Inhibition of therapeutically important polymerases with high affinity bis-intercalators

Article abstract of DOI:10.1016/j.bmcl.2012.05.041

We have previously demonstrated that polymerases such as telomerase can be inhibited by molecules (e.g., intercalators) that target the key RNA/DNA duplex substrate. In this work we show that this also holds true for reverse transcriptase, and show that the lead intercalators can be modified to increase inhibition efficacy. Specifically, we use the strategy of multiple simultaneous intercalation, by linking two intercalators with a variable linker. The rationale behind this design is that a specific linker has the potential to increase affinity and specificity for the target duplex. We have synthesized a library of 45 ethidium bis-intercalators in which the distance between intercalators is systematically varied. We observe that members of the dimer library have improved telomerase and reverse transcriptase inhibition, relative to the monomeric leads. We show that this improvement in inhibition over mono-intercalators is most prominent when non-productive sites of inhibitor binding are limited in the assay mix. When this is done, a 400-fold increase in inhibition efficacy is observed.

Full text of DOI:10.1016/j.bmcl.2012.05.041

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4844–4848
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of therapeutically important polymerases with high affinity
bis-intercalators
⇑
Nitin Jain, Subhashree Francis , Simon H. Friedman
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri, 2464 Charlotte Street, Kansas City, MO 64108-2718, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously demonstrated that polymerases such as telomerase can be inhibited by molecules
(e.g., intercalators) that target the key RNA/DNA duplex substrate. In this work we show that this also
holds true for reverse transcriptase, and show that the lead intercalators can be modified to increase inhi-
bition efficacy. Specifically, we use the strategy of multiple simultaneous intercalation, by linking two
intercalators with a variable linker. The rationale behind this design is that a specific linker has the poten-
tial to increase affinity and specificity for the target duplex. We have synthesized a library of 45 ethidium
bis-intercalators in which the distance between intercalators is systematically varied. We observe that
members of the dimer library have improved telomerase and reverse transcriptase inhibition, relative
to the monomeric leads. We show that this improvement in inhibition over mono-intercalators is most
prominent when non-productive sites of inhibitor binding are limited in the assay mix. When this is
done, a 400-fold increase in inhibition efficacy is observed.
Received 13 April 2012
Revised 2 May 2012
Accepted 8 May 2012
Available online 23 May 2012
Keywords:
Intercalators
Solid phase synthesis
Library
Reverse transcriptase
Telomerase
Ó 2012 Elsevier Ltd. All rights reserved.
Polymerases and other enzymes that process nucleic acids form
an important class of therapeutic targets. Members of this class in-
clude the anti-cancer target telomerase, and the reverse transcrip-
tase from HIV. Previously, we have developed a strategy to inhibit
telomerase by targeting small molecules to the RNA/DNA duplex
that forms during telomerase’s catalytic cycle.1 This duplex forms
when the template portion of telomerase’s RNA subunit binds to
the telomeric DNA, acting as a template for the DNA’s extension.
The rationale behind our approach is that molecules that bind this
duplex, for example intercalators, can distort it, thereby interfering
with the extension of the DNA and possibly its translocation. It
seems reasonable that other polymerases could be inhibited
through a similar mechanism. We previously demonstrated that
several intercalators act as inhibitors of telomerase, and that their
efficacy correlates with their affinity for the telomerase RNA/DNA
duplex.1,2 The most effective intercalator, ethidium, outcompeted
other tested intercalators for binding to an isolated RNA/DNA
duplex with the telomerase sequence. In addition, Ren and Chaires
demonstrated that ethidium, when allowed to compete simulta-
neously for 15 different nucleic acid structures, bound most avidly
to an RNA/DNA duplex.3 The IC50 we observed for ethidium was
While some intercalators are used as drugs, they represent a
challenging starting point for drug design. The cell contains high
concentrations of nucleic acids that can act as alternative binding
sites, leading to reduced concentrations of free intercalator, as well
as toxic effects. Our lab is therefore examining multiple strategies
for adding specificity to the inherent ability of these molecules to
target therapeutically important nucleic acid structures. Starting
from a high affinity intercalator, there are multiple strategies for
increasing its affinity and specificity. The approach we take in this
work is to attempt multiple, simultaneous intercalation. This has
the potential to increase affinity, through the multi-dentate effect.
