Compound Shape Effects in Minor Groove Binding Affinity and Specificity for Mixed Sequence DNA

Article abstract of DOI:10.1021/jacs.8b08152

AT specific heterocyclic cations that bind in the DNA duplex minor groove have had major successes as cell and nuclear stains and as therapeutic agents which can effectively enter human cells. Expanding the DNA sequence recognition capability of the minor

Full text of DOI:10.1021/jacs.8b08152

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Article
Compound Shape Effects in Minor Groove Binding
Affinity and Specificity for Mixed Sequence DNA
Pu Guo, Abdelbasset A. Farahat, Ananya Paul, Narinder Kaur Harika, David W. Boykin, and W. David Wilson
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08152 • Publication Date (Web): 15 Oct 2018
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Compound Shape Effects in Minor Groove Binding Affinity
and Specificity for Mixed Sequence DNA
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Pu Guo1, Abdelbasset A. Farahat1,2, Ananya Paul1, Narinder K. Harika1, David W.
Boykin1, W. David Wilson1*
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1. Department of Chemistry and Center for Diagnostics and Therapeutics Georgia State
University, 50 Decatur St Se, Atlanta, GA 30303, USA.
2. Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt
Abstract
AT specific heterocyclic cations that bind in the DNA duplex minor groove have had
major successes as cell and nuclear stains and as therapeutic agents which can
effectively enter human cells. Expanding the DNA sequence recognition capability of
the minor groove compounds could also expand their therapeutic targets and have an
impact in many areas, such as modulation of transcription factor biological activity.
Success in the design of mixed sequence binding compounds has been achieved with
N-methylbenzimidazole (N-MeBI) thiophenes which are preorganized to fit the shape of
the DNA minor groove and H-bond to the –NH of G·C base pairs that projects into the
minor groove. Initial compounds bind strongly to a single G·C base pair in an AT
context with a specificity ratio of 50 (KD AT-GC/KD AT) or less and this is somewhat low
for biological use. We felt that modifications of compound shape could be used to probe
local DNA microstructure in target mixed base pair sequences of DNA and potentially
improve the compound binding selectivity. Modifications were made by increasing the
size of the benzimidazole N-substituent, for example, by using N-isobutyl instead of N-
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Me, and by changing the molecular twist by introducing substitutions at specific
positions on the aromatic core of the compounds. In both cases, we have been able to
achieve a dramatic increase in binding specificity, including no detectible binding to pure
AT sequences, without a significant loss in affinity to mixed base pair target sequences.
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Introduction
Design and preparation of agents that can recognize mixed base pair (bp) sequences of
DNA containing combinations of A·T and G·C bps have been of long term interest.
Starting in the mid-1980s, this took a step forward with work in the Dickerson and Lown
laboratories to redesign the minor groove binding polyamides netropsin (Nt.) and
distamycin (Dst.), which specifically recognizing A·T bp, into synthetic compounds that
could also bind to a G·C bp.1,2 They proposed doing this by replacing one or more
pyrrole groups in Nt and Dst with an imidazole group. The extra N in imidazole relative
to pyrrole allows the imidazole group to form an H-bond with the G-NH2 in the minor
groove of a G·C bp.1-3 The proposal attracted attention in several additional laboratories
and success in GC recognition was achieved by connecting two recognition units to
make a hairpin polyamide.4-6 Although several groups have worked with these synthetic
polyamides, solution difficulties, aggregation, and poor cell uptake have limited their
applications.7-9
In contrast to netropsin (Nt.) and distamycin (Dst.), other types of AT specific
heterocyclic cations, such as, Hoechst dyes, furamidine, DAPI, pentamidine,
bisamidinium, as well as numerous compounds that covalently attach to the minor
groove, have had major successes as cell / nuclear stains and therapeutic agents.10-18
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Interestingly, however, no systematic effort to redesign these agents into mixed bps
recognition compounds, as with the polyamides, has been made. We felt that these
promising and successful agents could potentially be redesigned to have significantly
expanded use with broadened sequence recognition capability. We have now had
considerable success in the design of agents that can recognize G·C bps as well as
AT.19-21 Such new agents would have broad applications, for example, in targeting
transcription factors to modulate gene expression.22,23
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Mutations, protein fusions or other changes that modify the activity of
transcription actors (TF), which help control critical cell functions, can lead to a number
of different kinds of cancer.23-25 Given the variety of functions and diseases controlled
by TFs, a strong interest in targeting and controlling this activity with small molecules
has developed.23 The problem is that TFs have evolved to bind high molecular weight
nucleic acids, and not, in general, small molecules.26,27 This has made finding binding
sites on TFs to interact strongly and specifically with small molecules, a difficult task. As
a result, TFs are often defined as “undruggable”.27 Our concept is to target TF-DNA
complexes by binding of designed agents to specific DNA sequences. To do this, we
must design new types of cell-permeable DNA binding agents with broader sequence
recognition capabilities than the classical AT-specific agents.
