Modifications at the C-terminus to improve pyrrole-imidazole polyamide activity in cell culture

Article abstract of DOI:10.1021/jm900256f

Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of small molecule DNA minor groove binding compounds that have been shown to modulate endogenous gene expression in cell culture. Gene regulation by polyamides requires efficient cellular uptake and

Full text of DOI:10.1021/jm900256f

7380 J. Med. Chem. 2009, 52, 7380–7388
DOI: 10.1021/jm900256f
Modifications at the C-Terminus To Improve Pyrrole-Imidazole Polyamide Activity in Cell Culture
Claire S. Jacobs and Peter B. Dervan*
Division of Chemistry and Chemical Engineering, The California Institute of Technology, 1200 E. California Boulevard, Mail Code 164-30,
Pasadena, California 91125
Received February 28, 2009
Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of small molecule DNA minor groove
binding compounds that have been shown to modulate endogenous gene expression in cell culture. Gene
regulation by polyamides requires efficient cellular uptake and nuclear localization properties for
candidate compounds. To further optimize Py-Im polyamides for enhanced potency in cell culture, a
focused library of polyamides possessing various modifications at the C-terminus was synthesized and
tested. Comparison of polyamide biological activity in two cell lines revealed tolerance for structural
modifications and agreement in activity trends between cell lines. The use of an oxime linkage between
the polyamide and an aromatic functionality on the C-terminus resulted in a ∼20-fold increase in the
potency of polyamides targeted to the androgen response element (ARE) in LNCaP cells by measuring
AR-activated PSA expression.
Introduction
compounds.9-13 Subsequent studies aimed at eliminating
the fluorescent tag (FITC) utilized quantitative real-time
reverse transcriptase polymerase chain reaction (RT-PCR)
to measure mRNA levels of an endogenous inducible gene
as a biological readout of polyamide nuclear entry and
DNA-binding. A focused library of minimized FITC ana-
logues identified a smaller, stable replacement for fluores-
cein in the form of isophthalic acid (IPA), which rivaled the
activity of the original FITC-labeled polyamide in HeLa
(cervical cancer) and U251 (glioma) cells.8 Subsequent
work in other cell lines aimed at regulating different target
genes has validated this C-terminus modification as a
positive contributor to biological activity with negligible
detrimental impact on polyamide sequence specificity or
binding affinity.14,15
The discovery of new moieties that facilitate polyamide
cell uptake and nuclear localization for gene regulation
studies is an area of keen interest as we aim to enhance
further polyamide efficacy as we transition our studies
toward animal disease models. The present study investi-
gates the influence of linker, chemical linkage, and target
cell line on polyamide nuclear localization utilizing a small
library of C-terminus-modified polyamides (Figure 1).
Quantitative real-time RT-PCR of relative mRNA expres-
sion levels in cells treated with polyamides was used as a
measure of biological efficacy and the criterion for ranking
members of the polyamide library. Polyamides were
targeted either to the vascular endothelial growth factor
(VEGF) promoter in U251 (glioma) and LNCaP (prostate)
cells or to the prostate specific antigen (PSA) promoter
in LNCaP cells (Figure 1B), as these two transcription-
factor systems have been exploited for regulating such
medically relevant genes by our group.13-15 Although a
variety of structural parameters were evaluated, it was
discovered that a C3 aliphatic linker tethered to an aromatic
group through an oxime linkage resulted in enhanced
polyamide potency. This motif has been employed recently
Small molecules capable of regulating specific endogenous
gene expression pathways could find numerous applications
in molecular biology and human medicine.1,2 DNA-binding
polyamides comprising N-methylimidazole (Ima) and N-
methylpyrrole (Py) are a class of programmable small mole-
cules that bind the minor groove of DNA in a sequence
specific fashion and have been employed as regulators of gene
expression through DNA-binding and disruption of tran-
scription factor-DNA interfaces.3 Hairpin polyamides com-
prise two Im-Py strands linked via an aliphatic linker, with
sequence specificity resulting in a programmed fashion from
side-by-side heterocyclic amino acid pairings: Im/Py recog-
nizes G C over C G; Py/Py is degenerate for A T and T A;
3
3
3
3
3-chlorothiophene N-terminus cap/Py recognizes T A over
A T.4-6 Eight-ring Im-Py polyamides have been shown to
3
3
exhibit specific binding with binding affinities comparable to
those of natural DNA-binding transcription factors.7 Im-Py
polyamide regulation of gene expression presumably occurs
through either direct steric blockade of transcription factor
binding or allosteric modification of DNA topology. While the
sequence specificity and DNA-binding affinity of these small
molecules are well studied, investigations into their potential
therapeutic applications are active areas of investigation. We
have recently shown that the biological activity of Im-Py
polyamides can be improved via the incorporation of an
isophthalic acid (IPA) group at the polyamide C-terminus.
