Story of a Bisbenzimidazole Dye Fragment - ACS Publications

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Impact of Linker Length and Composition on Fragment Binding and Cell Permeation: Story of a Bisbenzimidazole Dye Fragment Nihar Ranjan, Patrick Kellish, Ada King, and Dev Priya Arya Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00929 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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Biochemistry

Impact of Linker Length and Composition on Fragment Binding and Cell Permeation: Story of a Bisbenzimidazole Dye Fragment. Nihar Ranjana, Patrick Kellisha, Ada Kingb and Dev P. Aryaa,b* a

Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, South Carolina (United States) 29634

b

NUBAD LLC, 900 B West Faris Road, Greenville, SC 29605

Corresponding author email address: [email protected]

Contact address: 461, Howard L. Hunter Chemistry Laboratories, Department of Chemistry, Clemson University, Clemson, South Carolina, United States 29634

Phone: +1-864-656-1106 Fax: +1-864-656-6613

KEYWORDS: Hoechst 33258, Linkers, DNA Binding, Minor groove, Isothermal Calorimetry

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Biochemistry

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Abstract:

Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common discovery platform-fragment based drug discovery, two fragments are identified that bind to adjacent sites on a target, and are then linked together using different linkers to identify the linkage for optimum activity. What is not known from these studies is the effects these linkers, that typically contain C, H, and O atoms, have on the properties of the individual fragment. Herein, we investigate such effects in a bisbenzimidazole fragment whose derivatives have a wide range of therapeutic applications in nucleic acid recognition, sensing, photodynamic therapy and as cellular probes. We report a dramatic effect of linker length and composition of alkynyl (clickable) Hoechst 33258 derivatives in target binding and cell uptake. We show that the binding of Hoechst 33258 modeled bisbenzimidazoles (1-9) which contain linkers of varying lengths (3-22 atoms) display length and composition dependent variation in BDNA stabilization using a variety of spectroscopic methods. For a dodecamer DNA duplex, the thermal stabilization varied from 0.3-9.0 ºC as the linker length increased from 3-22 atoms. Compounds with linker length up to 11 atoms (such as compounds 1 and 5) are localized in the nucleus while the compounds with long linkers (such as compounds 8 and 9) are distributed in the extra-nuclear space as well, with possible interactions with extra-nuclear targets. These findings provide insights for future drug design by revealing how linkers can influence the biophysical and cellular properties of individual drug fragments.


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Introduction: An important facet of modern drug discovery methods is achieving target selectivity and specificity at the binding site. One approach is to synthesize hybrid small molecules that combine fragments known to bind the target, in order to achieve strong affinity and specificity for the molecular target.1, 2 Within this context, we visit a well-established and historic nucleic acid recognition process using benzimidazole small molecules to dig deeper into the role of linker length and composition in target selectivity.3 The hybrid small molecules are generally chemically synthesized by connecting different binding moieties (e.g. A and B) through linkers of varying length and composition (e.g. L) to yield compounds such as A-L-B. Numerous studies have shown that activities, specificities and inhibitory effects of hybrid small molecules A-L-B are dependent on the length and compositions of the linkers (L) that connects them. 4-14 What is not well known is the effects these linkers L have on the properties of the individual fragments A and B. Such linkers are often chosen arbitrarily, and are, in part, guided by their commercial availability. In order to gain a deeper insight of the role of linkers/ side chains on the target binding, and uptake herein, we study the interaction of bisbenzimidazole Hoechst 33258 derivatives (Figure 1) with AT-rich duplex DNA using spectroscopic, calorimetric, microscopic and flow cytometry methods. We chose to study the binding of Hoechst 33258 derivatives for three main reasons: (a) because Hoechst 33258 is a well-known dye used for its nucleic acid binding, staining and the details of its DNA binding and uptake are well understood and validated by different methods (b) because it contains a bisbenzimidazole pharmacophore displaying anticancer,15 antiviral16 and antibacterial17 properties in its parent and derivatized forms as well as in the detection of β-amyloids,18 heavy metals19 and in pH-dependent sensing of nucleic acids20 and modulation of host-guest interactions21 (c) its usage as a multi-purpose


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fragment in developing hybrid molecules, as evident from its numerous application in asymmetric catalysis,22 multiple recognition,23,

24

sequence-specific nucleic acid targeting,25,

26

drug self-assembly on DNA27, recognition of RNA internal loops28, oncogenic non-coding RNAs29 as well as myotonic dystrophy type 1 RNA,30 live cell imaging31, 32 and in photodynamic therapy.33 Hoechst 33258 is one of the earliest small molecules used to stain and quantify nucleic acids due to its remarkable binding to B-DNA.34-36 In particular, it binds strongly to AT-rich DNA duplex and exhibits excellent fluorescent properties that allow for microscopic investigation in vivo.37 The X-ray structural studies of Hoechst 33258-B-DNA complex

38, 39

done in the late 1980s and

subsequent solution NMR studies with oligonucleotides showed that AATT bases were the core binding sites of Hoechst 33258.40 Hoechst 33258 binds in the minor groove of these oligonucleotides where it complements the groove curvature by binding snugly in the AT-tract38 excluding water from the interface of its DNA complex.

41

Calorimetric and spectroscopic

binding studies have reported Hoechst 33258 as a high affinity B-DNA binder with binding affinities in the nanomolar range. 42-44 Several studies with Hoechst 33258 based hybrid molecules containing a variety of linkers are reported in literature which have included triplex-forming oligonucleotides (TFO)- Hoechst 33258 conjugates, duplex targeting imidazole-pyrrole based polyamide-Hoechst 33258 conjugates1, 45-48, Hoechst 33258-porphyrin derivatives,49 Hoechst 33258-neomycin conjugates24, 50-52

as well as triple recognition agents. 23, 24 Surprisingly, none of these studies have investigated

in detail, the role of linker alone in the binding Hoechst 33258 derivatives to DNA targets. To understand the role of linkers in DNA binding, we report the binding of Hoechst 33258


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derivatives (1-9) to B-DNA duplexes. These Hoechst 33258 derivatives contain varying linker length at the phenolic end of the bisbenzindazole, and the linkers also vary in their oxygen content (monooxygen, bisoxygen and polyoxygen). The B-DNA binding was investigated using UV thermal denaturation, fluorescent intercalator displacement (FID) assay, circular dichroism (CD), UV-vis absorption, fluorescence spectroscopy, isothermal calorimetry (ITC) studies. We have also studied the cellular uptake and localization of these molecules from the perspective of linker length with a few cell lines.

