes containing inosine instead of guanine, which has no exocyclic amino group protruding from the groove floor, were used for the thermodynamic characterization of PBD complex formation. ITC studies113 showed that the PBD-DNA interactions are mainly enthalpically driven via hydrogen bonding and van der Waals forces, and the unfavorable entropies associated with these interactions are indicative



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of “induced fit” binding. These results are largely consistent with the ITC studies of amide linked MGBs (e.g. distamycin analogues).

6.3 Design of MGBs as potential antibacterial agents Tremendous efforts have been made to improve the anti-bacterial activity of polypyrrole MGBs. Many research groups were involved in the synthesis distamycin analogues as potential antibacterial agents.73, 82-83, 86, 114-115 These compounds were developed based on the molecular structure of the natural product distamycin A, which has been shown to exhibit antiviral and antibacterial activity.116 Distamycin and its analogues prefer to target A/T rich sequences within bacterial DNA close to the promoter regions which inhibit DNA replication and RNA transcription leading to bacterial cell death.117-119 GeneSoft Inc developed the earliest group of distamycin derivatives, for example, GSQ1530 (Figure 12) was able to inhibit various staphylococci including S. aureus, S. haemolyticus and S. epidermidis.82 The results of in vitro and in vivo assays of Gensoft’s compounds have shown to have potent activities against a broad spectrum of gram-positive bacteria. One molecule in particular, GSQ10547, was the most active compound both in in vitro and in vivo assays against MRSA peritonitis and neutropenic thigh infection model.114 Several other studies showed that the pyrrole tetraamides with two cationic guanidine groups (Figure 12) can inhibit both MRSA and vancomycin-resistant enterococci.83 The benzophenone-containing compounds (e.g. compound 1, Figure 12) developed at Wayne State University showed a very good activity against S. aureus and were as affective as vancomycin in the treatment of S. aureus infections.120


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O Cl

O

N

N

N

S GSQ1530

Cl

GSQ10547

HN

N

NH O H N

N

HN

H N

O

HN

HN

N

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NH2

O

O

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H N

H2N

HN

NH NH

Pyrrole tetramide

1

O

N

NH

HN

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H N

NH

O N

O

N

HN

N

O

H N O

N

O

HN N

O

O

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N

O

N

MGB-BP-3

HN NH

2

O HN H N

O

HN O

N O

N N

H N

O HN

N

N

O

O

Figure 12: Structure of some distamycin analogues developed as potential antibacterial agents.

The Strathclyde research group led by Professor Suckling has been engaged in the synthesis of distamycin analogues as potential antibacterial agents.115 The

main strategy that has been

followed is to enhance the antibacterial activity of these compounds by varying their lipophilicity, which led to enhanced hydrophobic interactions within the groove of DNA and thereby improved both antibacterial activity and uptake by the bacterial cells.

The main

modifications were made at the head group of distamycin structure (polar formyl group) which was altered to a range of more lipophilic head moieties (e.g. quinoline, benzene and pyridine) giving the enhanced lipophilicity required. The polar amide links between each heterocyclic monomer at the terminal positions were also modified to a range of more lipophilic linkers (e.g. 41 ACS Paragon Plus Environment


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alkene), while the amide links in the middle of structure were kept intact as they play a key role in both binding with DNA and keeping the crescent shape of these molecules, which is required to match the helical geometry in the minor groove of duplex DNA. Another major modification was the variation of the heterocyclic monomers by either varying the ring system, or its alkyl substituent by replacing methyl groups by larger alkyl groups (e.g. N-isopentylpyrrole, isopropylthiazaole, furan & thiophene). The polar amidine group of distamycin was also replaced with less polar groups having a range of pKas, to vary the degree of protonation at physiological pH, e.g. ethylmorpholine substituent has been linked to enhanced biological activity. As a result of applying this strategy, several compounds displayed significant activity against drug-resistant bacteria including MRSA and Clostridium difficile. For example compound 2 (Figure 12) had a significant MIC's for MRSA of 2.0 µM (amoxicillin:16.1 µM) and MGB-BP3 showed excellent activity against Clostridium difficile strains (MIC of 1 µg/mL) compared to vancomycin (MIC of 1 µg/mL).121 MGB-BP-3 is a successful example for a compound designed and synthesized in Professor Sucking’s laboratory using the strategy of enhanced lipophilicity for distamycin template structure. MGB-BP-3 is the first DNA minor groove binder that reached phase 2 clinical trials in humans and is currently being developed by MGB Biopharma for the treatment of Clostridium difficile infections.11

In the discovery and development of these compounds extensive use of biophysical techniques, such as isothermal titration calorimetry (ITC) and NMR spectroscopy was made to probe the binding affinity, sequence selectivity and the thermodynamic profile of these compounds with their DNA targets.4, 9, 122-123 These studies clearly revealed the relationship between the binding affinity, sequence selectivity and antibacterial activity, and showed the importance of using such 42 ACS Paragon Plus Environment

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biophysical techniques in guiding the medicinal chemistry optimization programme of lead compounds.