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Figure 1. A) Chemical structures of reported single G·C bp binders21 and the modified
compounds from DB2457 used in this study. B) The DNA sequences used in this study; DNA
sequences used for SPR studies were labeled with 5’-biotin.
Incorporating the N-MeBI thiophene module into the heterocyclic cations, as in
Figure 1, initially gave DB2429 which specifically binds to a target G·C bp with flanking
A·T bps.21 DB2429 was expanded to DB2457 to better cover the A·T bps that flank the
central GC. These compounds incorporate the σ-hole concept and are a significant step
forward in our molecular design and synthesis project for recognition of mixed bp DNA
sequences.19,20,28 The σ-hole interaction preorganizes the thiophene-N-MeBI unit for GC
interaction in the minor groove. Thiophene C-S single bonds present a relatively positive
electrostatic potential that can form an interaction with electron donating atoms such as
the unsubstituted N-MeBI N, a 1,4 N···S interaction. The interaction is based on the
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presence of low lying thiophene C-S σ* orbitals on S that give rise to the positive
electrostatic potential or a σ-hole.29
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Analysis of the importance of the N-MeBI-thiophene module to binding specificity
and selectively clearly shows that both the N-MeBI and thiophene are necessary for a
strong, specific interaction.21 Replacing the thiophene with a furan or pyridine, for
example, reduced both binding strength and specificity. The same was true for
replacing the N-MeBI with a benzoxazole group which in principle has the same H-
bonding capability as N-MeBI. Compounds with the N-Me substitution replaced by an
NH group bind much more strongly to pure AT DNA sequences than to any sequence
with a G·C bp.21,30 The remarkable reversal of binding specificity with a relatively small
change in the overall compound structure is an important observation in new ideas for
the design of agents to recognize specific DNA sequences. The –NH of benzimidazole
is a strong A·T bp recognizing element while the unsubstituted N of N-MeBI is a strong
G·C bp interacting element.
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The specificity ratio for binding to the single G·C bp sequence versus the pure
AT sequence is only about a factor of 10 for DB2429 while it increases to about 50 for
the larger DB2457 which binds more strongly to all sequences.21 These selectivity ratios
are too low to push these two initial compounds forward into biological testing and
additional development of the compounds is required. As described above, the strong
decrease of binding affinity and selectivity for most structural changes with DB2429 and
DB2457 raised the question of whether the selectivity ratio of these structures could be
enhanced by different types of substitutions. Because of the critical importance of high
selectivity in targeting DNA, we have investigated new modifications of these initial
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compounds that have the potential to increase the selectivity. Initial synthetic
modifications and DNA binding studies have been conducted with derivatives of the
larger, stronger binding compound, DB2457.
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From the first crystal structures of a minor groove binder to extensive structures
of heterocyclic diamidines by the Neidle group,31 and recent ideas about minor groove
from the microstructural analysis,32-36 the wider DNA minor groove in GC versus AT
sequences has been recognized. As a reference for the three binding sites of interest in
this report (Figure 1), we have used the DNA Shape algorithm of Rohs and coworkers
to estimate the minor groove widths.32 The program takes DNA sequences and predicts
a number of double helical properties, including minor groove width. The predictions are
based on an extensive compilation of DNA duplex properties from experimental
structure (PDB) as well as computational analysis by the Rohs and other groups.32-36
For AAATTT the predicted minor groove width in the center is 2.9 Å and widens to
almost 5 Å at the flanking G·C bps. At the AAAGTTT binding site, the predicted minor
groove width is between 3.3-3.4 Å, considerably wider on a molecular scale than the
AAATTT pure AT sequence. For AAAGCTTT the predicted minor groove width is over 4
Å. All sequences widen to near 5 Å at the flanking, terminal GC sequences. These
results agree with the general concept of widening of AT sequence minor grooves with
the incorporation of G·C bps.31-33
Based on these observations, the concept for the compound modifications
described here is to target DNA microstructural variations, such as the wide minor
groove in G·C bp containing regions. We reasoned that compounds with increased bulk
might favor the wider, mixed bp sequences over pure AT DNA. To test this idea the
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following general changes in the compound structure and functional groups were made
with the goal of increasing specificity: (i) by increasing compound bulk primarily by
replacing the N-Me group of N-MeBI with groups of increased size and complexity, and
(ii) by modifying the overall twist in the compound linked aromatic core structure with
appropriate substituents (Figure 1).
