Investigation of the factors that dictate the preferred orientation of lexitropsins in the minor groove of DNA

Article abstract of DOI:10.1021/acs.jmedchem.9b01534

Lexitropsins are small molecules that bind to the minor groove of DNA as antiparallel dimers in a specific orientation. These molecules have shown therapeutic potential in the treatment of several diseases; however, the development of these molecules to target particular genes requires revealing the factors that dictate their preferred orientation in the minor grooves, which to date have not been investigated. In this study, a distinct structure (thzC) was carefully designed as an analog of a well-characterized lexitropsin (thzA) to reveal the factors that dictate the preferred binding orientation. Comparative evaluations of the biophysical and molecular modeling results of both compounds showed that the position of the dimethylaminopropyl group and the orientation of the amide links of the ligand with respect to the 5′-3′-ends; dictate the preferred orientation of lexitropsins in the minor grooves. These findings could be useful in the design of novel lexitropsins to selectively target specific genes.

Full text of DOI:10.1021/acs.jmedchem.9b01534

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Article
An investigation of the factors that dictate the preferred
orientation of lexitropsins in the minor groove of DNA
Hasan Y. Alniss, Ini-Isabée Witzel, Mohamad H. Semreen, Pritam
Panda, Yogendra Kumar Mishra, Rajeev Ahuja, and John A. Parkinson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01534 • Publication Date (Web): 28 Oct 2019
Downloaded from pubs.acs.org on November 5, 2019
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An investigation of the factors that dictate the preferred orientation of
lexitropsins in the minor groove of DNA
a,b*
c
a,b
d
Hasan Y. Alniss , Ini-Isabée Witzel , Mohammad H. Semreen , Pritam K. Panda , Yogendra
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Kumar Mishra , Rajeev Ahuja , and John A. Parkinson
a
College of Pharmacy, University of Sharjah, P.O. Box 27272, Sharjah, United Arab Emirates.
b
Sharjah Institute for Medical Research, University of Sharjah, P.O. Box 27272, Sharjah, United
Arab Emirates.
c
Core Technology Platform, New York University of Abu Dhabi, P.O. Box 129188, Saadiyat
Island, Abu Dhabi, United Arab Emirates
d
Department of Physics and Astronomy, Uppsala University, Box 516, SE-75120, Uppsala
Sweden
e
Functional Nanomaterials, Institute for Materials Science, Kiel University, Kaiserstr. 2, D-
2
4143 Kiel, Germany
f
Department of Materials and Engineering, Royal Institute of Technology (KTH), SE-10044,
Stockholm Sweden
g
WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, 295
Cathedral Street, Glasgow G1 1XL, United Kingdom
Keywords: minor groove binders (MGB), lexitropsins, Androgen Response Element (ARE),
Isothermal Titration Calorimetry (ITC), Molecular modeling
*Corresponding author: Phone: +(971)-6-5057427. Fax: +(971)-6-5585812. E-mail address halniss@sharjah.ac.ae
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Abstract
Lexitropsins are small molecules that bind to the minor groove of DNA as antiparallel dimers in a
specific orientation. These molecules have shown therapeutic potential in the treatment of several
diseases; however, the development of these molecules to target particular genes requires revealing
the factors that dictate their preferred orientation in the minor grooves, which to date have not been
investigated. In this study, a distinct structure (thzC) was carefully designed as an analog of a well-
characterized lexitropsin (thzA) to reveal the factors that dictate the preferred binding orientation.
Comparative evaluations of the biophysical and molecular modeling results of both compounds
showed that the position of the dimethylaminopropyl group and the orientation of the amide links
of the ligand with respects to the 5’-3’-ends; dictate the preferred orientation of lexitropsins in the
minor grooves. These findings could be useful in the design of novel lexitropsins to selectively
target specific genes.
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1-Introduction
The cationic lexitropsins, which bind reversibly to the minor groove of DNA, have been shown to
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have therapeutic efficacy in the treatment of cancer, bacterial, viral, and fungal diseases. The
biological significance of these compounds arises from their ability to bind reversibly in the minor
groove of DNA and to control gene expression without inducing permeant DNA damage, which
is commonly encountered with the use of currently available toxic chemotherapy. The significance
of minor groove binders (MGBs) in anti-infective and anti-cancer therapy is currently being
investigated as a promising new class of antibiotics for the treatment of Clostridium difficile
infections, 2-4 and as novel transcription factor antagonists for the treatment of prostate cancer.5-7
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A remarkable advancement in the MGB research has come from the discovery that distamycin
binds to the minor groove as an antiparallel side-by-side dimer, in a head to tail fashion.8-10 This
opened new perspectives for developing novel MGBs with base-pair selectivity.11-14 Thiazotropsin
A (thzA), which is a synthetic analog of the natural polyamide distamycin A (Figure 1), can bind
selectively to the 5’-WCWWGW-3’ sequence (where W is A or T).15-17 Its structure has a crescent
shape that matches the curvature of the DNA minor groove. This geometrical fitness allows the
ligand to bind with the minor groove of duplex DNA as an antiparallel dimer via hydrogen bonding
with DNA bases (Figure 2B). This binding causes alterations in the DNA structure by widening
the minor grooves and bending the DNA helix toward the major groove leading to a compression
of the major groove. The ring slippage of the side-by-side minor groove binders (such as thzA,
Figure 2A) has enabled these ligands to span six base pair DNA sequences, contrary to the
expectation of four base pairs with respect to the ligand dimensions.12, 18, 19 The importance of this
in terms of gene targeting is that such ligands with small molecular weight and enhanced
lipophilicity can be used to disrupt the binding of transcription factors to the response element of
the target gene, which is composed of six base pair sequences. A response element is a short DNA
sequence within the promoter region of a gene, which can bind to a specific receptor complex
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(transcription factors) and therefore regulate transcription of genes. There are many examples of
transcription factors that recognize hormone response elements composed of a six base pair DNA
sequence, such as the androgen receptor (AR), progesterone receptor (PR), glucocorticoid
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receptors (GR), and mineralocorticoid receptor (MR). The androgen receptor recognizes the
androgen response element (ARE), which is organized as repeats of the consensus DNA sequence
5′-WGWWCW-3' (W is A or T), with a three-nucleotide spacer.20, 21 Malfunction of these
transcription factors can lead to dysregulated gene expression, which is observed in many human
cancers such as prostate cancer.22-24
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H
NH
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C)
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Distamycin A
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H
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Thiazotropsin A
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Figure 1. Structures of (A) distamycin, (B) thiazotropsin A and (C) NMR refined solution structure of a
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:1 complex of thiazotropsin A: DNA (PDB 1RMX). The figure was developed using PyMOL.