7. Conclusions Overall, this review provided clear evidences that the ligand structural heterogeneity is mainly responsible for dictating the thermodynamics of MGBS. The binding sites in the minor groove of DNA can provide all types non-covalent interactions which can enhance either enthalpy or entropy of binding. However, ligand structure is responsible for dictating the exact non-covalent forces that drive the association process and thereby it will determine whether the reaction is enthalpically or entropically driven. Thermodynamics of minor groove recognition is dependent on the molecular forces underlying the complex formation, and these forces are dictated by ligand structure. The analyzed data revealed that the interactions of amide-linked MGBs with DNA are mainly driven by enthalpy through hydrogen bonding and van der Waals forces, whereas the interactions of conjugated biaryl MGBs are mainly entropically driven via hydrophobic and electrostatic forces. Increasing the lipophilicity of MGBs improved both the transportation properties of these molecules and their antibacterial activity. However, although the hairpin structures developed by Dervan group are large and polar molecules, studies have shown that these molecules can cross biological membranes by poorly understood means and enter the nucleus where they disrupt transcription factors-DNA interaction.69-70 The hurdle which enthalpy/entropy compensation poses to the optimization of binding affinity cannot be avoided due to the intrinsic nature of both enthalpy and entropy which are driven by opposing forces. However, optimization of binding affinity requires first to determine whether the interaction is driven by enthalpy or entropy, ligand structural modifications will be then carried out to increase 43 ACS Paragon Plus Environment


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the number of non-covalent interactions with target to enhance enthalpy if the association process is mainly driven by enthalpy, or to enhance entropy if the complexation is mainly driven by entropy. This process requires also structural details about the complex, which can be obtained using other biophysical techniques e.g. NMR spectroscopy and x-ray crystallography, in addition to the molecular dynamic simulations methods which can provide more insight into the mechanism of binding, however, these are beyond the scope of this review. This review highlighted the advantages of developing MGBs as new class of antibacterial and anticancer agents, in particular, as selective transcription factor antagonists with special focus on their chemical structure, mechanism of action and the thermodynamic signature. This review also emphasized the advantages of using a biophysical tool such as ITC in directing the medicinal chemistry optimization programme of lead compounds. In the discovery of many novel MGBs that have reached clinical trials, extensive use of ITC was made to directly measure the binding profile of these molecules with short oligonucleotide sequences. Analysis of the resultant thermodynamic data and the dissection of the thermodynamic contributions to MGB binding helped to reveal the factors that drive minor groove recognition particularly the molecular forces and structural features that dictate the overall binding process and provided invaluable information for the optimization of binding affinity, selectivity and physicochemical properties of novel MGBs.

Ancillary Information Author information Corresponding Author: Hasan Alniss: phone, +(971)-6-5057427, fax, +(971)-6-5585812, email, [email protected]. ORCID: 0000-0001-8639-9531 44 ACS Paragon Plus Environment

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Acknowledgments This work was supported by a grant from Boehringer Ingelheim and the University of Sharjah Seed Grant (No.; 1601110113-P). Abreviations used MGBs, minor groove binders; ITC, isothermal titration calorimetry; ∆G, Gibbs free energy; ∆H, enthalpy changes; T∆S, entropy changes; K, binding constant; PIPES, 1,4-piperazinediethanesulfonic acid; ACES, 2-(carbamoylmethylamino)ethanesulfonic acid;

MES, 2-morpholin-4-

ylethanesulfonic acid; A, adenine; T, thymine; G, guanine; C, cytocine; ARE, androgen response element; ERE, estrogen response element; NF-κB, nuclear factor kappa-light-chainenhancer of activated B cells; HIF, hypoxia-inducible factor; Py, N-methylpyrrole; Im, Nmethylimidzole; Hp, 3-hydroxy-N-methylpyrrole; PBD, pyrrolobenzodiazepines; MRSA, methicillin-resistant Staphylococcus aureus; seco-CBI, 1-(chloromethyl)-5-hydroxy-1 ,2-dihydro -3H-benz[e]indole; IPA, isophthalic acid; GABA, gamma-Aminobutyric acid; mAb, monoclonal antibodies, CD70, cluster of differentiation 70.

Biography Hasan Alniss received his Ph.D. in Medicinal Chemistry (2011) from the University of Strathclyde-UK. In 2011, he was appointed Assistant Professor of Medicinal Chemistry at AnNajah National University (Palestine). In 2013, he was granted the Distinguished Scholar Award and joined the University of Toronto as a Visiting Professor in the Leslie Dan Faculty of Pharmacy, where he worked with Prof. Robert Macgregor on a project to characterize the factors that stabilize the G-quadruplex structures of nucleic acids. In 2015, Dr. Alniss took up his current 45 ACS Paragon Plus Environment


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position at the University of Sharjah (UAE) as an Assistant Professor of Medicinal Chemistry. In terms of research, his primary interest is cancer drug discovery and the biophysical characterization of nucleic acid structures and their complexes with drugs.

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Figure 13: Graphical abstract


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Figure 13: Graphical abstract

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Thermodynamics of DNA Minor Groove Binders - Journal of Medicinal

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