We present here further modifications to the hairpin polyamide
C-terminus that increase even further their biological activity.8
Early gene regulation studies in cell culture relied on
fluorescein-polyamide conjugates as cell permeable
*To whom correspondence should be addressed. Phone: (626) 395-
6002. Fax: (626) 683-8753. E-mail: dervan@caltech.edu.
a Abbreviations: Im, N-methylimidazole; Py, N-methylpyrrole;
VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible
factor; HRE, hypoxia response element; PSA, prostate specific antigen;
ARE, androgen response element; IPA, isophthalic acid.
r
pubs.acs.org/jmc
Published on Web 07/02/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7381
Results
Quantitative Real-Time RT-PCR Data for Polyamide-
Treated U251 and LNCaP Cells. The biological activity
against DFO-induced VEGF expression of HRE-targeted
match (6-11) and mismatch (12-22) eight-ring hairpin
polyamides was studied in U251 and LNCaP cell culture
and of ARE-targeted match (28-33) and mismatch (39-44)
eight-ring hairpin polyamides against DHT-induced PSA
expression in LNCaP cells. A schematic representation of
polyamide-DNA binding and an overview of the project are
shown in Figure 1. Structures of the polyamide cores and
C-terminus are shown in Figure 2, and results for cell culture
experiments are shown in Figures 3-6. Polyamides that
regulate VEGF expression under hypoxic (inducing) condi-
tions target the hypoxia response element (HRE) 50-
ATACGTG-30 of the VEGF promoter (compounds 1-11),
and those that target PSA expression following dihydrotes-
terone (DHT) induction target the androgen response ele-
ment (ARE) 50-AGAACAG-30 of the PSA promoter
(compounds 23-33). For each gene, a set of mismatch
control polyamides was included; mismatch polyamides
are designed not to bind to the HRE or ARE, though some
degree of biological activity is not unexpected, presumably
because of off-target effects. The mismatch polyamides for
the HRE series are 12-22 (target 50-WGGWCW-30
sequences) and for the ARE series are 34-44 (target 50-
WGWCGW-30 sequences). These compounds are linked
either through an amide linkage (6-8, 17-19, 28-30, and
39-41) or an oxime linkage (9-11 and 20-22, 31-33, and
42-44) to a C-terminal tail group. For each linker group,
unconjugated free amine control compounds lacking a tail
moiety (2-5 and 13-16, 24-27, and 35-38 (match and
mismatch polyamides, respectively) were included; because
of its historical use and known modest level of biological
activity, βDp tail (1, 12, 23, and 34) polyamides were
included for comparison. Polyamide-untreated uninduced
and polyamide-untreated induced conditions were used as
controls, and VEGF or PSA mRNA expression levels in
treated cells were normalized to those in the untreated,
induced controls. Induced and uninduced DMSO-treated
control conditions were included to rule out vehicle (DMSO)
as the source of relative mRNA expression changes (DMSO
concentration of e0.1% for all biological experiments).17
Unless otherwise noted, U251 cells were dosed with poly-
amides at 1, 0.2, and 0.02 μM, and LNCaP cells were dosed at
10, 2, and 0.2 μM. For both the VEGF and PSA systems,
cells were dosed with control unconjugated control poly-
amides only at the highest concentration.
Figure 1. Project overview. (A) Schematic illustration of poly-
amide-DNA recognition through formation of hydrogen bonds
with the floor of the DNA minor groove. The γ-turn, polyamide
heterocycle core, C-terminus linker, C-terminus linkage, and
C-terminus tail of the polyamide are indicated by braces and labeled
for clarity. (B) Schematic illustration of the mechanism by which
polyamides affect gene expression. Sequence-specific binding of a
polyamide core to a gene response element (RE) blocks transcrip-
tion factor (TF) binding to the RE, thus blocking up-regulation of
gene product expression under inducing conditions. Relative
mRNA expression levels under inducing conditions were employed
as a biological readout of polyamide cell uptake nuclear localiza-
tion. Displayed in tabular form are the relevant transcription
factors, gene products, DNA RE binding sequence, and cell lines
for each set of match and mismatch polyamides.