In addition, it also has the potential to increase specificity, if the
specific distance and geometry of the linker allows for simulta-
neous (and hence high affinity) intercalation with the target nu-
cleic acid, while preventing bis-intercalative binding to unwanted
sites (such as genomic DNA). We apply this approach both to telo-
merase and HIV reverse transcriptase (RT). Specifically, we tested
both commercially available bis-intercalators (a bis-ethidium and
a bis-acridine), as well as a library of bis-intercalators that we
synthesized, where the linker distance and geometry was system-
atically varied. This resulted in the identification of several low
nano-molar inhibitors of reverse transcriptase, and mid-nano-
molar inhibitors of telomerase. Interestingly, differences between
different library members were only observable when competing
nucleic acids were limited, either by reducing substrate concentra-
tion in the case of RT, or by purifying telomerase from other cellu-
lar components found in cellular homogenate.
3.3 lM. The aim of our subsequent work has been to increase the
efficacy of inhibitors against telomerase by increasing their
affinity.
⇑
Corresponding author. Tel.: +1 816 235 2224; fax: +1 816 235 5779.
E-mail address: friedmans@umkc.edu (S.H. Friedman).
Present address: Institute for Therapeutics Discovery and Development,
University of Minnesota, United States.
To identify potentially high affinity and high specificity bis-
intercalator inhibitors of telomerase, we built a library, based on
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.041

N. Jain et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4844–4848
4845
1.2
1
our lead, ethidium. Specifically, we used ethidium benzylic acid
(EBA), a monomer developed in our lab that allows for easy conju-
gation to amines.7 We previously have shown that both the parent
EBA and simple derivatives maintain telomerase inhibition ability.
We constructed a library of scaffolds that present two amines to be
acylated by EBA (Scheme 1). The library of 45 compounds was de-
signed to vary both the distance between the intercalators as well
as the potential conformations. The first and third positions were
either di-amino propionic acid (Dap), ornithine (Orn), or lysine
(Lys). The second or linker position was either glycine (Gly), beta
alanine (b-Ala), gamma amino butyric acid (Gaba), epsilon amino
hexanoic acid (Ehx), or no linker. We used an orthogonal Mtt pro-
tection scheme that allowed us to construct the scaffold on resin,
and in the late stages of the synthesis install the EBA. Representa-
tives of the library were characterized by ESI-MS, and showed the
expected molecular weights (see Supplementarydata for these ESI-
MS and complete experimental detail, including assay setup).
The library was then screened using a single point PCR assay of
telomerase activity.4 The results show a narrow range of low
(a)
Dap-β-Ala-Lys
Raw homogenate
IC = 0.60µM (.15)
R 5=0 .98
0.8
0.6
0.4
0.2
0
micro-molar estimated IC50 values from 2.1 to 13.6 lM, similar to
0
5
10
15
20
25
the parent EBA inhibition value (data not shown). This was
somewhat surprising, as we expected that molecules capable of
bis-intercalation would have greater efficacy compared to mono-
intercalators if in fact they bis-intercalated. To confirm the low
affinity indicated during single point screening, we determined a
full IC50 of eight of the compounds. The determined IC50 values for
the eight compounds varied from 0.58 to 1.81 lM, somewhat lower
than the single point estimates, but close to the original lead
mono-intercalator. A representative inhibition curve of compound
1, Dap-b-Ala-Lys, with an IC50 of 0.60 lM is shown in Figure 1a.
One explanation for the lack of improvement in these bis-
intercalators over the lead mono-intercalator was that the library
members were simply incapable of simultaneous bis-intercalation.
This seemed less likely as the library covered many possible
distances and conformations. A second explanation was that the
putative concentration of free bis-intercalator in the system was
actually significantly less than the amount added, due to non-
specific binding of the bis-intercaltor by non-telomerase species
in the assay. This hypothesis was supported by the shape of the
inhibition curve, which showed signs of tight binding (sharp transi-
tion in the curve). The assay as performed uses HeLa cell homoge-
nates that include many components of the cellular contents,
including other nucleic acids. These could potentially bind to the
bis-intercalator in the assay mix, and reduce their concentration.
Concentration (µM)
1.2
1
(b)
Dap-β-Ala-Lys
Partially purified telomerase
IC = 0.10 µM (.02)
R 5=0 .99
0.8
0.6
0.4
0.2
0
0
2
4
6
8
10
Concentration (µM)
Figure 1. Telomerase inhibition assay of compound 1, Dap-b-Ala-Lys, using whole
cell homogenate (a, top), or partially purified telomerase (b, bottom). Error values
are indicated in parenthesis.