Figure 1. Chemical structure of the compounds used in this study.


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EXPERIMENTAL SECTION: General Methods. Unless otherwise specified, chemicals were purchased from Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) and used without further purification. UV spectra were collected on a Varian (Walnut Creek CA) Cary 100 Bio UV-Vis spectrophotometer equipped with a thermoelectrically controlled 12-cell holder. Fluorescence Intercalator Displacement (FID) experiments were performed on Cary Photonics fluorimeter equipped with a 96 well plate reader. Circular Dichroism studies were performed on JASCO J-810 spectropolarimeter equipped with a thermo-controller. Fluorescence microscopy experiments were done using Nikon Ti-Eclipse inverted fluorescence microscope. Nucleic Acids. Nucleic acids were purchased from Eurofins MWG Operon (Huntsville, AL) and used without further purification. The nucleic acid concentration was determined using the extinction coefficient provided by the supplier. All nucleic acid stock solutions were prepared in buffer 10 mM sodium cacodylate, 0.1 mM EDTA and100 mM NaCl at pH 7.0. The nucleic acid solutions were prepared by heating them at 95 C for fifteen minutes and then slowly allowing it ο

cool back to room temperature. After two days of incubation, the duplex formation was checked by UV thermal denaturation experiments. The stock solutions were stored at 4 C and diluted to ο

desired concentrations as required. Ultra Violet (UV) Thermal Denaturation Experiments. All UV experiments were performed on a Cary 1E UV-Vis spectrophotometer (12 cell holder) equipped with a temperature controller. Quartz cells with 1 cm path length were used for all the experiments. Spectrophotometer stability and wavelength alignment were checked prior to initiation of each melting point experiment. The


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melting of DNA with and without the compound was performed at a heating rate of 0.2 °C/min. Samples were brought back to 20 °C after the experiment.

All UV melting experiments were

monitored at 260 nm. For the Tm determinations, first derivatives were used. Data points were recorded every 1.0 °C. The DNA concentration used was 1 µM /duplex while the compound was mixed at 1:1 DNA to compound ratio. All compound solutions were prepared in dimethyl sulfoxide (DMSO) as concentrated solutions and were diluted (0.2% final DMSO concentration). Fluorescent Intercalator Displacement (FID) Experiments. Fluorescence experiments were performed on Cary Photonics fluorometer (Walnut Creek, CA) equipped with a 96 well plate reader. All experiments were performed at room temperature i.e; 21-23 C. The experiments ο

were performed in the 96 well plates as triplicates. The DNA solution was prepared at 1 µM / duplex in buffer 10 mM sodium cacodylate, 0.1 mM EDTA, 100 mM NaCl at pH 7.0 and mixed with thiazole orange (TO) at a concentration of 6 µM. The compounds were added to the DNATO complex solution at 1:1 ratio which was followed by a 5 minute equilibration time before the fluorescence emission was recorded. All the compounds used the study were prepared in DMSO and the final DMSO concentration used in the experiment was 2%. The TO excitation was performed at 501 nm and the emission was recorded at 534nm. The change in the fluorescence was plotted as % fluorescence change (% ΔF) = (ΔF/IF) × 100 where, ΔF = Change in fluorescence upon compound addition and IF = Initial fluorescence of the DNA- TO complex


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Fluorescence Emission Experiments. Fluorescence emission experiments were performed in a 3.0 mL quartz cuvette on a Photon Technology International instrument (Lawrenceville, NJ). The fluorescence emission spectrum of the compound solution (100 nM) was recorded in the absence of DNA in buffer 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0. This was followed by the addition of the DNA duplex solution (so that the ratio of compound:DNA was 1:1). All the compounds used the study were prepared in DMSO and the final DMSO concentration used in the experiment was 0.1%. After 5 minute equilibration, the emission spectrum was recorded. All experiments were performed at 20 C. ο

Isothermal Titration Calorimetry (ITC).

All ITC measurements were performed on a

MicroCal VP-ITC (MicroCal, Inc., Northampton, MA) instrument. The DNA concentration was 10 µM/duplex. In every titration, a 10 µL aliquot of compound solution (in DMSO) was injected into a sample cell containing 1.42 mL of nucleic acid solution. The injection spacing was 240s, syringe rotation rate was 260 revolutions per minute (rpm) and duration of each injection was 20s. For each titration, an identical control experiment was performed by titrating the compound solution (in DMSO) into the buffer. The resulting data was processed using Origin 5.0. Integrating the area under each heat burst curve yielded the enthalpy upon compound injection. The corresponding enthalpy of dilution was subtracted to obtain the actual enthalpy changes associated with the compound-nucleic acid binding. The final DMSO concentration used in the experiments was < 4%. Differential Scanning Calorimetry (DSC). The nucleic acid melting temperature and enthalpy changes were obtained using aMicroCal VP-DSC (MicroCal, Inc., Northampton, MA). The DNA concentration was 50 µM/duplex. The scan rate was 1 ºC/min, and the operating


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Biochemistry

temperature range was 5- 110 ºC.

After each DSC experiment, a corresponding control

experiment was conducted with only the buffer in the sample cell. The corrected DSC profile was obtained by subtracting the control data from the sample data. The enthalpy of melting of the duplex DNA was calculated by integrating the area under the heat capacity curves using Origin version 5.0. Fluorescence Microscopy: Fluorescence microscopy experiments were performed using J744.A1 cells on a Nikon Ti-Eclipse inverted fluorescence microscope. Cells were plated in a 24 well plate on 12 mm cover-slips plated at 12,000 cells/well and incubated at 37oC, 5% CO2 for 24 hours before treatment with each compound. Each compound was added at a final concentration of 1 µM followed by incubation at 37oC, 5% CO2 for 15 minutes. Subsequently, 16% paraformaldehyde (PFA) was added in, to reach a final concentration of 4% followed by incubation for 12 minutes at room temperature (protected from light). All the compounds used the study were prepared in DMSO and the final DMSO concentration used in the experiment was 0.1%. Cells were then washed twice with 1X PBS, and permeabilized for actin staining with 0.1% Triton X-100 in PBS for 5 minutes at room temperature then washed twice with 1X PBS. Actin staining was performed using Alexa Fluor 594 phalloidin (165 nM in PBS) added to each well and incubated at room temperature for 20 minutes, following actin staining cells were washed twice with 1X PBS before mounting on slides for imaging. Fluorescence micrographs were overlaid on differential interference contrast (DIC) micrographs to illustrate cellular localization of each compound. All image processing and associated microscopy data analysis was performed using Nikon NIS-Elements software. Flow Cytometry: Flow cytometric experiments were performed using DU145 and HEK293 cells. Cells were harvested by trypsin dissociation and suspended in DMEM supplemented with


Biochemistry

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2% FBS at 1x106 cells/mL.