A)
B)
Figure 2. A) Schematic showing the alignment of thiazotropsin A dimer relative to the DNA sequence.
B) The arrangement of hydrogen bonding between thiazotropsin A and the DNA
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sequenced(CGACTAGTCG) . Color coding: magenta diamond, formyl head; yellow pentagon,
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isopropyl thiazole; red pentagon, N-methylpyrrole; blue triangle, dimethylaminopropyl group.
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The ability of thzA and its analogs to recognize a DNA sequence very similar to that of the ARE
(for instance, thzA can recognize the 5′-WCWWGW-3′ sequence W is A or T) is encouraging',
and the development of these ligands is therefore of value in order to target the ARE sequence and
other promoter sequences. The small molecular weight of the lexitropsins, which are composed of
only three aromatic rings, and their enhanced lipophilicity via the isopropyl thiazole system, could
offer them additional beneficial physical properties in terms of absorption and oral bioavailability
compared with the large molecular weight cyclic polyamide ligands or hairpin structures, which
were developed by the Dervan group.5, 18, 25 Moreover, although one might expect that the larger
polyamide ligands (composed of 8 heteroaromatic rings) would have much higher binding affinity
as they could form larger number hydrogen bonds with DNA, however, the fact that lexitropsins
are able recognize the minor grooves as dimers enable them to form comparable number of
hydrogen bonds with same binding site (5’-WGWWCW-3’). Tremendous research efforts have
focused on revealing the factors that drive thiazotropsins-DNA associations, particularly the
ligand's structural features that dictate DNA sequence selectivity, and significant progress was
achieved in exploring structure-activity relationships (SARs) of these compounds, which is vital
for the rational design and development of new MGBs. For instance, the binding properties of
thzA16, 17 (Figure 1) and its analogs, P22, P36, thiazotrobcin B and AIK 18-51 (Figure 3) are well
characterized. 15-17, 26, 27 NMR, circular dichroism (CD) and isothermal titration calorimetry (ITC)
studies revealed that thzA and its analogs recognize the minor groove of DNA as a slipped dimer
where two of the three aromatic rings from each ligand stack on top of each other, allowing the
ligand to read six base pairs (Figure 2 & 4). The ligand binds specifically to the central four base
pairs (CWWG, W is A or T) while the other two bases at the edge of the binding site are covered
by the dimethylaminopropyl (DAP) tail, which cannot differentiate between the four DNA bases.
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The pairs of Thz/Pyr or Thz/Fmyl recognize G·C; Pyr/Thz or Fmyl/Thz recognize C·G but not
A·T or T·A; the Pyr/Pyr pair does not distinguish
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P36
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Figure 3. Structures of the thiazotropsin A analogs.
Figure 4. A schematic shows all the possible DNA recognitions by the slipped dimer of
thiazotropsin A. * A steric clash between the NH of guanine and the DAP of ligand reduces the
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affinity. Fmy, formyl “head”; Pyr, N-methylpyrrole; Thz, thiazole; DAP, dimethylaminopropyl
“
tail.”
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T·A and A·T but will not bind C·G or G·C; and the DAP tail covers the flanking bases, A, T, C,
or G. A reduced affinity was observed when the DAP tail lies adjacent to G’s because of a steric
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clash with the exocyclic NH group. These findings are consistent with Dervan’s pairing rules
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for minor groove recognition.28
The development of these small molecules to selectively target specific DNA sequences requires
an understanding of the factors that dictate the preferred ligand orientation in the minor groove
concerning the 3’ and 5’ ends, which have not been thoroughly investigated to date. In all the
studies that have been conducted, thzA and its analogs (P22, P36, and AIK 18-51) bind to 5′-
WCWWGW-3′ in the minor groove as dimers in an anti-parallel side-by-side fashion and only a
single orientation of the ligand dimer has been observed: the positively charged DAP groups are
orientated towards the 3’-ends of the DNA strands (Figure 4). The role of isopropyl thiazole in
guanine recognition is clearly understood; however, the question that remained unanswered is the
role of the DAP group in dictating the preferred dimer orientation toward the 3’-ends, and whether
reversing the position of the DAP group (from N- to C-terminus) in the ligand structure will change
the preferred dimer orientation toward the 5’-ends or not?