Linker
Intercalator
site A
a = 1,3,4
Intercalator
site C
site B
b = 1,2,3,5
or no linker
c = 1,3,4
To test for this effect, we then repeated the inhibition assay
using partially purified telomerase, prepared using a procedure de-
rived from Szatmari et al.5 This method likely limits the amount of
extra nucleic acids present. When we retested the IC50 of com-
pound 1 it showed a significantly lower IC50 value of 103 nM
(Fig. 1b).
In a parallel effort, we also examined the ability of bis-interca-
lators to inhibit reverse transcriptase from HIV-1. Because HIV-RT
also depends on a duplex substrate, it seemed reasonable that it
could also be inhibited by intercalators. The distortion of the
duplex substrate that would result from intercalator binding was
anticipated to limit enzyme activity.
()
b
()
()
a
c
We initially screened a range of 14 mono-intercalators using a
single point assay of HIV-RT activity.6 This showed estimated IC50
values of 2 lM and above (Table 1). This was consistent with our
previous screening results of mono-intercalators against telome-
Scheme 1.
rase. It is of interest to note that ethidium had the greatest inhibition

4846
N. Jain et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4844–4848
efficacy, as it previously showed the greatest efficacy against telo-
merase. With both HIV-RT and telomerase, our target is an RNA/
DNA heteroduplex. Previously Ren and Chaires have shown that of
all the common intercalators, ethidium has the greatest preference
for RNA/DNA duplexes over other nucleic acid structures.3 In addi-
tion, we have shown that when multiple intercalators simulta-
neously compete for an RNA/DNA duplex, ethidium preferentially
binds.2
To improve the inhibition of HIV-RT by ethidium, we then
tested a commercially available ethidium dimer (Fig. 2, ethidium
homodimer-1). The rationale behind this approach was to increase
inhibition through bis-intercalation. While the binding curve was
not particularly ‘clean’ it gave an IC50 fit value of 2.3 lM (Fig. 3a).
This was surprising, as we originally anticipated that a molecule
capable of bis-intercalation would have improved inhibition effi-
cacy through higher affinity. Because of the poor fit of the binding
curve, we suspected that we were in a tight-binding situation,
where the actual concentration of ligand in solution was in fact
much lower than the theoretical, due to sequestering of the ligand
by high concentrations of binding sites in the assay mix. A poten-
tial cause of this was the high concentration of nucleobases found
in our standard HIV-RT RNA/DNA substrate. Intercalators can bind
both to duplex and single stranded basepairs affecting the true free
concentration of intercalator. The standard conditions used for this
Figure 2. Structures of commercially available bis-intercalators tested.
substrate are 25
lg/mL which corresponds to a total concentration
concentration dependent inhibition, with fit IC50 values of
of substrate of 64.5
base pairs, a concentration high enough to impact the true free
l
M single strand bases, and 1.86 M duplex
l
12.9
0.05
l
M, 400 nM and 221 nM at 25
lg/mL, 0.5 lg/mL, and
l
g/mL, respectively (Fig. 5 depicts high and low substrate
concentration of ligand in solution.
To test this hypothesis, we repeated the analysis of the ethi-
dium dimer in the presence of 500-fold lower concentration du-
conditions).
Given this improvement in HIV-RT inhibition with bis-intercala-
tion, we then examined whether or not we could identify unique
bis-intercalators from our library that had particularly high efficacy
due to a close match of the bis-intercalator structural properties
(e.g., distance between the intercalators, conformation) with the
structural properties of the target duplex (e.g., pitch, helicity). To
examine this, we screened the library of bis-intercalators we previ-
ously synthesized to target telomerase. This collection of 45 differ-
ent molecules was analyzed in single point assays at 100 nM
plex substrate (0.05 lg/mL). At this concentration of substrate
there are 3.7 nM duplex base pairs, and 130 nM single stranded
bases present. This lower substrate concentration gave a much
cleaner inhibition curve and a fit value of 10.8 nM, 213-fold lower
than the apparent inhibition constant at the higher substrate con-
centration (Fig. 3b). In addition, we tested the ethidium dimer at an
intermediate concentration of duplex substrate (0.5 lg/mL) and it
gave an intermediate fit value (85 nM, data not shown). To confirm
that the improvement in apparent inhibition was due to the
hypothesized mechanism of tight binding and not something else,
we also tested the inhibition of monomeric ethidium under all
three duplex substrate conditions. Ethidium gave an IC50 value that
was essentially unchanged at all three substrate conditions
concentration of drug and 0.05 lg/mL of RNA/DNA substrate. We
observed a very similar range of estimated IC50 values ranging from
45 to 173 nM. Because of the inherent limitation of single point
screening, we retested five of these using full concentration range
IC50 determinations. These included compounds with two of the
best estimated IC50 values, two of the worst, and one intermediate.