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As we move to design new compounds to inhibit specific transcription factors,
two things are very important: the selectivity ratio and the ability of the compounds to
enter different cell types. At least some substituent changes at the N-BI position should
have positive effects on both of these critical factors. We were able to successfully
synthesize compounds with a wide variety of substituents at the N-BI position and three
compounds with substituents on the aromatic groups of DB2457 that modified the
effective steric bulk of the compounds. With these modifications, we were able to make
significant improvements in compound specificity without a decrease in binding affinity.
Compound Design
The first set of compounds that were prepared had modifications at the N-BI
position of DB2457 (Table 1). We wanted to increase the length and complexity of the
alkyl group, for example, methyl – ethyl – isopropyl – isobutyl – neopentyl substituents.
There are also linear groups with more polar substituents as in DB2728. To significantly
change the shape of the alkyl group, compounds with cyclobutyl (DB2726), cyclopentyl
(DB2714) and cyclohexyl (DB2727) substituents were prepared. Finally, the chemistry
of the substituent was changed dramatically with different phenyl (Ph) substituents on
the BI group, N-Ph (DB2740) and N-ortho-Me phenyl (DB2747). A control compound
without an N-BI substituent, a benzimidazole, was also prepared. Two compounds, with
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an N-MeBI and an N-isopropyl-BI were prepared with the thiophene S replaced by Se to
evaluate the effect of size and changes in polarizability at that position.37,38 Four
additional compounds were prepared with substitutions designed to increase the twist of
the aromatic rings of the core structure. DB2754 has a Me on the thiophene, DB2753
has a Me on the Ph attached to the BI while DB2759 has a chloro and DB2762 has a
trifluoromethyl group adjacent to the amidine on the same phenyl ring. We were unable
to synthesize a compound with a Me adjacent to the amidine. Brief synthetic details are
given here for the synthesis of the new compounds along with Scheme 1. Full synthetic
details for all compounds are given in the Supporting Information.
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Results and Discussion
Chemistry
Scheme 1. The general synthetic approach to the target mixed bp binding diamidine
compounds
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The general synthetic approach to the target diamidine compounds 7 is outlined
in Scheme 1. Nucleophilic aromatic substitution reactions between 4-bromo-1-fluoro-2-
nitrobenzene 1 and various aliphatic and aromatic amines yields the 4-bromo-N-alkyl
and N-aryl-2-nitrobenzenes 2 in reasonable yields (41-79%), which ultimately leads to
the N-alkyl and N-aryl benzimidazoles. The nucleophilic aromatic substitution reaction
with aliphatic amines was achieved at room temperature in ethanol whereas the less
basic aryl amines required heating in dimethylacetamide in the presence of Cs2CO3 at
160 oC. Suzuki-Miyaura coupling of 4-cyanophenylboronic acids with the various 4-
bromo-N-alkyl and N-aryl-2-nitrobenzenes under standard conditions provides the nitro
biphenyl analogues 3 in good yields (51-79%). The arylphenylene diamines 4 required
for formation of the N-substituted benzimidazoles were obtained by stannous chloride
reduction of the nitro groups of 3 and were subsequently allowed to react directly
without characterization. Sodium metabisulfite mediated the oxidative condensation and
cyclization of the phenylene diamines 4 with the previously reported21 5-(cyanophenyl)-
2-formylthiophenes 5 to yield the bis-nitriles 6 in acceptable yields (39-76%). Lithium
bis(trimethylsilyl)amide in THF reacts with the bis-nitriles 6 at room temperature to yield,
after acidic work-up, the diamidines 7 generally in acceptable yields (30-79%).
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DNA thermal melting: Screening for mixed DNA sequence binding
Changes in DNA thermal melting temperature (Tm) provide a rapid way for initial ranking
of compounds for binding affinity with different DNA sequences and are shown in Table
S1. We have used the three selected sequences shown in Figure 1 for comparative
experiments. Most heterocyclic cations prepared to this time have been pure AT
sequence binders and a DNA with the binding site -AAATTT- was used to test for pure
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AT sequence binding affinity. The target DNA of primary interest has a single G·C bp
with the binding sequence –AAAGTTT-. The third sequence with two G·C bps was also
used to test for binding affinity and selectivity versus AAAGTTT.