53, 54

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Each compound was added to 1 mL cell suspension for a final

concentration of 4 µM and incubated at 37oC, 5% CO2 for 90 minutes.

53

All the compounds in

the study were prepared in DMSO and the final DMSO concentration used in the experiment was 1%.

Following incubation, cells were centrifuged and the media/compound solution was

removed followed by washing twice in cold 1× PBS with 2% FBS, then it was suspended again in 1× PBS with 2% FBS and kept on ice until analyzed. Samples were analyzed using a CyAn flow cytometer with the 405 nm excitation laser and data analysis was performed using FlowJo v10. All treated samples were compared to an untreated control.

RESULTS AND DISCUSSION Design and Synthesis of Alkynyl Derivatives of Hoechst 33258. Hoechst 33258 modified compounds (1-9) were designed so that that they cover the linker length from short (3 atoms) to long (22 atoms). The derivatization of Hoechst 33258 was performed at the phenolic end of the bisbenzimidazole moiety using aliphatic linkers of variable length. These derivatives are broadly categorized in three categories as those containing monooxygen (1-3), bisoxygen (4-8) or polyoxygen atoms (9) (Figure 1) that end with an alkyne which is chemically rather ‘inert’ but extremely useful for click chemistry. The details of the synthesis of these compounds have been recently reported by us.55, 56 UV Thermal Denaturation Experiments. The UV thermal denaturation experiments allow us to observe binding related stabilization or destabilization of the DNA. denaturation experiments of compounds (1-9) were performed with

The UV thermal

a self-complementary

dodecamer d(CGCAAATTTGCG)2.57, 58 The results obtained from these experiments are shown


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in Figure 2 and Table 1. The dodecamer oligonucleotide d(CGCAAATTTGCG)2 is well-known for Hoechst 33258 binding to its AT-rich region.57, 58 The DNA duplex formation was checked using both UV- thermal denaturation and differential scanning calorimetry (DSC) experiments (see supporting information Figure S2). Figure 2a shows a representative thermal melting profile of the dodecamer DNA in the absence and presence of compound 1. Figure 2b shows the thermal stabilization of compounds (1-9) with the oligonucleotide d(CGCAAATTTGCG)2 as a bar graph plot.

The thermal denaturation experiments with compounds (1-9) show very

interesting thermal stabilization trend with respect to the length of the alkyl linkers. As depicted in Figure 2a, in the absence of compound, the oligonucleotide duplex exhibited a relatively broad hyperchroism 1

25

(b)

0.8

2

50

60

70

80

o

90

0

3

6

11

Temperature ( C)

8

10

12

Compound 8

40

Compound 7

30

Compound 6

5

Compound 5

0.2

10

Compound 4

(Compound 1)

Compound 3

66.1 C

Compound 2

o o

(Control)

0.4

15

m

o

57.8 C

Compound 1

0.6

0 20

d(CGCAAATTTGCG) duplex

20

14

16

Compound 9

(a)

ΔT ( C)

Normalized Absorbance

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

21

Linker Length

Figure 2. (a) A representative UV thermal denaturation plot of the d(CGCAAATTTGCG)2 duplex in the absence and presence of compound 1. (b) A bar graph plot showing the increase in the thermal denaturation temperature of DNA duplex d(CGCAAATTTGCG)2 in the presence of different compounds The DNA duplex (2 µM/duplex) was mixed with various compounds in 1:1 compound to DNA ratio and denatured in the temperature range 20 C- 98 C (at a rate of 0.2 C/min) in buffer 10 mM sodium ο

ο

cacodylate, 0.1 mM EDTA, 100 mM NaCl at pH 7.0.


ο

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indicating the dissociation of the duplex into single strands at 57.8 C. The thermal denaturation ο

of the oligonucleotide was then performed in the presence of compound 1 at 1:1 drug:DNA ratio. The binding of compound 1 leads to 8.3 C increase in the thermal stability of the DNA duplex ο

and also results in a sharp thermal melting profile suggesting a cooperative transition. The thermal stabilization was found to be clearly dependent on the length and composition of the linker. As depicted in Figure 2b, the thermal stabilization afforded by compounds 1-9 are shown

Table 1. Thermal denaturation temperatures (Tm and ΔTm) of DNA duplex 5’d(CGCAAATTTGCG)-3’ in the absence and presence of the ligands studied (10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0). Compound

Linker Length

5’-d(CGCAAATTTGCG)3’ Tm ( C)

ΔTm ( C)

ο

ο

None

--

57.8

--

1

3

66.1

8.3

2

6

66.5

8.7

3

11

59.2

1.4

4

8

66.8

9.0

5

10

66.1

8.3

6

12

62.6

4.8

7

14

58.5

0.7

8

16

58.1

0.3


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Biochemistry

9

21

59.5

1.7

as a bar graph plot and categorized in the three categories. The black bars represent compounds containing one oxygen atom, red bars represent compounds containing two oxygen atoms and the blue bar represents the compound containing seven oxygen atoms on their linker arm. For the monooxygen linker derivatives, as the linker length increased from three atoms to eleven atoms, a 6.9 C drop in the thermal stabilization was observed for the oligonucleotide duplex. The large ο

decrease in the thermal stabilization clearly showed the role of linker length in the binding of compounds 1-3 to the oligonucleotide duplex. For the compounds with bisoxygen linkers (3-8), similar linker length dependent thermal stabilization was also observed. For compound 4 (linker length = 8 atoms) 9.0 C increase in the thermal melting temperature was observed. However as ο

the linker length increased from 8 to 16 atoms, a huge drop (8.7 C) in the thermal stabilization ο

was noticed. The results obtained from the bisoxygen containing linkers of Hoechst 33258 derivatives again showed that there is a clear relation between the length of linker of the Hoechst 33258 derivatives and their DNA binding. We then tested another compound 9, with the longest linker length of all the compounds studied, and whose linker arm contains hexaethylene glycol (22 atoms long linker containing a total of seven oxygen atoms). A reversal of thermal stabilization was seen in this case (1.7 C) as compared with other longer linkers (7 and 8; 0.7 C ο