The IC50 values for these and their identities are shown in Table 2.
The best inhibition was observed with Orn-Ehx-Orn which gave
an IC50 of 4.9 nM (Fig. 6). The consistency of low nM inhibition indi-
cates that the range of structural parameters explored in the library
were all capable of effecting bis-intercalation. In addition, the shape
(2.2
lM, 1.8
lM, 2.1 lM, respectively at 25 lg/mL, 0.5 lg/mL,
and 0.05
l
g/mL. Fig. 4 depicts high and low substrate conditions).
A second commercially available bis-intercalator, acridine dimer
(Fig. 2), was tested and showed a pattern of improvement similar
to ethidium dimer. Specifically it showed a duplex substrate
Table 1
Estimated IC50 values for mono-intercalators against reverse transcriptase.
S. No.
Molecule
IC50 estimates
1
2
Ethidium bromide
Propidium iodide
2
3
l
l
M
M
3
4
5
6
7
8
9
10
11
12
13
14
Rivanol
Acridine yellow
Doxorubicin
7-amino actinomycin D
Daunorubicin
Ellipticine
Acridine orange
Actinomycin D
9-Amino-6-chloro-2-methoxy acridine (ACMA)
2-Anthracene carboxylic acid
1-Anthracene carboxylic acid
9-Acridine carboxylic acid
20
32
52
93
139
182
200
270
l
l
l
l
l
l
l
l
M
M
M
M
M
M
M
M
No inhibition observed
No inhibition observed
No inhibition observed
No inhibition observed

N. Jain et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4844–4848
4847
1.2
1
1.2
(a)
(a)
1
Ethidium homodimer-1
Ethidium
RT assay, high substrate
IC = 2.3 µM (.49)
R 5=0 .90
RT assay, high substrate
IC50 = 2.2 µM (0.24)
R = .99
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
0
5
10
15
20
Concentration (µM)
Concentration (µM)
1.2
1
1.2
(b)
(b)
1
0.8
0.6
0.4
0.2
0
Ethidium homodimer-1
Ethidium
RT assay, low substrate
IC = 10.8 nM (2.3)
R 5=0 .94
RT assay, low substrate
IC = 2.1 µM (0.42)
R 5=0 .97
0.8
0.6
0.4
0.2
0
0
0.1
0.2
0.3
0.4
0.5
0
20
40
60
80
100
120
140
Concentration (µM)
Concentration (µM)
Figure 3. Reverse transcriptase inhibition by ethidium homodimer-1 at high
substrate (a) and low substrate (b) concentrations. Error values are indicated in
parenthesis.
Figure 4. Reverse transcriptase inhibition by ethidium at high substrate (a, top) and
low substrate (b, bottom) concentrations. Error values are indicated in parenthesis.
of the inhibition curve still suggests tight binding. This is consistent
with the actual concentration of 3.7 nM duplex base pairs found in
the duplex substrate, as if this is the target of binding, we could not
observe an apparent binding constant lower than this number. It
may well be that there are significant binding/inhibition differences
between individual members of the library, however they have
been effectively leveled by the relatively high concentration of sub-
strate needed to practically assay RT activity.
Finally, we tested the ethidium dimer inhibition efficacy against
HIV-RT in the presence of competitor calf thymus DNA. The purpose
of this DNA was to simulate the effect that competitor cellular nu-
cleic acids would have on the inhibition efficacy of bis-intercaltors.