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The reference compound, DB2457, with an N-Me substituent was previously
reported21 and has preferential binding to the single G·C bp sequence and weaker
binding to the pure AT and two G·C bps sequences (Table S1). The compounds with
the N-ethyl and N-isopropyl substituents had similar binding to AAAGTTT and weaker
binding to the pure AT sequence for improved selectivity as desired. At the level of
evaluation of Tm, most of the other derivatives at the N-X position had very weak binding
to the pure AT sequence but variable binding to the single and double G·C bps
sequences. A –Cl substituent ortho to an amidine group, DB2759, had relatively strong
binding to AAAGTTT and very low binding to the pure AT and two G·C bps sequences.
It provides a potential new lead for biological studies. Two compounds were prepared
with S in thiophene replaced with Se, but they showed no improvement in ΔTm for
AAAGTTT or in selectivity. The compounds will be of a long term interest, however, in
cell uptake studies.
Biosensor-SPR: Methods for quantitative binding
Table 1. Summary of Binding Affinity (KD, nM) for the Interaction of All Test Compounds
with Biotin-labeled DNA Sequences using Biosensor-SPR Methoda
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a. All the results in this table were investigated in Tris-HCl buffer (50 mM Tris-HCl, 100 mM
NaCl, 1 mM EDTA, 0.05% P20, pH 7.4) at a 100 µL min-1 flow rate. NB means no detectible
binding. The listed binding affinities are an average of two independent experiments carried out
with two different sensor chips and the values are reproducible within 10% experimental errors.
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A biacore sensorchip (CM5) was functionalized with streptavidin and used to
immobilize 5’-biotin labeled DNA sequences (Figure 1) in flow cells 2-4 and flow cell 1
was left as a blank, for background subtraction. With different compounds in the flow
solution, we were able to determine comparative binding constants for all of the
derivatives (Table 1). Sensorgrams were obtained for all compounds and are shown for
representative compounds with the different DNAs in Figure 2. With the AAATTT and
AAAGCTTT sequences, all compounds showed significantly weaker binding to AAATTT
than with AAAGTTT with generally fast on and off rates. No kinetics and limited KD
values could be accurately determined for those DNA sequences (Table 1). With the
AAAGTTT sequence, however, excellent sensorgrams were obtained at low
concentrations for most derivatives (Figure 2). Kinetics fits with a one site model were
excellent and allowed a determination of KD values (Table 1). Kinetics fits were
generally required to determine KD values since many sensorgrams did not reach a
steady-state level, especially at the lower concentrations.
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Figure 2. Representative SPR sensorgrams for A-C, DB2714, D-F, DB2753 in the
presence of AAATTT, AAAGTTT and AAAGCTTT hairpin DNAs. In A, C, D, F, the
concentrations of sensorgrams is 2-500 nM of each compound from bottom to top. In B,
the concentrations of DB2714 from bottom to top are 15, 20, 30, 50, and 100 nM; In E,
the concentrations of DB2753 from bottom to top are 30, 70, 100, 200, and 500 nM. In B
and E, the solid black lines are best-fit values for the global kinetic fitting of the results
with a single site function.
DB2429, the original compound (Figure 1), has a KD for AAAGTTT of 50 nM with
10 fold higher KD value for AAATTT and 20 fold higher KD value for AAAGCTTT.21 The
parent N-Me compound in this study, DB2457, has a 10 fold lower KD value for
AAAGTTT, 4 nM, with significantly better, 50 fold higher, KD values for AAATTT and
AAAGCTTT relative to DB2429. The N-ethyl and N-isopropyl compound have KD values
similar to the N-Me for single G·C bp sequence but higher values for AAATTT and
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impressive improvement in selectivity ratio of KD s of near 250. Unfortunately, they have
similar selectivity for G·C bp to the N-Me compound and it was necessary to synthesize
additional compounds for a full increase in specificity. The N-isobutyl (DB2711) has
slightly weaker binding to the single G·C bp sequence (KD = 13 nM) but the desired high
selectivity with no measurable binding to both the AAATTT and AAAGCTTT sequences.