ο

and 0.3 C respectively) suggesting that the number of oxygen atoms present in the linker may ο

also have a role in the thermal stabilization. These results closely mirrored our previous findings with a B-DNA duplex dA60. dT60 where, a sharp decrease (15.9 C) in the thermal stabilization by monooxygen linkers (1-3) was ο

observed as the linker length increased from 3 to 11.56 This trend of decreased thermal stabilization with increased linker length was seen again with the bisoxyegn liner where a 22.2 C ο


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decrease in the thermal stabilization was found as the linker length increased from 8 to 16 atoms. Here again, a reversal in the thermal stabilization was observed for compound 9 (linker length = 22 atoms) which afforded 12.2 C thermal stabilization, similar to the thermal stabilization ο

obtained with compound 6 (a twelve atom linker).56 This observation suggested that it is not just the length but also the composition of the linker that plays a role in the binding to the B-DNA duplex. The results obtained from thermal denaturation studies lead to a few important conclusions (i) the thermal stabilities afforded by compounds 1-9 are clearly dependent on the linker length (ii) linkers which are up to 8 atoms long provide numerically similar thermal stabilization. The thermal stabilization starts to decrease as the linker length increases beyond eight atoms. Hoechst 33258 derivatives with longer linkers (14 and 16 atoms containing two oxygen atoms) lead to very poor thermal stabilization (iii) a reversal in the thermal stability of the B-DNA duplex was seen with a linker that contained polyoxygen atoms. What could possibly be hindering the interaction of bisbenzimidazoles, which have longer linkers, with the B-DNA? Increasing the length of the linker causes these molecules to be increasingly hydrophobic as determined from their distribution coefficient in octanol-buffer mixtures (Table 2). However, in polar media, as used in our experiments, these long alkyl chain linkers are highly prone to experience hydrophobic effect which could possibly lead to micelle type structures.59 This could prevent the bisbenzimidazoles from entering the minor groove of the B-DNA duplex due to the steric effects. Recently, Hoechst 33258 derivatives that carry bulky groups on the phenolic ring of Hoechst 33258 have been reported in an effort to develop RNA


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Biochemistry

selective compounds due to the inability of these sterically hindered derivatives to bind AT-rich DNA duplexes.28 Table 2. A table showing the experimentally determined distribution coefficients of compounds 1-9 against octanol -buffer (10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0) system.

Compound

Linker length

UVmax (nm)

Distribution coefficient

1

3

348

0.43

2

6

348

0.41

3

11

348

0.92

4

8

348

1.00

5

10

348

1.62

6

12

348

2.29

7

14

348

2.20

8

16

348

2.47

9

21

348

2.50

Credence to the hypothesis of hydrophobic effect driven diminished binding of Hoechst 33258 derivatives with long linkers to B-DNA duplex is further provided by enhanced thermal stabilization of compound 9. The compound 9, despite having the longest linker, is able to have appreciable thermal stabilization in comparison to other derivatives of similar linker length (such as compound 7 and 8). The main difference between compound 9 and compound 7 or 8 is the presence of more oxygen atoms (five extra oxygen atoms in compound 9 as compared to


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compound 7 or 8). The hydrophobic effect works primarily by breaking the hydrogen bonding network in the polar media. In the case of compound 7 or 8, much greater hydrophobic effect would be expected than that seen in compound 9, since the larger number of oxygen atoms present in compound 9 would enable it to make more hydrogen bonding contacts in the aqueous media, thus preventing self-association of the compound. This lessened hydrophobic effect in compound 9 would, in turn, allow for the bisbenzimidazole unit to interact more favorably in the minor groove of B-DNA. Additionally, oxygen atoms on the linkers may also contribute to thermal stability by providing hydrogen bonding interactions in the proximity of G/C base pairs. While it is possible that the stacking of aromatic units of bisbenzimidazole contributes to the weakened binding to DNA, polarity of the linker should not affect this aggregation and additionally no biphasic transition in the thermal denaturation profiles was observed. Hoechst 33258 has been known to stack in solution in the presence of DNA. However such complexes result in biphasic transitions which arise from the dissociation of stacked aromatic rings of the bisbenzimidazole and the dissociation of the duplex DNA.60

The structural differences in the linker component of these compounds can have significant bearing on the entropic contributions to binding in the minor groove of the DNA. AT-rich oligonucleotides such as d(CGCAAATTTGCG)2 are known to possess highly ordered spine of hydration and these water molecules contribute favorably to entropy when displaced by the minor groove binder.61 Since compounds with short linker lengths might still fit in the available space in the minor groove, their binding in the minor groove is likely to have more favorable entropic contribution than long linkers because they may extend beyond the minor groove. Accommodation of a longer linker within the minor groove would likely result in a much larger 16 Environment ACS Paragon Plus

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Biochemistry

opening of the groove, thereby, disrupting overall base pairing of the duplex and other electrostatic interactions with the cations vital to the stability of the duplex. Therefore, it is likely that the large alkyl linkers protrude out in the bulk solvent where they are afforded greater flexibility to aggregate and or form micelle type structure, further reducing the binding in the minor groove. Fluorescence Intercalator Displacement (FID) Assay. FID assay was developed to rapidly screen sequence-specific DNA binding agents62 and it can also be utilized to obtain DNA binding affinities of the ligands.63 It has also been utilized for the identification of triplexforming oligonucleotides64 as well quadruplex DNA binders.65 We have previously used this assay to screen duplex66, triplex67 and quadruplex binders.68, 69 This assay relies on changes in the fluorescence emission exhibited by a DNA-intercalator (usually ethidium bromide or thiazole orange) complex upon compound binding. DNA intercalators such as ethidium bromide or thiazole orange have very poor fluorescence emission in the absence of DNA. However, when bound to DNA, fluorescence emission increases multi-fold, which can be used to study the DNA binding of compounds. For FID experiments, nucleic acid affinity of compounds can be determined either by obtaining the DC50 values (DC50 value refers to concentration of compound required to displace 50 % of the bound intercalator) or by a single point screen to gauge their DNA binding affinities by adding compounds (at a fixed compound-nucleic acid ratio) to the DNA-intercalator complex solution.

66

The resulting fluorescence change is then plotted to rank

the affinities of those compounds.