As higher duplex substrate concentrations ‘leveled’ the apparent
inhibition constant of bis-intercalators, we expected that under cel-
lular conditions, genomic DNA as well as RNA of different forms
could act as reservoirs and soak up the ligands leading to lower
apparent efficacy. We selected a concentration of 340 lM base pairs
of calf thymus DNA, as this is ꢀ1/10th the total concentration of
genomic DNA in a cell and would represent a significant challenge
to a completely non-specific nucleic acid binding compound.8 In
addition, higher concentrations of calf thymus DNA introduced sol-
ubility issues. The IC50 curve for ethidium dimer produced a fit va-
lue of 8.5 lM (data not shown). While this does confirm a degree of
a ‘leveling’ effect of the competitor DNA, it does suggest some spec-
ificity of the bis-intercalator for the HIV-RT RNA/DNA duplex over
the DNA/DNA competitor duplex. This is illustrated by the fact that
a >90,000-fold molar excess of competitor DNA/DNA duplex base
pairs to RNA/DNA substrate duplex base pairs did not eliminate
inhibition, which would be expected if the two nucleic acid forms
had identical affinity for the intercalator.
The aim of this work was to explore the strategy of bis-
intercalation for improving the efficacy of intercalator based

4848
N. Jain et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4844–4848
1.2
1
1.2
(a)
Acridine dimer
1
RT assay, high substrate
IC = 12.9 µM (3.8)
R 5=0 .82
Orn-Ehx-Orn
RT assay, low substrate
IC50 = 4.9 nM (1.8)
R = .99
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Concentration (µM)
Concentration (µM)
Figure 6. Reverse transcriptase inhibition at low substrate of Orn-Ehx-Orn from the
bis-intercalator library. Error value is indicated in parenthesis.
1.2
1
(b)
dimers, we demonstrated that we could increase the efficacy of
inhibition by greater than 400-fold. This improvement was only
seen when we eliminated extraneous sources of intercalator bind-
ing sites from the assay mixture. These included cellular homoge-
nate components in the case of telomerase assay, and duplex
substrate in the case of HIV-RT assay. Within our library members,
most showed improved efficacy in comparison to a monomeric
intercalator. This was likely due to the relatively high concentra-
tion of substrate duplex. The fact that they can retain inhibition
efficacy even in the presence of >90,000-fold molar excess of com-
petitor DNA/DNA duplexes suggests that they have structural
specificity for the target RNA/DNA duplex. It is possible that this
differential affinity can be further optimized to result in molecules
with even greater specificity for the important therapeutic target
of the RNA/DNA duplex.
0.8
Acridine dimer
RT assay, low substrate
IC50 = 0.22 µM (0.051)
R = .91
0.6
0.4
0.2
0
Acknowledgments
0
0.5
1
1.5
2
2.5
3
3.5
Concentration (µM)
The support of NIH Grant RO1CA090410 is gratefully acknowl-
edged. We thank Professor William Gutheil for curating the mass
spectrometers used in this study.
Figure 5. Reverse transcriptase inhibition by acridine dimer at high substrate (a,
top) and low substrate (b, bottom) concentrations. Error values are indicated in
parenthesis.
Supplementary data
Table 2
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.041.
IC50 values from full inhibition studies, determined against reverse transcriptase for
five members of bis-intercalator library
S. No.
Molecule
IC50 (nM)
References and notes
1
2
3
4
5
EBA-Orn-Ehx-Orn-EBA
EBA-Orn-b-Ala-Dap-EBA
4.9 (1.8)
9.2 (3.2)
10.5 (3.7)
22.3 (9.4)
30.7 (17.3)
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2. West, C.; Francis, R.; Friedman, S. H. Bioorg. Med. Chem. Lett. 2001, 11, 2727.
3. Ren, J.; Chaires, J. B. Biochemistry 1999, 38, 16067.
4. Francis, R.; Friedman, S. H. Anal. Biochem. 2003, 323, 65.
5. Szatmari, I.; Tokes, S.; Dunn, C. B.; Bardos, T. J.; Aradi, J. Anal. Biochem. 2000, 282,
80.
EBA-Lys-
c
-Abu-Lys-EBA
EBA-Dap-Orn-EBA
EBA-Dap-Ehx-Lys-EBA
6. Tramontano, E.; Cheng, Y. C. Biochem. Pharmacol. 1992, 43, 1371.
7. Rangarajan, S.; Friedman, S. H. Bioorg. Med. Chem. Lett. 2007, 17, 2267.
8. Leyva, A., Jr.; Kelley, W. N. Anal. Biochem. 1974, 62, 173.
inhibitors of telomerase and HIV-RT. By testing with both com-
mercially available intercalator dimers and a library of ethidium