This compound, with its broad, high selectivity in binding validates the idea of N-
substitution to enhance selectivity, a key feature for designing any new potential drug
molecules in this series. Most of the remaining substituents at the N-position in Table 1
are longer than isopropyl (DB2708) and these compounds have no detectible binding to
the narrowest full AT sequence, AAATTT, binding site. They generally have satisfactory
binding to the AAAGTTT single G sequence with only one above 21 nM KD. Most of
these compounds, however, do not have satisfactory selectivity ratios with the two GC
sequence, only two with selectivity ratio >100. It seems likely that these compounds are
able to induce a narrowing of the AAAGCTTT minor groove to provide a minor groove
that is a reasonable, but not as good as AAAGTTT, binding site.
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The isobutyl compound, as with all of the larger N-R substituted derivatives does
not fit well into the narrowest AAATTT groove and give no detectible binding to this
sequence under our conditions. The N-isobutyl compound, DB2711, is not able to
reshape the AAAGCTTT minor groove into a conformation that supports significant
minor groove binding. Relative to the N-Me, -ethyl, and -isopropyl derivatives DB2711
has only about a 3 fold increase in KD and still binds very strongly to AAAGTTT.
The second type of major substitution changes were made on the core aromatic
system. Methyl substitution on a phenyl (DB2753) and the thiophene (DB2754)
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apparently causes too much twist in the compound structure with the N-isopropyl group
and the KD values for AAAGTTT increase are 20 fold relative to the unsubstituted N-
isopropyl compound (DB2708). We also tried to increase the twist at a phenyl amidine
position (DB2759 –Cl and DB2762 –CF3) with mixed results. The –CF3 derivative
apparently has too much twist and binds relatively weakly to all DNAs. The derivative
with the smaller –Cl substitution, however, apparently has near perfect twist on the
amidine to fit the DNA microstructure at the end of the AAAGTTT sequence where there
are G·C bps and some expansion of the groove. This compound has negligible binding
to AAATTT and AAAGCTTT but only a 3 fold increase in KD relative to unsubstituted N-
isopropyl derivative (DB2708). These excellent binding results with AAAGTTT coupled
with effectively infinite selectivity over the classical recognition sequence AAATTT as
well as the two GC sequence make the –Cl (DB2759) and N-isobutyl derivatives our
current lead compounds for biological development. This entire compound set and
especially the two lead compounds provide excellent ideas for future design efforts of
minor groove binding compounds.
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The two derivatives with Se in place of S do not have results that are more
attractive than the equivalent thiophenes. The N-isopropyl derivative has about a 2 fold
increase in KD for the single GC sequence. It has weaker binding to both the AT and
two GC sequence. In other minor groove binders series, we have found that Se has
some attractive cell and nuclear uptake properties and the N-isopropyl Se compound
will be an important one for biological testing.22
Each of the compounds in Table 1 can also induce changes in the minor groove
structure of DNA and at this time an experimental analysis is the best way to find the
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optimum compound for affinity and selectivity to a specific sequence. The most
important finding, however, is that modification of DB2457 can dramatically increase
binding selectivity.
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Biosensor-SPR Kinetics
Table 2. Comparison of SPR Binding Kinetics Results for Selected Compoundsa
a. Kinetics analysis was performed by global fitting with a 1:1 binding model. The ka value is the
association rate constant, while the kd value is the dissociation rate constant obtained from the
fit. KD is given by kd / ka. The kinetics errors are based on ka and kd values obtained from three
separate, independent SPR experiments with different DNA functionalized CM5 chips. For KD,
ka, and kd the errors represent the standard errors of the mean.
A selection of compounds with a range of KD values and a variety of chemical
substitutions and structures was used to evaluate the kinetics of association, ka, and
dissociation, kd, for the AAAGTTT sequence. As can be seen in Figure 2, the binding
kinetics were generally too rapid to fit accurately with the AAATTT and AAAGCTTT
sequences. To help compare the kinetics results for AAAGTTT, log ka is plotted versus
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log kd in Figure 3 and results are summarized in Table 2. The green dashed lines in
Figure 3 plot the binding affinities (KD) in logarithmic jumps. The parent compound
DB2457 and the strongest binding compounds, DB2740 and DB2708, are located on a
KD line with a slope of 3~4 nM. Below these, there are six compounds with different, but
similar sized N-alkyl substitutions that are closely distributed around the 10 nM KD green
dashed line. The on-rates and off-rates for this group change in a correlated manner to
give similar KD values.