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Figure 3. A bar graph plot showing percent change in fluorescence emission of thiazole orange upon DNA binding. The DNA d(CGCAAATTTGCG)2 duplex (1µM/duplex) was mixed with TO (6µM). The DNA-TO complex was added with the compounds at 1:1 compound:DNA duplex ratio. The experiment was performed in buffer 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0 at room temperature (23 C). Each entry represents an ο

average of three experiments. In the present study, we used thiazole orange (TO) as our intercalating agent. The DNATO complex was added with compounds in 1:1 duplex: compound ratio. The resulting fluorescence change was then plotted to obtain the rank of DNA binding affinities of these compounds. On the Y-axis, % ΔF is the percent change in the fluorescence emission of thiazole orange upon binding to the DNA duplex in the present study. As shown in Figure 3, the change in the fluorescence emission afforded by the compounds followed a similar trend as observed with UV thermal denaturation experiments where a general decrease in the DNA binding affinity with the increasing linker length of the Hoechst 33258 derivatives (1-9) was observed for all subclasses. However, the percent change in fluorescence for compounds 7 and 8 are not appreciably


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Biochemistry

lower and distinct when compared to the results obtained with the UV thermal denaturation experiments. This difference could be attributed to the fundamental differences in the two sets of experiments conducted; as UV thermal denaturation reflects the outcome of a direct interaction at the binding site while FID reflects fluorescent intensity changes of the intercalator due to allosteric effects of a minor groove binder disrupting TO intercalation by changing DNA shape. The change in the fluorescence is quite low as TO and Hoechst do not compete for the same binding sites. Nevertheless, the results obtained from this assay reaffirmed the findings of the UV thermal denaturation experiments. Circular Dichroism (CD) Studies. In order to investigate if the compounds with long linkers (such as compound 3, 8 and 9) were able to form similar complexes with the DNA, we performed CD studies. Circular dichroism studies help in obtaining crucial details regarding the compound-nucleic acid interaction.70 Studies using CD have been conducted previously to describe Hoechst 33258-DNA interactions.71 The binding of Hoechst 33258 in the DNA minor groove results in a positive induced CD indicating the compound-DNA interaction. A large positive induced CD typically results from the binding of chromophores in the DNA groove.72 To identify the complex formation, we chose compounds 1, 3, 8 and 9 to observe the changes in the chromophore absorption region of the CD spectrum. We chose compounds 1 as well as 3 and 8 as they represented the strong and the weak binders of DNA duplex while compound 9 had shown an improved binding amongst all compounds that contained long linkers. As a control, we also performed the experiments with Hoechst 33258. As depicted in Figure 4, the binding of different compounds to dodecamer DNA d(CGCAAATTTGCG)2 is shown. Figure 4a shows the interaction of Hoechst 33258 to the


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DNA duplex. At 1:1 compound-DNA ratio, an induced positive CD signal at 352 nm results as a consequence of DNA binding by Hoechst 33258. Compound 1 also showed a positive induced CD of similar magnitude at 352 nm (Figure 4b). However, both compounds 3 and 8 showed no induced CD spectrum between 300-400 nm and their CD spectra was identical and superimposable to CD spectrum of the DNA in the absence of compound. However, for compound 9, we observed the positive induced CD spectrum similar in magnitude to the same obtained in the presence of Hoechst 33258.

Figure 4.

Circular dichroism studies of DNA duplex d(CGCAAATTTGCG)2 with various

compounds (a) Hoechst 33258 (b) compound 1 (c) compound 3 (d) compound 8 (e) compound 9 as indicated on each graph. The duplex DNA sample (10 µM/duplex) was mixed with the compounds (10 µM) and equilibrated for five minutes before the CD scan was taken. Each spectrum is an average of three scans. All experiments were conducted in buffer 10 mM sodium cacodylate, 0.5 mM EDTA, 100 mM NaCl at pH 7.0 at 20 C. In each graph, the CD spectrum of ο

DNA in the absence of compound is shown in black while the same in the presence of compound is shown in blue.

These CD studies provide some insight into the compound-DNA complexation of the compounds studied. First, compound 1 binds in the same manner to Hoechst 33258 leading to an


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induced CD signal of similar magnitude. The absence of induced CD in the 300-400 nm for compounds 3 and 8 (Figures 4c and 4d) indicate that the bisbenzimidazole moiety of these molecules is unable to reach the minor groove of B-DNA (as evident by the lack of induced CD). The presence of positive induced CD in the case of compound 9, which was of nearly equal magnitude as obtained with Hoechst 33258, shows that it also interacts in a similar way as Hoechst 33258. These results also support our initial hypothesis of hydrophobic effect based binding of these molecules. As discussed earlier, the presence of large alkyl groups on the linkers of compounds 3 and 8 make them likely to self-aggregate due to the hydrophobic effect, leading to their inaccessibility of the minor groove of B-DNA. The restoration of minor groove binding in the case of compound 9 (Figure 4e) also supports this hypothesis, as the presence of multiple oxygen atoms in its linker is likely to allow lesser self-aggregation because of the ability of oxygen atoms to engage in hydrogen bonding with the solvent. UV-Vis Absorption Studies. The binding of compounds 1, 3, 8 and 9 was also evaluated using UV-Vis absorption spectroscopy. The changes in the absorbance of Hoechst 33258 upon DNA binding has been previously reported by several other laboratories.42, 43, 60, 71, 73 In our studies we studied the binding of the above-mentioned compounds with the dodecamer DNA duplex d(CGCAAATTTGCG)2.As shown in Figure 5a, upon binding, the absorption maximum of the Hoechst 33258 spectrum underwent a red shift by 15 nm and was accompanied by a slight hyperchroism. However, two clear isosbestic points (at 300 nm and 344 nm) were observed suggesting the complexation between the DNA and Hoechst 33258. Compound 1 exhibited similar UV spectrum (Figure 5b) to the one obtained with Hoechst 33258 where the absorption maximum was red shifted by 16 nm. Again, we observed two isosbestic points at 301 nm and 21 Environment ACS Paragon Plus

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343 nm suggesting the complex formation between the DNA duplex and compound 1. However, the binding of compounds 3 and 8 to DNA duplex (Figures 5c and 5d) resulted in much lower red shifts (~ 8 nm each). The two compounds, also, did not show the presence of any isosbestic points suggesting lack of a tight binding complex. However, compound 9 resulted in the red shift of the maximum absorption by 17 nm (Figure 5e). Isosbestic points at 301 nm and 345 nm were also observed. All of these findings corroborated the results obtained with the UV thermal denaturation and CD studies which also showed the decreased binding of compounds 3 and 8 and reversal of the binding with compound 9 to the DNA duplex.