In the current work, a new compound, thiazotropsin C (thzC), Figure 5, was carefully designed
and synthesized to investigate the role of the DAP tail in dictating the preferred orientation of the
lexitropsins anti-parallel dimer in the minor groove of DNA with respect to the 5’- and 3’-ends.
The proposed structure, thzC, was developed from the structure of thiazotropsin A. In the new
structure, the positions of both the DAP tail and the isopropyl thiazole in thzA were changed: the
DAP group is attached to the C-terminus, and the isopropyl thiazole replaces the pyrrole ring
located towards the C-terminus. Studying the DNA binding behavior of this compound compared
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to that of thzA helps in both revealing the factors that dictate the preferred orientation of
lexitropsins anti-parallel dimers in the minor groove of DNA, and the development of these small
molecules to target specific genes such as the androgen response (ARE).
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C-terminus
NH
H N
2
C-terminus
NH
NH2
N
O
O
HN
S
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N
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H
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Flip 180°
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Thiazotropsin C
NH
H
N-terminus
NH
O
HN
H
N
N
N
S
O
HN
O
N
O
Thiazotropsin A
Figure 5. Structure of thiazotropsin A (DAP at the N-terminus) and its proposed analog, thiazotropsin C
DAP at the C-terminus).
(
3
3
. Results and discussion
.1 Chemical Synthesis
The synthesis of the target compound, thiazotropsin C, was achieved by using convergent
synthesis. The detailed synthetic PDB pathway is summarized in Scheme 1. To synthesize
thiazotropsin C, several MGB monomers were prepared (1, 2, 3, 4, and 5, Scheme 1). Methyl-2-
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amino-5-isopropyl-1,3-thiazole-4-carboxylate 1 was prepared in good yield (70 %) by reacting
methyl dichloroacetate with isobutyl aldehyde at 0 ºC in diethyl ether in the presence of sodium
methoxide as a strong base to give the α-chloro glycidic ester intermediate 1a (Darzens reaction,
3
0
Supporting Information S2). Since the α-chloro glycidic ester is highly reactive, it was directly
used for the second step without further purification. The second step involves the coupling
between the α-chloro glycidic intermediate 1a and thiourea in dry methanol to give the methyl 2-
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amino-5-isopropyl-1,3-thiazole-4-carboxylate 1. The reaction proceeds via nucleophilic attack of
the thiourea sulfur atom on the epoxide carbon which leads to epoxide ring-opening and the
formation of new α-carbonyl moiety. The formation of the isopropylthiazole ring was achieved by
subsequent nucleophilic attack of the thiourea amino group on the α-carbonyl carbon in a common
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Schiff’s base reaction to form the thiazolidine ring (Scheme 2). Two dehydration steps of the
thiazolidine ring then restore the aromaticity of the thiazole ring. 2-Trichloroacetyl-1-
methylpyrrole (2) was prepared in good yield (77%) by the reaction of trichloroacetylchloride and
1
-methylpyrrole. 2-Trichloroacetyl-1-methyl-4-nitropyrrole (Compound 3) was prepared by the
nitration of 2-trichloroacetyl-1-methylpyrrole (2) with nitric acid in acetic anhydride, in good yield
70%). The presence of the deactivating group, trichloroacetyl, attached to the pyrrole ring plays
(
an important role in improving the yield of nitro product. Nitration of the carboxylic acid derivative
of 3 produced much lower yield (25 %). These results suggest that the trichloroacetyl group is
more efficient than the carboxylic acid in deactivating the electron-rich pyrrole nucleus, thus
reducing the poly-nitration of N-methylpyrrole ring at position C3 and C5. The amide product 4
was obtained after preparing 3, which was reacted with ammonium hydroxide to give 4 in a very
good yield (81%).
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4
4
5
5
5
5
5
5
5
5
5
5
6
N
O
Cl
Cl
Cl
Cl
2
O
O N
2
CCl3
H
Cl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
HNO Ac O
3/ 2
O
N
CCl3
O
Cl
O
N
3
O
NH OH
4
Cl
O
O N
2
O
N
OH
1a
NH2
O
N
8
O
4
O
S
Oxalyl chloride
Reflux
H N NH2
2
Pd/C, H2
N
N
O N
Cl
O
H N
2
CO Me
2
2
9
H N
2
CCl3
S
1
NH2
N
3
O
N
5
O
H
N
N
N
O N
2
CO Me
2
O
S
H
NH2
O
N
N
6
1
0
O
N
LiOH
Pd/C, H2
H
OH
O
N
H N
2
N
N
S
H
N
NH2
O
11
N
7
HBTU
O
O
N
N
H N
2
O
NH
O
N
N
H
HN
S
N
12
O
O
N
Scheme 1: Synthetic pathway for thiazotropsin C.