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KD
1 nM
10 nM
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DB2728
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DB2711
DB2740
DB2457
DB2764
100 nM
DB2712
1 uM
DB2753
-3
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-1
Log kd
Figure 3. Kinetic plot showing the relationship between on-rates (ka), off-rates (kd) and
binding affinities (KD) (Green dash line) represented on the diagonal axis for compounds
in Table 2, and determined by SPR. On-rates and off-rates and are plotted as
logarithmic values.
For the N-substituted compounds with an unsubstituted aromatic system, the KD
values with AAAGTTT in Table 2 cover a narrow range, from around 4 to 13 nM with
only two compounds above 10 nM. The kinetics values for these compounds also cover
a relatively small range for ka, from around 2 to 5 x 106 m-1s-1. The kd values cover a
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somewhat larger range, from 0.008 to 0.05 s-1 but as noted above, a general correlation
of ka and kd values accounts for the small variation in KD. The N-Me (DB2457) and N-Ph
(DB2740) compounds have the lowest KD values in this set and this arises from their
similar low kd values. DB2715 with n-butyl and DB2711 with isobutyl have the fastest
disassociation rates with the weakest binding (12.6 and 9.6 nM) to AAAGTTT among
the compounds with an unsubstituted aromatic system. These results suggest that the
very dynamic structure of the butyl substituents interfere with binding in the minor
groove giving them fast association and rapid dissociation with the highest KD values in
this set. These observations indicate that the fast association results from a lack of
optimum penetration into the minor groove and, as a result, the compounds also do not
make strong contact with the groove and dissociate rapidly. DB2764 with 2-
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methoxyethyl substituent, which is the approximately same length and flexibility as n-
butyl (DB2715), also has a relatively high kd and KD. The Se N-isopropyl derivative
DB2712 has a ka value similar to the equivalent thiophene but a higher kd and as a
result, a KD that is around 2 fold weaker. Clearly, the larger, more polarizable Se is a
slight disadvantage in DNA complexes in this set. A methyl-substituted phenyl, N-
isopropyl derivative, DB2753, has much weaker binding, KD > 100 nM, that is due to a
very low ka of 0.073 x 106 m-1s-1. The kd for this compound, however, is in the range of
other compounds in Table 2. The core aromatic system of this compound is significantly
twisted due to repulsion between the –Me substituent and the adjacent phenyl ring
(Figure 4). To bind into the minor groove, the twist must be reduced at an energy cost
that slows association with lowering of ka and an increase in KD. Once bound it appears
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that the compound can form interactions similar to the compounds with an unsubstituted
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aromatic system to give a kd in the same range.
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CD: Binding mode
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CD titration experiments are an effective and convenient method of evaluating the
binding mode and the saturation limit for compounds binding with DNA sequences. CD
spectra monitor the asymmetric environment of the compounds binding to DNA and
therefore can be used to obtain information on the binding mode.39 There are no CD
signals for the free compounds but on the addition of the compounds into DNA,
substantial positive induced CD signals (ICD) arose in the absorption region between
300 and 450 nm. These positive ICD signals indicate a minor groove binding mode by
these ligands, as expected from their structures. As can be seen from Figure S1, all
tested compounds form complexes in the minor groove of the AAGTTT sequences with
a 1:1 stoichiometry, in agreement with SPR results. In summary, the CD titration results
confirm a minor groove binding mode for the compounds in Figure 1.
Molecular Structure: Calculations
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Figure 4. (A, B, C, green label) Torsional angle maps of a Phenyl (Ph)-amidine (Am) bond for
DB2708, DB2759, and DB2762; (D, E, blue label) a Ph-BI bond for DB2708 and DB2753; and
(F, G, purple label) a Thiophene (Thio)-Ph bond for DB2708 and DB2754. A comparison of the
KD values and torsional angles/rel. Es for the three aromatic substituted compounds versus the
unsubstituted control compound, DB2708 is shown in H. The torsional angles at minimum
relative energies and the energies are also shown in H. The energy required to move the
compounds from the structurally most stable state to a 0° torsional angle is the minimum relative
E in H and the plots. All calculations are performed at the B3LYP/6-31G* level of theory. The
range of dihedral is from 0°-100°. The scanned dihedral angle calculations are shown as the
bold red line at 0° in each plot.