Figure 5.

UV-Vis absorption studies of DNA duplex d(CGCAAATTTGCG)2 with various

compounds (a) Hoechst 33258 (b) compound 1 (c) compound 3 (d) compound 8 (e) compound 9 as indicated on each graph. The DNA sample (5 µM/duplex) was mixed with the compounds (5 µM) and equilibrated for five minutes before a scan was taken. All experiments were performed in buffer 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0 (T = 20 C). In ο

each graph, the UV spectrum of DNA duplex in the absence of compound is shown in black while the same in the presence of compound is shown in blue. Isosbestic points are indicated by arrows on each plot. Fluorescence Emission Studies of Compounds 1, 3, 8 and 9. Hoechst 33258 and related compounds have been known for excellent fluorescence emission properties when bound to a BDNA duplex even at very low concentrations (1 ng/mL).37 This unique highly fluorescent nature


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of Hoechst 33258 and similar compounds made them ideal for nuclear staining and chromosome sorting.34 The fluorescence emission enhancement of these compounds results from reduced loss of energy to the bulk solvent when bound in the minor groove of B-DNA. We, therefore, conducted fluorescence emission studies of compounds 1, 3, 8 and 9 with the dodecamer duplex DNA sequence d(CGCAAATTTGCG)2. The results from these studies are depicted in Figure 6. In each graph, the fluorescence emission spectrum of compound in the absence of DNA duplex is shown in the black while the blue lines represent the fluorescence emission profile of each compound in the presence of DNA duplex. Both Hoechst 33258 and compound 1 show similar fluorescence enhancement upon complexation (Table 3)

Figure 6. Fluorescence emission spectrum of various compounds in the absence and presence of DNA duplex d(CGCAAATTTGCG)2 (a) Hoechst 33258 (b) compound 1 (c) compound 3 (d) compound 8 (e) compound 9 as indicated on the graph. The experiment was conducted at 100 nM/compound concentrations and the appropriate amount of the DNA duplex was added to achieve 1:1 DNA-compound ratio. The experiments were done in buffer 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0 (T = 20 C). Each entry in the spectrum ο

represents an average of two scans.

with DNA duplex (~49 fold increase). However, both compound 3 and 8 display very poor enhancement in the fluorescence emission when bound to DNA duplex (~ 2 fold in both cases).


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In the case of compound 9, however, the enhancement in the fluorescence emission is restored back to be nearly half of the magnitude ( ~27 fold) of the same found with Hoechst 33258 and compound 1. The results also show clearly that the minor groove binding is missing or weakened in the cases compound 3 and 8. Restoration of fluorescent enhancement for compound 9 is in accord with the results discussed in the previous sections.

Figure 7. (a) Circular dichroism studies of DNA duplex d(CGCAAATTTGCG)2 with compounds 1, 3 and 8 as indicated on each graph. The DNA sample (10 µM / duplex) was mixed with the compounds (10 µM) and equilibrated for five minutes before a scan was taken. Each spectrum is an average of three scans. All experiments were conducted in 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl buffer at pH 7.0 mixed with 1,4 dioxane (20 % v/v) at 20 C. In each graph, the CD spectrum of DNA in the absence of DNA is shown in black. ο

(b) The fluorescence spectrum of various compounds in the presence of DNA as indicated on the graph. The experiment was conducted at 100 nM/duplex DNA concentration and appropriate amounts of the compounds were added to achieve 1:1 DNA / compound ratio. The experiments were conducted in buffer 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0 mixed with 1,4 dioxane (20 % v/v) at 20 C. ο

Testing the Role of Hydrophobic Effect. The DNA binding studies discussed so far suggest the role of hydrophobic effect in the compound binding. To test this hypothesis, we decided to


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reduce the solvent polarity of our buffer by adding a less polar solvent 1,4 dioxane (20 % v/v). The reduction in the solvent polarity is expected to lead to a decrease in the hydrophobic effect, which, in turn, would improve the minor groove binding of the compounds 3 and 8. As shown in Figure 7a, both compounds 3 and 8 show an induced CD signal in the bisbenzimidazole absorption region, which is in contrast to their CD spectrum shown in Figure 4, where we did not observe any induced CD with the same compounds. This data shows that increasing the non-polar character of the solvent does lead to increased minor groove binding of these compounds, which could be an outcome of loss/reduction of the hydrophobic effect. In the case of compound 3, the magnitude of the induced CD was slightly lower than compound 1, while the same for compound 8 was much lower. These results indicate that even though increasing the solvent polarity leads to a decrease in the hydrophobic effect, it is not fully reversed. We further tested these results using the fluorescence emission experiments. The fluorescence emission spectrum for each compound is shown in Figure 7b. Both compounds 3 and 8 showed improved fluorescence emission in the presence of 20 % (v/v) 1,4 dioxane. A comparison of their fluorescence emission properties is given in Table 3. In the absence of 1,4 dioxane, the ratio of increase in the fluorescence emission for both Hoechst 33258 and compound 1 is nearly 24 folds higher than compounds 3 or 8. However, the same ratio in the presence of 1,4 dioxane is ~ 3 and ~8 folds for compound 3 and compound 8 respectively (Table 3). These studies clearly show that reducing the polar character of the solvent results in enhanced fluorescence emission of compound 3 and 8, due likely to the reduced hydrophobic effect experienced by the alkynyl termini of these novel Hoechst 33258 derivatives.


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Table 3. Fluorescence emission properties of various compounds in the absence and presence of 1, 4 dioxane (reduced hydrophobic effect).