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NH
H
thiazolidine
O
Cl H
Cl
Cl
O
NH
H
N
S
O
H
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
S
S
H
HO
O
CH3
O
H N
2
NH2
H C
3
O
O
CH3
H
O
H
H
H
O
NH
NH
S
NH
Cl
H
H
N
S
N
N
S
H
H O
2
O
O
O
N
O
O
O
CH3
CH3
CH3
NH2
S
O
O
H C
3
Scheme 2: Mechanism of thiazole ring formation by the coupling between α-chloro glycidic ester
and thiourea.
Compound 5 was prepared by reducing the nitro compound 4 by catalytic hydrogenation. This
compound was directly used for the next step of synthesis due to the lack of stability of the amino
pyrrole product. The amide-linked pyrrole dimer 6, N-(5-carbonyl-1-methyl-1H-pyrrol-3-yl)-1-
methyl-4-nitro-1H-pyrrole-2-carboxamide, was prepared by the coupling between 3 and 5. The
reaction mixture was heated to 70 ºC overnight, and that was enough to form the amide linkage
directly by releasing the trichloromethyl group (it leaves as chloroform) from the carbonyl group.
This route was efficient, quick and less laborious. It saved the time required for the synthesis of
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the carboxylic acid derivative of 3 and the use of coupling agents to activate the carboxylic acid to
form the amide link. Compound 7 was prepared by reducing the nitro group of 6 via catalytic
hydrogenation using Pd-C/H . The amino compound 7 was used directly for the next step of
2
synthesis due to the lack of stability of the amino group attached to the pyrrole ring.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-(Dimethylamino) butanoyl chloride 9 (Scheme 1) was prepared by refluxing 8 in oxalyl chloride
to produce the acid chloride, which was directly coupled with the amine compound 1 to obtain 10
in good yield (70 %). Compound 11 was obtained by the basic hydrolysis of the methyl ester in
compound 10 to corresponding carboxylic acid in good yield (80%). Mild basic hydrolysis
conditions (lithium hydroxide at room temperature) were used for the hydrolysis of the methyl
ester in order to avoid the hydrolysis of amide group in the compound.
Thiazotropsin C, 12, N-(5-(5-carbonyl-1-methyl-1H-pyrrol-3-ylcarbomyl)-1-methyl-1H-pyrrol-
3
-yl)-2-(4-(dimethylamino)butanamid)-5-isopropylthiazole-4-carboxamide, was prepared by
coupling of the pyrrole dimer amine 7 with the carboxylic acid 11 using HBTU as a coupling
1
13
agent. Structures of all intermediates and final products were characterized by using H and C
NMR spectroscopy and mass spectrometry.
3
.2 The rationale of ligand design and interpretation of possible binding orientation
Thorough structural and biophysical studies have been previously conducted for the
characterization of thzA-DNA associations. These studies revealed that thiazotropsin A binds to
the 5′-WCWWGW-3' sequence (W is A or T) in the minor groove of DNA as a dimer in an anti-
parallel side-by-side fashion, and most interestingly, only one orientation of the ligand dimer was
observed: the positively charged DAP groups are orientated towards the 3’-ends of the DNA
strands (Figure 2A). However, the ligand's structural features that dictate the preferred dimer
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orientation with respect to the 5’-3’-ends, especially the role of the DAP group, are still not
understood.
The proposed ligand structure, thiazotropsin C, was therefore specifically designed to investigate
the factors that dictate the preferred orientation of lexitropsin anti-parallel dimers in the minor
groove of DNA by changing the positions of both the DAP tail (dimethylaminopropyl) and the
isopropyl thiazole in thzA (Figure 5). In theory, there are four possibilities for the binding of
thiazotropsin C to the minor groove if it is compared with the binding properties of the lead
compound thzA with 5′-WCWWGW-3′. Each possibility would suggest certain ligand structural
features responsible for DNA minor groove recognition, which might reveal the factors that dictate
the preferred orientation of lexitropsins anti-parallel dimers with respect to 5′-3′ ends.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1- ThzC recognizes only the 5′-WCWWGW-3′ sequence as a dimer, with its positively
charged DAP group lying towards the 3’-end, and the carbonyl group of each amide
(colored blue) linking the aromatic rings is closer to the 3’-end than the partnering NH
(Figure 6A). If the biophysical experiments confirm the binding of thzC to the 5′-
WCWWGW-3' sequence, such results would suggest that the orientation of lexitropsins in
the minor groove is dictated by the positively charged DAP group of the ligand. In this
case, the DAP group always prefers to point towards the 3′-end regardless of its position
in the ligand structure at the N- or C- terminus, and regardless of the two possible
orientations of the amide links (blue/red colored amides) with respect to the 5’-3’-ends.
These conclusions are based on the fact that the DAP group in thzA, which is linked to the
N-terminus, always points toward the 3’-end (Figure 7A). Consequently, if thzC, which
has its DAP group linked to the C-terminus, also prefers to point toward the 3’-end, such
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B)
Figure 6 Representation of the possible binding orientations that may be adopted
by thiazotropsin C in the minor groove of DNA. A) The ligand is aligned in a 5’-3’
direction and recognizes the 5’-WCWWGW-3’ sequence. B) The ligand is aligned
in a 3’-5’ direction and recognizes the 5’-WGWWCW-3’ sequence.