To help to understand the large differences in binding affinities between the N-
isopropyl compound DB2708, with no substitutions on the core aromatic system, and
the aromatic substituted N-isopropyl compounds DB2753, DB2754, DB 2759 and
DB2762, torsional angle maps were constructed in the SPARTAN 16 software
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package40 (Figure 4). The relative E in these plots is the energy required to convert the
compound torsional angle from the lowest energy angle to a planar conformation at 0°.
This gives an approximate idea of the relative energy input into the compound for a
structural change to bind to the minor groove. This analysis assumes that the torsional
angle must be between about 0-10° for optimum binding to the minor groove. The
substituted compounds all have larger KD values than DB2708 and both their lowest
relative E torsional angles and the energies required to reach 0° increase in a correlated
manner with the KD values (Figure 4H). These results clearly show why the –Cl
compound (DB2759) has a much lower KD than the –CF3 compound (Figure 4). In the
same manner, the methyl substituted compounds have large KD values and correlated
large low energy torsional angles. It seems highly likely that these conformational
energy costs are responsible for at least part of large binding constant differences for
DB2708, DB2759, DB2762, DB2753, and DB2754. It should be noted that choosing a
specific minimum torsional angle, such as 5°, leads to the same conclusions.
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Molecular Structure: Molecular Dynamics
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Figure 5. Molecular Dynamics (MD) model of DB2708 bound to an AAAGTTT site: (A) A
space-filling model viewed into the minor groove of the AAAGTTT binding site with bound
DB2708. The DNA bases are represented in tan-white-red-blue-yellow(C-H-O-N-P) color
scheme and DB2708 is light pink-white-blue-yellow (C-H-N-S) color scheme with the N-
isopropyl group facing out of the minor groove in a brighter-pink. The important
interactions between different sections of the DB2708-DNA complex are illustrated in (B)
and (C). The terminal amidine group forms a strong hydrogen bond with the carbonyl
group of a dT (yellow dashed lines), and another direct stabilizing interaction is observed
from a Ph–CH group of DB2708 which points to N3 of a dA at the floor of the minor groove
(B). In (C), the imidazole-N makes a strong hydrogen bond interaction with the exocyclic
NH of dG. The BI-C-H that points to the floor of the minor groove forms strong interactions
with the carbonyl group of a dT in the minor groove and this unit also points towards the
carbonyl group of a dC with close distances.
To help better understand the structural basis of molecular recognition of DNA
sequences with a single G·C bp in an AT context, molecular dynamics (MD) simulations
for the representative N-isopropyl compound, DB2708, were conducted41. Force
constants for DB2708 were determined as described previously and in the methods
section, and added to the force field for the simulations. After 500 ns of MD simulation,
25 PDB structures for the complex of DB2708 in the AAAGTTT binding site (Figure 5)
were collected. Structures were evaluated in detail every 20 ns (25 structures for 500
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ns) to determine what major features of the DB2708-DNA complex are primarily
responsible for the excellent stability (KD) and specificity ratios (KD of AAAGTTT/ KD of
AAATTT). There are three optimum H-bonds in the complex. Both amidine groups form
–N-H to T=O H-bonds (Figure 5B) that are an average of 2.8 Å in length (SD = 0.1 Å).
The amidines also form numerous highly dynamic H-bonds to water molecules that
move in and out of the minor groove.42 These water molecules frequently also form H-
bonds with A·T bps at the floor of the minor groove and help link the compound to the
specific binding site in the groove and stabilize the complex. The third strong H-bond is
from the central G-NH that projects into the minor groove to the unsubstituted imidazole
N in the BI group of DB2708, Figure 5C, to account for much of the binding selectivity of
DB2708. Additional selectivity in binding is provided by the –CH group of the six-
member ring of BI that points into the minor groove. This -CH forms a dynamic close
interaction with the –C=O of the dC base of the central G·C bp as well as to a T=O on
an adjacent A·T bp (Figure 5C).