Biological Activity of HRE-Targeted Polyamides in U251
and LNCaP Cells. The data obtained for the amide-linked
HRE series in U251 and LNCaP are shown in parts A and B
of Figure 3, respectively. The biological activity measured
for 6 is comparable to previously obtained data for 6 at 1 μM
in U251 cells.8,15 The general trend in this cell line indicates
that the unconjugated control compounds lacking the IPA
tail moiety all exhibited a modest degree of activity but that
activity is improved through IPA conjugation. IPA conjuga-
tion resulted in a greater increase in activity for compounds
bearing the triamine linker than for compounds bearing the
C7-linker or C3-linker. The βDp tail was the least active of all
C-terminus modifications in this series. In LNCaP cells, the
unconjugated controls were more active than in U251 cells,
with the unconjugated parent compound 4 as active as IPA
for the facile 18F-labeling of polyamides for in vivo biodis-
tribution by positron emission tomography.16 This parti-
cular modification, as well as a variety of structural
perturbations to the hairpin oligomer C-terminus, is dis-
cussed in detail.

7382 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Jacobs and Dervan
Figure 2. Schematic illustration of the polyamide cores and tail groups employed. (A) At left are given the HRE match (1-11) and HRE
mismatch (12-22) cores employed in U251 and LNCaP cells, and on the right are the ARE match (23-33) and ARE mismatch (34-44) cores
employed in LNCaP cells. Below the chemical structure is a schematic “ball-and-stick” shorthand representation of each core, in which pyrrole
(Py) is symbolized by an open circle, imidazole (Im) by a filled circle, and chlorothiophene cap by an open square. (B) The chemical structures of
the tail groups “R” for compounds 1-44 are shown. Structures 45-48 are the “coreless” oxime-linked control compounds.
conjugates 6-8. Conjugation of the IPA tail increased the
activity of the triamine and C7-linked compounds, but the
difference in activity between unconjugated parents and the
IPA derivatives was more modest than in U251 cells. The
βDp tail polyamides (1 and 12) were more active in LNCaP
than U251.
The activity of the HRE oxime-linked series was measured
in U251 and LNCaP, and the results are shown in parts A
and B of Figure 5, respectively. Compounds 9-11 were
active in U251 cells under hypoxic conditions: VEGF
mRNA expression was down-regulated ∼60% in U251 cells
treated with 1 μM 9-11, which is comparable to the activity
of 6 at 1 μM in U251 (Figure 3A). Mismatch polyamide
compounds 20-22 were as active in U251 cells as their
respective match polyamides 9-11. In LNCaP cells, com-
pounds 9-11 were also active: VEGF mRNA expression
levels measured for cells treated with 10 μM 9-11 were
down-regulated ∼80%. Mismatch oxime-linked polyamides
were significantly active in LNCaP cells.
Biological Activity of ARE-Targeted Polyamides in LNCaP
Cells. The relative PSA mRNA levels in LNCaP cells induced
with 1 nM DHT and dosed with polyamides 23-44 were
measured (Figure 4 and Figure 5C). In the amide series, the
unconjugated free amine control compounds 23-26 were more
active that the mismatch controls 34-37. The amide-linked ARE
compounds 28-30 were all appreciably active, and a modest
linker effect was observed (C7 > C3 > triamine); mismatch
compounds 39-41 were less active than their match mates. The
difference in activity between each match 28-30 and respective
mismatch 39-41 was greater than measured for VEGF regula-
tion in both U251 and LNCaP.
The activity of the oxime-linked series 31-33 and 42-44
in LNCaP cells was measured at 5, 1, and 0.1 μM, and that
for unconjugated controls 27 and 38 was measured at 5 μM;
the data are shown in Figure 5C. A significant increase in
biological activity was measured for match polyamides 31-
33; all three compounds decreased PSA mRNA levels at
5 μM below untreated, uninduced levels. PSA mRNA levels
were reduced to untreated basal levels at 1 μM 31 and 33.