Compound

1,4 Dioxane

λ max (nm)

Fluorescence Increase (fold)

Hoechst 33258

0%

457

47.4

1

0%

457

49.7

3

0%

457

2.0

8

0%

457

1.6

9

0%

457

26.8

1

20 %

456

24.9

3

20 %

454

7.2

8

20 %

456

3.1

Calorimetric Insights into DNA Binding. To understand the enthalpy changes associated with DNA-compound complexation, we performed excess site ITC titrations74 at various temperatures (Supporting information, Figure S3). The resulting enthalpy values are given in Table 4. For compound 1, we obtained a positive enthalpy of interaction at temperatures 20 ºC and 25 ºC, while a negative enthalpy of interaction at 30 ºC was observed. This resulted in a negative heat capacity value of -134 cal/mol/K for the DNA-compound interaction. However, for both compound 8 and 9, we observed negative enthalpy of interaction at all studied temperatures as well as negative heat capacity values of –117 cal/mol/K and -380 cal/mol/K respectively (Figure


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8). The negative enthalpy values obtained could be ascribed to weak DNA binding resulting in reduced transfer of these compounds from the bulk solvent to the specific binding site of the duplex. Such differences can lead to changes in the interaction of DNA with the metal cations, altered hydrogen binding patterns as well as changes in the burial of the hydrophobic surface resulting in overall negative enthalpy values. The differing heat capacity values of the three compounds studied can be attributed, in part, to the overall effect of compounds on the displacement of water molecules from the bulk solvent and the DNA minor groove, as binding of Hoechst 33258 is known to alter the hydration patterns of the minor groove.41 As shown by Petty,41 the net enthalpies of interaction for Hoechst 33258 are similarly negative at most temperatures whereas the overall heat is generally positive, as it includes a large endothermic heat of dilution of the benzimdiazole ligand. Depending upon the ligand aggregation and resulting dilution heats, the net enthalpies of these compounds can therefore vary significantly in magnitude and sign. For compound 9, the observed heat capacity change was -380 cal/mol/K, which was close to the experimentally determined heat capacity change for Hoechst 33258 interaction with d(CGCAAATTTGCG)2 duplex under similar conditions.44 The differences in the heat capacity values obtained with compound 9 with the same reported in the literature likely originate from changes in the structure of the two compounds (Compound 9 having a much larger alkyl chain tail) which may contribute to bulk water displacement as well differences in the experimental conditions (different buffer and higher salt in study done by Chaires and coworkers44). For compound 8, the heat capacity change was less negative and could largely be contributed by hydrophobic effect exerted by the long alkyl linker, as it does not enter the minor groove of DNA duplex. Alkyl groups have also previously been known to contribute to negative enthalpy in 27 Environment ACS Paragon Plus

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systems that are prone to micelle formation.75 However, in the case of compound 1, the heat capacity value was -134 cal/mol/K. This value was nearly two and half times lower than the experientially determined negative heat capacity values of compound 9, as well as Hoechst 33258 interaction with d(CGCAAATTTGCG)2 duplex, and suggests that minor perturbations in compound structure can have profound effects on its interaction with the solvent network and target site interactions, much different from the effects of compound binding on DNA as observed by UV thermal denaturation temperatures.

Figure 8. Heat capacity plots of (a) Compound 1 (b) Compound 8 and (c) Compound 9 in buffer 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0.

Table 4. A table showing the enthalpies of compound 1, 8 and 9’s interaction with d(CGCAAATTTGCG)2.

Compound

ΔH (Kcal/mol)

ΔH (Kcal/mol)

ΔH (Kcal/mol)

(20 C)

(25 C)

(30 C)

ο

ο


ο

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1

1.15 + 0.30

0.62 + 0.01

-0.19 + 0.10

8

-0.68 + 0.04

-1.28 + 0.04

-1.85 + 0.03

9

-3.50 + 0.10

-4.83 + 0.13

-7.30 + 0.10

Besides the changes in the experimental conditions, the binding stoichiometry obtained for compound 1 binding was found to be different than the same obtained with Hoechst 33258. Hoechst 33258 binds to d(CGCAAATTTGCG)2 duplex with a 1:1 compound: duplex binding stoichiometry, while compound 1 binds to the same duplex in a 2:1 compound:duplex ratio as determined by the Job plot analysis (Figure 9). This “anomalous” binding stoichiometry has previously been described by Bruice and coworkers with a Hoechst 332258 related compound Hoechst 33377.76 Hoechst 33377 differs from Hoechst 33258 just by a phenyl ring on the phenolic end which is able to self-stack by a π-π stacking of terminal phenyl ring on Hoechst 33377 and the central benzimidazoles in a side by side binding manner. Due to presence of the alkyne functionality at the phenolic terminal, we believe that a similar π-π


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Figure 9. A Job plot of compound 1 binding to d(CGCAAATTTGCG)2 duplex showing 2:1 binding stoichiometry. The experiment was done in buffer 10 mM sodium cacodylate, 0.5 mM EDTA and 100 mM NaCl at pH 7.0 (T = 20 C). ο

interaction between the terminal alkyne and the benzene rings of central benzimidazoles could drive the 2:1 binding in this case (Figure 9). Fluorescence Microscopy Studies of Cellular Localization. Since compounds 1-9 are modifications of the DNA staining dye Hoechst 33258, which is well known for its large fluorescence enhancement in the presence of DNA, we determined their cellular localization using fluorescence microscopy. Figure 10 shows the cellular localization of bisbenzimidazoles which represent the short (compound 1), mid (compound 5) and long (compounds 7 and 8) linker lengths of compounds used in this study. Figure 10 shows the DIC, bisbenzimidazole and actin stained J774A.1 macrophage cells as well as DIC-bisbenzimidazole and actin-bisbenzimidazole overlay images to delineate their cellular localization. The driving force for the nuclear staining of Hoechst 33258 is its affinity for B-DNA. We observed that compound 1, having a short linker, localized exclusively in the nucleus. The nuclear localization was also observed for compound 5 which contained a mid-length linker (11 atoms). Interestingly, it was a different story for the compounds with long linkers (compounds 7 and 8) which displayed poor thermal stabilization of the DNA. These compounds showed both nuclear and extra-nuclear distribution throughout the cell. Employing a set exposure time revealed two interesting observations: (a) the compounds that show significant thermal stability upon DNA binding resulted in higher fluorescence with their localization limited to the nucleus (b) the compounds with long linkers show lower


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fluorescence and distribution in the extra-nuclear space of the cells. The distribution of compounds 7 and 8 in the extra-nuclear space indicates the possibility of extra-nuclear targets for these compounds.

Figure 10.

Side chain dependent cellular distribution of bisbenzimidazole compounds in

J774A.1 macrophage. DIC (differential interface contrast), Hoechst, and actin shown independently, DIC/Hoechst and Actin/Hoechst overlays illustrate the differential cellular distribution with shorter side chain compounds that bind DNA with higher affinity localizing within the nucleus and longer side chain compounds with lower affinity to DNA show extranuclear distribution. Scale bar: 50 microns.

Flow Cytometry Studies.