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results will indicate that the DAP group is responsible for dictating the preferred orientation
of lexitropsin dimers in the minor groove of DNA.
2
- ThzC recognizes only the 5′-WGWWCW-3′ sequence as a dimer, with its positively
charged DAP group lying toward the 5’-end and the carbonyl group of each amide (colored
red) linking the aromatic rings is closer to the 5’-end than its partnering NH (Figure 6B).
If the biophysical experiments confirm the binding of thiazotropsin C to the 5′-
WGWWCW-3′ sequence, such results would suggest that the factor that dictates the
orientation of lexitropsin ligands in the minor groove is the orientation of the amide links
with respects to the 5’-3’-ends. In this case, the amide link always prefers an orientation
where its carbonyl group is closer to the 5’-end than the partnering NH regardless of the
DAP group position in the ligand structure, whether it is attached to the N- or C- terminus.
This conclusion is supported by the fact that thzA recognizes 5′-WCWWGW-3′, where its
carbonyl group is closer to the 5’-end than the partnering NH (Figure 7A).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
- ThzC exhibits both orientations. If the biophysical experiments confirm the binding of thzC
to both the 5′-WGWWCW-3′ and 5′-WCWWGW-3′, such results would indicate that the
DAP tail and the orientation of the amide links with respects to the 5’-3’-ends play no role
in dictating the orientation of lexitropsin dimers in the minor groove of DNA. This lack of
sequence selectivity could happen if the ligand dimer is highly flexible and can adopt
multiple conformations to match the helical structure of the DNA minor grooves at
different binding sites. However, based on the flexibility of thzC, which is similar to that
of thzA, this scenario is not expected, because the biophysical studies on thzA have shown
that lexitropsins prefer to adopt a single orientation (Figure 7).
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
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5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
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9
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A)
B)
Figure 7 Representation of the confirmed single binding orientations adopted by thiazotropsin
A in the minor groove of DNA A) The ligand aligned in a 5'-3’ direction, recognizing the 5’-
WCWWGW-3’ sequence B) Biophysical studies showed that thzA does not recognize the 5’-
WGWWCW-3’ sequence, thus ligand alignment in a 3’-5’ direction is not possible.
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4- In the fourth scenario, the ligand does not recognize both orientations. If the biophysical
experiments show that thzC neither binds to 5′-WGWWCW-3′ nor 5′-WCWWGW-3′ or
very weak binding is observed, such results would suggest that both the position of the
DAP group and the orientation of the amide links with respects to the 5’-3’-ends dictate
the preferred orientation of lexitropsin ligands in the minor groove of DNA. The inability
of thzC to recognize the 5’-WGWWCW-3′ presumably indicates a significant steric clash
between the DAP group and the backbone of DNA when the DAP, which is attached to the
C-terminus, is lying toward the 5’-end (Figure 6B). These results would suggest that the
orientation of lexitropsins in the minor groove is dictated by the position of the positively
charged DAP of the ligand. In this case, the DAP group should be attached to the N-
terminus and always prefers to point towards the 3′-end. The inability of thzC to recognize
the 5’-WCWWGW-3′ sequence would indicate that the orientation of the amide links with
respects to the 5’-3’-ends also dictates the preferred orientation of lexitropsin dimers in the
minor groove. In this case, the amide link always prefers an orientation where its carbonyl
group is closer to the 5’-end than the partnering NH, which implies that this orientation is
crucial for the formation of hydrogen bonds with DNA bases.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Recent data from Dervan’s group have shown that the hairpin and cyclic polyamide minor groove
binders, comprised of 8 imidazole/pyrrole rings which bind to the ARE (5′-WGWWCW-3′), can
5
regulate the expression of androgen receptor target genes in cell culture studies. Although it was
concluded from these studies that the relatively large and polar polyamide structures are cell-
permeable, absorption studies carried out on cyclic polyamide compound showed low Caco-2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
permeability, suggesting that it might not be orally bioavailable.25 The ability of lexitropsins to
1
7
recognize a DNA sequence very similar to that of the ARE (for instance, thzA can recognize the
5
′-WCWWGW-3′ sequence, where W is A or T) is encouraging and understanding the factors that
dictate the preferred dimer orientation of lexitropsin in the minor groove is crucial for the
development of these ligands in order to target the ARE sequence. The small molecular weight of
the lexitropsins, which are composed of only three aromatic rings and their enhanced lipophilicity
via the isopropyl thiazole system, could offer them additional beneficial physical properties
compared with the large molecular weight polyamide ligands. Furthermore, additional lipophilic
functional groups could be introduced without sacrificing binding affinity as these substituents
point out from the minor grooves.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.3 Biophysical testing and evaluation of thiazotropsin C-DNA association
The interaction of thiazotropsin C with ten dodecamer ODNs (5’-GCGAGTACTCGC-3’)d, (5’-
GCGAGATCTCGC-3’)d, (5’-GCGTGATCACGC-3’)d, (5’-GCGTGTACACGC-3’)d, (5’-
GCGACTAGTCGC-3’)d, (5’-GCGACATGTCGC-3’)d, (5’-GCGTCATGACGC-3’) d, (5’-
GCGTCTAGACGC-3’)d, (5’-GCGAGAACACGC-3’)d, (5’-GCGTGTTCTCGC-3’)d were