Mismatch polyamides 42-44 were less active than their
respective match polyamides 31-33. The hydroxylamine
match control compound 27 down-regulated PSA mRNA
levels by ∼50%, while the hydroxylamine mismatch com-
pound 38 showed very little activity. LNCaP cells treated
with 5 μM 31-33 showed noticeable cytotoxic response
upon incubation for 48 h, an effect that was more pro-
nounced for 32 and 33 compared to 31.
DNA Melting Temperature (Tm). The effect of polyamide
binding on 14-mer oligonucleotide melting temperature (Tm)
was measured (Table SI 2 in Supporting Information) to
confirm that match polyamides possess appreciable DNA
binding affinities and thus that lower levels of biological
activity are not due to poor DNA binding affinities.18 Tm
measurements require minimal materials and present a rapid

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7383
Figure 4. Quantitative real-time RT-PCR data measuring the ef-
fect on relative PSA mRNA expression levels under DHT induction
in LNCaP cells dosed with compounds 23-26, 28-30 and their
mismatch congeners 34-37 and 39-41. Induced and uninduced
control conditions are indicated by black bars, match core com-
pounds by light-gray bars, and mismatch core compounds by dark-
gray bars. Uninduced and induced control compounds without
DMSO are on the left and with 0.1% DMSO on the right. Errors
shown are the fractional standard deviation. Cells were treated with
match core control compounds 23-26 and mismatch core control
compounds 34-37 as cell media solutions 5 μM in polyamide. Cells
were treated with match compounds 28-30 and respective mis-
match compounds 39-41 as 5, 1, and 0.1 μM solutions in cell culture
media.
the HRE of VEGF and ARE of PSA, respectively. For both
the HRE and ARE 14-mers, the melting temperature increased
appreciably upon match compound binding (1-11 and 23-
33, respectively). Tm values also generally increased upon
mismatch binding (12-22 and 34-44, respectively) but to a
more modest degree. Melting temperatures of the free amine
parent compounds were higher than those of the IPA con-
jugates, with the exception of 36 versus 40. The ΔTm values
calculated for match compounds ranged from an increase of
9.0 to 14.8 °C for the HRE polyamide series (1-12) and from
an increase of 10.4 to 17.3 °C for the ARE series (23-33).
Cell Viability Effects of ARE-Targeted Match Compounds
28-33 and 39-44 in LNCaP Cells. LNCaP cells were dosed
with each compound at concentrations spanning 30-0.01
μM, and metabolic activity was measured with a standard
WST-1 colorimetric assay. Metabolic activity decreased with
increasing concentrations of 28-30 but did not significantly
surpass 50% viability; therefore, IC50 values for growth
inhibition could not be calculated. Cells treated with the
mismatch mates (39-41) had little effect on cell growth. The
three oxime-linked match compounds had measurable IC50
values for growth inhibition: 31, 1.7 ( (0.3) μM; 32, 0.6 (
(0.1) μM; 33, 0.4 ( (0.0) μM. The mismatch oximes displayed
a similar pattern; the growth inhibition of the fluorine-
bearing oximes 43 and 44 was more pronounced than the
3-carboxy tail mismatch 42, but the effect on cell growth of
the mismatch polyamides was less pronounced than that of
the match polyamides. The cell growth data for 28-33 and
39-44 in LNCaP are shown in Figure 6 and Table 1.
Figure 3. Quantitative real-time RT-PCR data showing the effect
of HRE-targeted match compounds 1-4, 6-8 and their mismatch
congeners 12-15, 17-19 on DFO-induced VEGF expression in
cultured cells. Induced and uniduced control conditions are indi-
cated by black bars, match core compounds by light-gray bars, and
mismatch core compounds by dark-gray bars. Uninduced and
induced control compounds without DMSO are on the left and
with 0.1% DMSO on the right. Errors shown are the fractional
standard deviation. (A) Expression of VEGF in cultured U251 cells
under DFO-induced hypoxic conditions of match core control
compounds 1-4 and mismatch core control compounds 12-15 at
1 μM concentration. Cells were treated with match compounds 6-8
and respective mismatch compounds 17-19 as 1, 0.2, and 0.02 μM
cell culture media solutions. (B) Expression of VEGF in cultured
LNCaP cells under DFO-induced hypoxic conditions of match core
control compounds 1-4 and mismatch core control compounds
12-15 at 10 μM. Cells were treated with match compounds 6-8 and
respective mismatch compounds 17-19 as 10, 2, and 0.2 μM cell
culture media solutions.