After observing the differences in fluorescence intensity and

distribution in eukaryotic cells by microscopy, we sought to quantify the cellular uptake and fluorescence observed due to binding in treated cells. Quantification of the cellular fluorescence 31 Environment ACS Paragon Plus

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observed after treatment allows determination of how linker length influences the cellular uptake. To accomplish this task, we performed flow cytometry experiments with cell lines DU145 and HEK293 (Figure 11). Both cell lines show a similar trend in fluorescence intensity for all compounds (1-9), with DU145 showing higher fluorescence post-treatment than HEK293 for all compounds including Hoechst 33258 and Hoechst 33342. While microscopy studies revealed that compounds 1 and 5 with linker length ≤11 atoms showed nuclear localization similar to Hoechst 33258, the flow cytometry analysis resulted in 5.9-9.0 fold higher fluorescence in DU145 cells and 4.8-8.2 fold higher fluorescence in HEK293 cells compared with Hoechst 33258. Given that compound 1 and Hoechst 33258 both retain DNA binding, with similar fluorescence enhancement, the difference between these compounds is an example of how the linker can increase cellular uptake. Interestingly, compounds 6-9, with linker length > 11 atoms, had similar fluorescence to Hoechst 33258 in flow cytometry analysis. These results also shed light on possible differences in the cellular uptake of these compounds. Since the fluorescence observed in the flow cytometry experiments is a function of both the cellular uptake as well as the DNA binding linked fluorescence enhancement; and that the fluorescence enhancement when bound to all extra-nuclear targets is unknown, quantification of the absolute cellular uptake for all compounds could not be determined with confidence. The differences in the cellular uptake of these compounds are not likely to impact their intrinsic propensity for nuclear or extra-nuclear targets. DNA binding affinity and cellular uptake are two unrelated aspects of compound binding. While cell permeation of the test compound is highly dependent on their interaction with the cell membrane and subsequent internalization; DNA binding of the compounds presented in this study is directly dependent on how efficiently they access the minor


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groove. Therefore, compounds (e.g. 9) with long linkers, despite having appreciable DNA binding may not permeate cell and nucleus efficiently. Compounds that retain B-DNA binding (such as compounds 1, 2, 4 and 5) are expected to show large fluorescence change due to the quantum yield increase when complexed with the B-DNA duplex. Up to linker length 11, compounds show fluorescence enhancement and nuclear distribution similar to Hoechst 33342. Compounds having long linkers that showed poor binding to B-DNA duplex (such as compounds 7 and 8) and extra-nuclear distribution, may have additional binding with proteins since their large linkers have been recently reported to interact with and impact enzymes such as E.coli DNA and RNA topoisomerase.

While the new

eukaryotic cellular targets of these compounds are yet to be examined in detail, the decreased DNA binding resulting in lower cytotoxicity, as seen in dermal fibroblast cells, and enhanced E.coli DNA and RNA topoisomerase activity illustrates that these compounds provide a scaffold for future drug development. For example, the combined ability of these compounds to penetrate into eukaryotic cells and the selective targeting of bacterial enzymes could make these compounds good pharmacophores for further refinement and targeting of intracellular bacterial infections.


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Figure 11. Flow cytometric quantification of cellular uptake. Quantification of cellular uptake and Hoechst dependent fluorescence of compounds 1-9, Hoechst 33258, and Hoechst 33342 relative to an untreated control. MFI (mean fluorescence intensity) of HEK293 cells (A) and


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DU145 cells (B) following treatment with compounds 1-9, Hoechst 33258, and Hoechst 33342. (C) Representative histogram plots for DU145 cells with treated cells (blue) shown relative to the untreated control (red) illustrating the shift in fluorescence observed due to cellular uptake of the indicated compound.

CONCLUSIONS New drugs are continuously needed to combat diseases whose current therapies are increasingly getting limited due to toxicity and resistance issues. Nucleic acids offer a validated target system for development of new drugs such as anticancer agents, antiviral agents and as antibacterial agents. Therefore, understanding of small molecule fragment binding with the nucleic acids, such as DNA, the influence of core structural moieties and linkers on DNA binding and features that dictate selectivity is necessary to further advance such efforts. The studies presented here highlight the importance of linker length and composition on the DNA binding ability, cellular uptake, and localization of derivatives of a well-known DNA minor groove binder Hoechst 33258. Since the development of Hoechst 33258 as a molecular tool to study DNA minor groove interactions, growing literature reports have indicated that by covalently attaching the nucleic acid binding moieties to Hoechst 33258, dual binding compounds with enhanced nucleic acid binding can be obtained and their altered properties can be utilized in a variety of applications. The composition of atoms, the presence of ring structures in linkers determine flexibility between the two covalently attached nucleic acid binding compounds and the nucleic acid selectivity.17,

18

The findings of this study lead to following

important conclusions for the study of bisbenzimdazole fragment: (a) Up to a linker length of 8 atoms, the thermal stability of B-DNA duplexes are numerically similar (b) linker length and its


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composition beyond 8 atoms play a role in the thermal stability of B-DNA duplexes (c) increasing length of linkers consisting carbon, hydrogen and oxygen atoms lead to increased lipophilicity of the molecule (d) compounds with long aliphatic linkers do not enter B-DNA minor groove; however minor groove binding can be restored by addition of non-polar solvents such as 1,4 dioxane (e) interaction of these compounds with DNA leads to negative heat capacity values whose magnitude is dependent on the linker length (f) compounds with short linkers localize inside the nucleus only while those with long linkers occupy both the nuclear and extranuclear space within human cell lines. (g) Flow cytometry analysis of these compounds 1-5 resulted in ~5-9 fold increase in the fluorescence intensity as compared to Hoechst 33258 for both HEK293 and DU145 human cancer cell lines. Since compounds with long linkers (such as 3, 7, and 8) are known to bind bacterial enzymes where their functions are modulated by interaction of long linkers with the helical domains of the protein, we postulate that compounds with long linkers likely have extra-nucleic acid targets within the eukaryotic cells studied. Overall, these results show that bisbenzimidazole with short linkers are excellent B-DNA binders which permeate human cells and enter the nucleus easily, while those with long linkers show diminished B-DNA binding but may be good tools for additional targets in the extra-nuclear space.

ASSOCIATED CONTENT Supporting Information.

All UV thermal denaturation profiles of compounds 1-9, DSC

melting plot and excess site ITC titration plots for determination of enthalpy changes. This material is available free of charge at pubs.acs.org.


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Biochemistry

Acknowledgement. We thank National Institutes for Health for financial support (grants CA125724, R42GM097917, AI114114) to D.P.A.

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Story of a Bisbenzimidazole Dye Fragment - ACS Publications

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