evaluated using Isothermal Titration Calorimetry (ITC). ITC is used to directly determine the
thermodynamic profile of intermolecular binding at constant temperature and that includes the
determination of enthalpy (ΔH), entropy (ΔS), binding affinity (K), stoichiometry (N), and free
energy of binding (ΔG).32-35 ITC experiments were carried out at low salt concentration in order
to avoid the competition between the ions of salt and thzC molecules, which would be more
pronounced at higher salt concentration. The ODNs were selected based on the 5’-WCWWGW-
3
’ sequence, the binding sequence of thzA, and its complementary sequence, 5’-WGWWCW-3’,
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which might be recognized by thzC if its orientation is reversed in the minor grooves. The ITC
results showed that thzC does not recognize both the 5’-WGWWCW-3’ and 5’-WGWWCW-3’
frame (Figure 8). A control experiment was first performed by titrating the ligand into the buffer
solution, in which endothermic peaks appeared as a result of ligand dilution in the buffered
solution. ThzC was then titrated to solutions containing ten different DNA dodecamers (supporting
information S3); however, no change in the heat was observed; only endothermic peaks appeared
which were identical to those of ligand dilution in the buffer. The inability of thzC to recognize
both 5’-WGWWCW-3’ and 5’-WGWWCW-3’ suggests that the factors that dictate the orientation
of lexitropsin dimers in the minor grooves are both the position of the DAP group in the structure
of the ligand and the orientation of the amide links with respect to the 5’-3’-ends. This implies that
the DAP group must be attached to the N-terminus and point toward the 3’-ends to avoid a steric
clash with the backbone of DNA, and the carbonyl of amides must be closer to 5’-ends than its
partnering NH to effectively form hydrogen bonds with the DNA bases.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For the 5’-WGWWCW-3’ sequence, given that the heterocyclic units of thzC dimer are
compatible with the central GWWC sequence, the inability of thzC to recognize this sequence
indicates a significant steric clash between the DAP group and the backbone of DNA or DNA
bases. To evaluate this conclusion, the active conformation of the DAP group in the resolved NMR
1
9
structure of the complex between d(CGACTAGTCG) and thzA was inspected. The NMR
2
structure of thzA-DNA complex shows that DAP is folded in a way to enable the protonated
tertiary amine to form an intra hydrogen bond with the adjacent carbonyl oxygen (Figure 9). This
folded conformation makes the DAP group of each ligand in the dimer lie in the middle of the
minor grooves, deviated toward the opposite DNA strand. The folded conformation of the DAP
group may explain why there is one preferred orientation for the thzA dimer to recognize the minor
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
groove with respect to the 5-’ and 3’-end. Reversing the position of ligand 1 and 2 of the thzA
dimer (flipped thiazotropsin A dimer-FthzA), as shown in Figure 10B is expected to allow FthzA
to read the 5’-WGWWCW-3’ sequence, however biophysical studies showed that this recognition
is not possible17, 19 and that this is presumably due to a significant steric clash between the DAP
group and the walls of the minor groove when the DAP group points toward the 5’-end. Moreover,
in the FthzA dimer, the orientations of the ligand amide links with respect to the 5’ and 3’ ends
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(1) Control Exp.: ThzC-Buffer
(2) ThzC/5’-AGTACT-3’
(3) ThzCl5’-ACTAGT-3’
Figure 8. ITC titrations of thzC to (1) PIPES Buffer (control exp.) (2) d(5’-GCGAGTACTCGC-3’) (3)
d(5’-GCGACTAGTCGC-3’). No change in the heat was observed upon ligand titration to the DNA
dodecamers suggesting the absence of interaction.
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Figure 9. Representation of the NMR refined solution structure of thzA complex with 5′-ACTAGT-3′
(1rmx) showing the confirmation of the DAP group. The protonated nitrogen of the DAP group is attracted
to the carbonyl oxygen to form a strong intra hydrogen bond.
are reversed, and this might disrupt the hydrogen bonds formed between the ligand amide groups
and DNA bases. Hydrogen bonds are directional, and their strength is also profoundly affected by
the distance between hydrogen bond acceptor and donor. These assumptions were supported by a
molecular modeling study, as shown in the next section.
The inspection of the NMR structure of thiazotropsin B-DNA complex also shows that the DAP
1
5
group of thzB adopts the folded conformation. If we assume that the DAP group of thzC, which
is attached to the C-terminus, adopts the folded confirmation, this can explain why thzC does not
recognize both 5’-WGWWCW-3’ and 5’-WCWWGW-3’. The alignment of the thzC dimer in the
minor groove of 5′-WGWWCW-3′ is shown in Figure 10C. Here the heterocyclic units of
ligand dimer are compatible with the central GWWC. The carbonyl of the amide groups is closer
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Thiazotropsin A dimer
Thiazotropsin C dimer
C)
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A)
B)
D)
Figure 10 Illustrations of the feasible and unfeasible dimer orientations of thzA and thzC in the minor
groove of DNA. A) thzA-WCWWGW (confirmed recognition): the DAP group is attached to the N-
terminus and points toward the 3’-ends, and the carbonyl of amides is closer to 5’-ends than its partnering
NH. B) Flipped thiazotropsin A (FthzA)- WGWWCW (no recognition): the DAP group points toward the
5
’-ends (steric clash), and the carbonyl of amides is closer to 3’-ends than its partnering NH (disruption of
hydrogen bonds). C) thzC-WGWWCW (no recognition): the DAP group points toward the 5’-ends (steric
clash). D) thzC-WCWWGW (no recognition): the carbonyl of amides is closer to 3’-ends than its partnering
NH (disruption of hydrogen bonds).