method for determining the rank-order of binding affinities,
although a quantitative Ka value is not obtained. The 14-mer
oligonucleotide sequences (Materials and Methods) are based on

7384 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Jacobs and Dervan
Figure 5. Gene-regulation effects measured by quantitative real-time RT-PCR for the oxime-linked polyamides 9-11, 20-23, 31-33, and
42-44 and the hydroxylamine tail control compounds 5, 16, 27, and 38. Induced and uninduced control conditions are indicated by black bars,
match core compounds by light-gray bars, and mismatch core compounds by dark-gray bars. Uninduced and induced control compounds
without DMSO are on the left and with 0.1% DMSO on the right. Errors shown are the fractional standard deviation. (A) Effect on
DFO-induced expression of VEGF by HRE-targeted match and mismatch polyamide cores in U251 cells at 1, 0.2, and 0.02 μM. (B) Effect on
DFO-induced expression of VEGF by HRE-targeted match and mismatch polyamide cores in LNCaP cells at 10, 2, and 1 μM. (C) Effect
on DHT-induced expression of PSA mRNA by ARE-targeted match and mismatch polyamide cores in LNCaP cells at 5, 1, and 0.1 μM.
Cell Viability Effects of Control Compounds 45-48 in
U251 and LNCaP Cells. IC50 values for growth inhibition
in U251 cells treated with oxime-linked polyamide coreless
compounds 45-48 at a range of 10-0.002 μM and LNCaP
cells at 50-0.02 μM were measured by a standard WST-1
colorimetric assay. Negligible cell viability effects were mea-
sured in either U251 or LNCaP cells after a 48 h treatment
course with 45-48 at the concentrations used. These data are
shown in Supporting Information Figure SI 2.
Gene Regulation IC50 Values for ARE-Targeted Oxime-
Linked Compounds in LNCaP Cells. IC50 values for PSA
mRNA expression levels were measured for 28, 31, and 33 by
quantitative real-time RT-PCR in LNCaP cells. 28 was used
as a benchmark, and 31 and 33 were selected on the basis
of their high biological activity and to provide variety in the
tail substitution. Cells were dosed with 28 at a range of 30-
0.1 μM and 31 and 33 at 10-0.03 μM. Isotherms were
generated for each of three independently performed real-
time quantitative RT-PCR experiments in LNCaP cells for
28, 31, and 33. An IC50 value was generated for each run, and
the IC50 value given in Table 1 for each compound is the
average value of three independent measurements (error
given is the standard error of the mean (SEM) between the
three independent runs). Isotherms (Figure 6B) were gener-
ated from the average value of the independent runs at each
concentration (error bars are the SEM for each data point).
Average IC50 values for gene regulation were as follows: 28,
6.0 ((2.6) μM; 31, 0.28 ((0.1) μM; 33, 0.6 ((0.3) μM.
Discussion
A focused library of polyamides was synthesized with
different modifications at the C-terminus, in which the linker,
linkage, and tail group were varied and tested in two separate
cell lines for gene regulation activity of two different target

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7385
effort to evaluate whether modifications in tail or linker
structures can have differing effects on biological activity in
a cell-line dependent fashion. The polyamide dose required to
effect gene regulation activity was found to be cell-line
dependent, but the trend in relative activities of polyamides
did not vary greatly between different cell lines. Two encoura-
ging results of this study were the degree to which changes in
the linker composition are well tolerated and the significant
increase in biological activity achieved upon replacement of
an amide linkage with an oxime linkage between the linker
and tail groups.
The biological activity of a series of polyamides targeted to
the HRE of VEGF was measured under hypoxic (inducing)
conditions in two different cell lines, U251 (glioma) and
LNCaP (prostate cancer) lines. Polyamide regulation of
DFO-induced VEGF expression in LNCaP cells has not
previously been published. In the HRE-targeted amine series
(1-8and12-19), a slightlinkereffect wasobservedbut varied
between the two cell lines. Overall, changes in the linker group
did not dramatically affect biological activity in either cell line.