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Additional direct stabilizing interactions are formed by –CH groups that point to
the floor of the minor groove from the two phenyls of DB2708. These –CH groups form
significant dynamic interactions with A-N3 groups on the bases at the floor of the groove
(see Figure 5B for an example). DB2708 tracks optimally along the minor groove with
appropriate twist to match the minor groove curvature. Extensive interactions are
formed by the conjugated aromatic-system of DB2708 with the sugar-phosphate walls
of the minor groove. In addition to H-bonds with the A·T bps, the amidines of DB2708
also form electrostatic interactions with the backbone phosphates that result in Na+
release and an entropy increase that is another stabilizing component, along with water
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release from the minor groove, on complex formation. The sum of these numerous,
relatively weak, stabilizing interactions results in strong binding of DB2708, a KD of 4 nM
(Table 1) with a ΔG of binding of 11.5 kcal/mol. The isopropyl group of DB2708 forms a
tight interaction with the DNA backbone in the G·C bp region. As noted above the
AAATTT sequence has a narrower minor groove in this central region of the binding site
than AAAGTTT and, the isopropyl group would have increased steric hindrance to
increase the KD for AAATTT binding. The KD for binding to the AAATTT sequence, for
example, increases from around 200 nM with DB2457, the N-Me derivative, to around
1000 nM with the isopropyl compound DB2708 (Table 1) to provide a significant
increase in selectivity. Clearly, many of the N-X derivatives in Table 1 can match the
minor groove microstructure in AAAGTTT better than with AAATTT. Simulation of
additional N-X derivatives and more detailed analysis will be presented in a future
paper.
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Conclusion
Here we have synthesized a series of DNA sequence-specific recognition ligands based
on the structure of DB2457 with N-MeBI-thiophenes which are preorganized by a σ-hole
interaction to fit the shape of the DNA minor groove and H-bond to the –NH of G·C bps
that project into the minor groove. Over a long period of time it has been observed that
adding a G·C bp into an entire AT DNA sequence widens the minor groove at the G·C
bp. We show some examples of the widened groove width from AAATTT to AAAGTTT
and AAAGCTTT in this report. Based on these observations, we prepared a series of
compounds with different sized substituents to determine if we could take advantage of
the difference in groove width to increase the sequence specificity of minor groove
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binder. The compounds were localized to the G·C bp in the AT sequence by an H-bond
acceptor group on an N-X substituted BI-thiophene module in all of the compounds.
Varying the X substituent on the N-X BI produced a remarkable enhancement of
selectivity for the single G·C bp sequence (Table 1). Molecular dynamics simulation
showed that the strong binding of the compound in the minor groove was the result of
numerous relatively weak interaction that can sum to give an excellent ΔG of binding.
These interactions include H-bonds, including a critical H-bond from the G-NH2 to the
unsubstituted N of the N-X BI group that accounted for a major part of the sequence
selectivity of the compounds in this series. Other important interactions are listed in the
MD section.
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A second major change in the structure of some pounds in Table 1 was caused
by adding substituents to the core aromatic system of the compound. The goal of this
modification was to increase the aromatic system twist to match the sequence-
dependent microstructural change in the minor groove. Most of the substituents
apparently caused too much twist and caused major decrease in the binding affinity.
Computational studies indicated that the minor groove of the AAAGTTT sequence
increased in width at the ends of the sequence (Figure 1). This suggested that an
increase in twist at a terminal amidine might again select for minor groove width
difference for the different sequences. A –Cl and a –CF3 substituent were placed next to
one phenyl-amidine to investigate this possibility. The –CF3 caused too much twist and
decreased binding for all sequences. The –Cl substituent, however, slightly decrease
binding to the single GC sequence but essentially abolished binding to the full AT and
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two G·C bps sequences under our conditions (Table 1) and is now one of our lead
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compounds for biological studies.
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In summary, the compound design concept described and illustrated in this
report provide now the idea for the field. The compounds in Table 1 show that high
specific binding to DNA can be obtained for minor groove binders. It seems likely that
more complex sequence recognition can be obtained by combining recognition modules
together such that many different target sequence can be selectively targeted. Similar
idea for targeting RNA could greatly enhance therapeutic targeting of RNA.
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Associated Content
Supporting Information. Experimental details, DNA melting experimental data table, CD
spectrums, Molecular structure with specific atom types of DB2708, Frcmod file of the
DB2708, 1H NMR spectra of key Intermediates and final products
Author Information
Corresponding Author: W. David Wilson, wdw@gsu.edu
Notes: The authors declare no competing financial interest.
ORCID:
Pu Guo: 0000-0002-4599-2037
Abdelbasset A. Farahat: 0000-0003-0664-5670
Ananya Paul: 0000-0003-4592-3442
Narinder K. Harika: 0000-0002-3637-6697
David W. Boykin: 0000-0001-9712-8278
W. David Wilson: 0000-0001-5225-5089
Acknowledgment
We thank the National Institutes of Health Grant GM111749 (W.D.W. and D.W.B.) for
financial support. We also thank the Leukemia & Lymphoma Society (LLS) award:
Leukemia & Lymphoma Society 6504-17 for support.
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