to the 5’-end than its partnering NH, which is similar to the known and confirmed orientation of
amide groups in the thzA complex with 5′-WGWWCW-3′ (Figure 10A). However, although
this dimer configuration is supposed to enable the amide groups of thzC to form hydrogen bonds
with DNA bases, the folded conformation of the DAP group, which is lying toward the 5’-end,
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causes significant steric clash with the backbone of DNA thus preventing the recognition of the
5’-WGWWCW-3’ sequence (Figure 10 C).
Reversing the position of ligand 1 and 2 of thzC dimer (flipped thiazotropsin C dimer- FthzC), as
shown in Figure 10D, is expected to make it compatible with the 5’-WCWWGW-3’ sequence. In
this dimer configuration, the DAP group is lying toward the 3’ end in the middle of minor grooves
with no steric clash with the backbone of DNA. However, the inverted configuration of FthzC
dimer changes the orientation of amide groups with respect to the 5’ and 3’ ends, which is expected
to disrupt the hydrogen bonds responsible for the ligand -DNA interactions, thus preventing the
recognition of 5’-WCWWGW-3’ sequence.
1
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.3 Molecular modeling
.3.1 Static structural analysis
An insight into the intrinsic atomic structure of a docked complex to depict and analyze its stability
can reveal inter- and intramolecular bonding, which contributes to its binding affinity and stability.
Hydrogen bonds play a key role in the intermolecular interactions, and in predicting the shapes
36
and properties of biomolecules. UCSF Chimera program was used for the analysis of hydrogen
bonds and steric clashes associated with thzA-DNA and FthzA-DNA complexes (Supporting
Information, S4) respectively. The formation of different types of intermolecular hydrogen bonds
between H-bond donor (D) and acceptor (A) (D...A and D–H...A) are well established. The average
distances of hydrogen bond in nucleic acid structures are close to 3.0 Å according to the Watson
and Crick pairing model. The strong hydrogen bonds with D...A distance of 2.2–2.5 Å are mostly
covalent, 2.5–3.2 Å are moderate and mostly electrostatic, and 3.2–4.0 Å are weak and mainly
electrostatic.37, 38 The availability of a refined NMR structure for thzA association with 5’-
1
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ACTAGT-3’ (1rmx) facilitated the assessment of potential steric clashes and the formation of
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hydrogen bonds in the complexes under investigation. Analysis of hydrogen bonds for thzA and
FthzA complexes with DNA were depicted in Table 1 and Figure 11. Ten hydrogen bonds were
formed in the thzA-DNA complex, whereas, only two weak hydrogen bonds were observed in the
FthzA-DNA complex indicating the disruption of hydrogen bonds in this complex when the
carbonyl of amide links of the ligand is closer to the 3’-ends of DNA than its partnering NH (Figure
10 B).
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In ligand-bound complexes, the unnatural overlap of any two non-bonding atoms or moieties in
a complex structure often leads to steric clashes that may affect the stability of the macromolecule.
Such a steric clash was observed in case of FthzA-WGWWTW complex (Table 2), whereas in
1
rmx (thzA-ACTAGT complex), no steric clashes were noticed. Both the formyl head and DAP
tail of thzA encountered steric clashes with C7-G14 and G4-C17 base pairs regions respectively
(Figure 12, Table2) , and this may explain the inability of the flipped thzA dimer to recognize the
5
’-WGWWCW-3’ sequence when the DAP tail is lying toward the 3’-ends (Figure 10 B).
Table 1: Hydrogen bond analysis using Chimera
H-bonds
rmx
Donor
Acceptor
Hydrogen
D..A dist (Å)
D-H..A dist (Å)
1
DG 7.A N2
AIK 11.A N16
AIK 11.A N2
AIK 11.A N26
AIK 11.A N9
AIK 2.B N16
AIK 2.B N2
AIK 2.B N26
AIK 2.B N9
DG 17.B N2
AIK 11.A N21
DG 7.A N3
DT 5.A O2
DG 7.A H22
AIK 11.A H16
AIK 11.A H2
AIK 11.A H26
AIK 11.A H9
AIK 2.B H16
AIK 2.B H2
2.89
2.864
3.437
2.703
3.464
2.97
2.084
1.854
2.601
1.912
2.492
1.954
1.701
1.814
1.884
1.922
DT 8.A O2
DA 6.A N3
DG 17.B N3
DT 15.B O2
DT 18.B O2
DA 16.B N3
AIK 2.B N21
2.713
2.646
2.888
2.876
AIK 2.B H26
AIK 2.B H9
DG 17.B H22
FtzhA
AIK 11.A N2
AIK 2.B N9
DT 18.B O2
DA 6.A N3
AIK 11.A H2
AIK 2.B H9
3.368
3.242
2.596
2.388
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Table 2: Steric clashes in FthzA-DNA complex
Atom1 (ligand)
AIK 2.B H2
Atom2 (DNA)
DG 14.B N2
DG 14.B N2
DC 7.A O2
DC 17.B O2
DG 14.B C4'
DC 17.B O2
DG 14.B N2
DG 14.B N2
DG 14.B C2
DC 7.A O2
DT 15.B H5'
DC 7.A O2
DG 4.A N2
DG 4.A N2
DG 14.B C4'
DG 14.B C2
DG 14.B C1'
DG 14.B C5'
DG 14.B C5'
DC 7.A C2
Overlap (Å)
2.106
2.012
1.675
1.289
1.203
1.181
1.16
Distance (Å)
0.534
1.253
1.505
1.191
1.677
1.999
2.18
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AIK 2.B N2
AIK 2.B C7
AIK 11.A H7
AIK 11.A 2H32
AIK 11.A C7
AIK 2.B C3
AIK 2.B C1
1.159
1.136
1.075
1.069
1.02
2.181
1.564
2.105
1.631
1.46
AIK 2.B H2
AIK 2.B C3
AIK 11.A C29
AIK 2.B H7
AIK 11.A H2
AIK 11.A N2
AIK 11.A C32
AIK 2.B N2
0.996
0.907
0.868
0.834
0.824
0.813
0.796
0.792
0.759
0.736
0.73
1.644
2.358
2.712
2.491
2.056
2.067
2.784
2.608
2.421
2.664
1.97
AIK 11.A 1H28
AIK 11.A 2H32
AIK 11.A C32
AIK 2.B C7
AIK 2.B C6
DC 7.A O2
DT 15.B C5'
DT 5.A H5'
DT 5.A H5'
DT 15.B H5'
DG 14.B C1'
DG 14.B N2
AIK 11.A C29
AIK 2.B C28
AIK 2.B 1H28
AIK 11.A C28
AIK 11.A C28
AIK 2.B C7
0.693
0.652
0.603
0.601
1.307
2.048
2.977
2.739
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A)
B)
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Figure 11: Intermolecular hydrogen bonding analysis. A) H bond analysis in thzA-DNA complex
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(PDB 1RMX). . B) H bonds in FthzA-DNA complex (PDB FthzA, Supporting Information).
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Figure 12: A) Steric clashes in FthzA-DNA complex (PDB 1RMX) B) A drawing of thzA structure
showing the moieties that encountered a steric clash with DNA and the numbering scheme used for the
1H-atoms of thzA in the PDB file of FthzA-DNA complex (PDB FthzA, Supporting Information).
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If we compare the DNA binding results of thzC with thzA, and supposing that the DAP group
of thzC adopts a similar conformation to that of thzA, similar explanations can be reached: the
inability of thzC to recognize the 5’-WGWWCW-3′ is due to a steric clash with the DAP group
when it is attached to the C-terminus and point toward the 5’-ends (Figure 10 C), and the
inability of thzC to recognize the 5’-WCWWGW-3′ is due to the disruption of hydrogen bonds
with DNA when the carbonyl of the amide links of thzC is closer to the 3’-end than the
partnering NH (Figure 10 D).
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3
.3.2 Stability analysis
To assess the stability of the structures (DNA-ligand), Root Mean Square Deviation (RMSD’s) of
both complexes were analyzed for the total equilibrated system containing ions and solvent and
individual trajectories without solvent and ions. From the stability point of view, the whole system
containing solvent, ions, DNA & ligand were observed to be stable during the course of 20 ns
simulation and the average RMSDs were obtained for both thzA-DNA and FthzA-DNA
complexes. The average RMSDs for the whole system were 3.58 ± 0.1 nm and 3.59 ± 0.1 nm for
thzA-DNA & FthzA-DNA respectively (Figure 13A). Variation in RMSDs of individual DNA
decamer duplex trajectories, ligand dimers and complex systems (DNA-ligand) were depicted in
(
Figure 13 B-D). It was evident that when thzA and FthzA dimers were bound to DNA, a
significant difference in RMSDs was observed, thus, from the viewpoint of stability, the preferred
binding orientation of lexitropsins in the minor grooves of DNA can be established, and that is
consistent with experimental results. Based on the MD results, consistent stability was achieved
for the preferred orientation of lexitropsin dimers (thzA) in the minor groove of the DNA whereas
for the flipped orientation (FthzA), the stability seems to be diverged from the original orientation.
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Figure 13: A) RMSD for the whole system containing all the trajectories of solvent, ions, DNA and ligand
dimers. B) the individual trajectories for the DNA decamer duplex. C) the complex DNA-ligand dimers.
D) the individual ligand dimers.
In order to establish the significant conformational rearrangements of thzA and FthzA bound to
the DNA duplex, the energy profiles have been obtained during 20 ns simulation. The total energy
of the complex systems obtained were averaged around -2.04e+05 ± 0.01 kJ/mol for both the
systems, however, a little deviation has been observed in case of FthzA (Figure 14A). Similarly,
the potential energy profile of both systems were observed to be -2.46e+05 ± 0.1 kJ/mol (Figure
14B). Root mean square fluctuations (RMSF) were analyzed for thzA-DNA and FthzA-DNA
complex along with individual ligand dimers. The greater oscillations were observed for the
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