The most pronounced effect was conjugation of the IPA tail,
which resulted in an increase for each linker group in both cell
lines, with the exception of the C3 linker in LNCaP cells. IPA
conjugation affected biological activity of the triamine-linked
compounds most significantly in both U251 and LNCaP.
The amide-linked ARE series was assayed for activity
against DHT-induced, AR-regulated PSA expression in
LNCaP cells. Removal of the tertiary amine in the linker
resulted in an increase in biological activity, as seen for 29 vs
28, while reduction in linker length from a C7 to a C3 linker led
to a modest loss of activity (29 vs 30). Although the mechan-
ism by which mismatch polyamides affect gene expression is
unknown, removal of the tertiary amine reduces mismatch
activity (e.g., 39 is more active than 40 and 41). These results
again indicate that there is tolerance of linker group modifica-
tion and that the ARE system in LNCaP cells benefits from
removal of the amine group in the linker. The Tm data indicate
that the absence of biological activity is not due to an inability
of the polyamides to bind to DNA, as each of the IPA
conjugates has a lower Tm than their respective free-amine
parent compounds but higher biological activity. The tria-
mine-bearing polyamides 24 and 28, in fact, have higher Tm
values than the neutral, aliphatic linker-bearing polyamides
25and29(C7 linker) and 26and 30 (C3linker)(free amine and
IPA, respectively, for each pair). These data suggest that the
presence of the tertiary amine and the positive charge increase
the binding affinity of these polyamides.
Figure 6. Cell viability data and IC50 values for gene regulation for
in LNCaP cell culture. (A, top) Cell viability was measured by a
WST-1 colorimetric assay for LNCaP cells dosed for 48 h with
polyamides 28-33 at the following concentrations: 30, 10, 3, 1, 0.3,
0.1, 0.03, and 0.01 μM. For 31-33, IC50 values for cell viability were
extracted from isotherms generated in KaleidaGraph from the data.
(B, bottom) Quantitative real-time RT-PCR data for 28, 31 and 33
in LNCaP cells to establish IC50 values for PSA gene regulation
under inducing conditions. Concentrations employed for 28 were
30, 10, 3, 1, 0.3, and 0.1 μM, and for 31 and 33 they were 10, 3, 1, 0.3,
0.1, and 0.03 μM. IC50 values were extracted from isotherms
generated in Kaleidagraph from the RT-PCR titrations. (C) IC50
values (μM) for cell viability estimated or calculated for 28-33, and
IC50 values (μM) for PSA gene regulation for 28, 31, and 33 shown
in tabular format. Values given are average values of three inde-
pendent experiments; the standard error of the mean is given in
parentheses.
Oxime-linked polyamides were synthesized for both the
HRE (9-11, 20-22) and ARE (31-33, 42-44) systems. The
oxime had been introduced as an efficient means of conjugat-
ing a polyamide core to 18F-labeled 4-fluorobenzaldehyde for
PET studies, but the ability of polyamides possessing this
motif to regulate endogenous gene expression was not known.
The aldehyde tail groups selected were 4-fluorobenzaldehyde,
3-fluorobenzaldehyde, and 3-carboxybenzaldehyde; 3-fluor-
obenzaldehyde was selected to probe position effects, and 3-
carboxybenzaldehyde provided an oxime-linked mimic of the
amide-linked IPA tail.
In U251 cells (Figure 5A), the oxime linked match (9-11)
polyamides had activity comparable to the triamine- or C7-
linker amide-linked IPA conjugates (6 and 7, respectively).
Introductionofthe oxime linker hada moresignificantimpact
on the biological activity of the mismatch polyamides 20-22,
which were as active as their oxime-linked counterparts 9-11.
genes. Prior to the current study, no direct comparison of the
gene regulatory activity of a polyamide series between multi-
ple cell lines had been performed. Measurement of VEGF
mRNA expression levels in both U251 and LNCaP for the
entire amide- and oxime-linked series was performed in an

7386 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Jacobs and Dervan
Table 1. Table of IC50 Values (μM) for Growth Inhibition and Gene
Regulation in LNCaP Cells for 28-33a
these concentrations did not show g50% decrease in cell
growth, so an IC50 value for growth inhibition could not be
calculated. IC50 values for growth inhibition were calculated
for 31-33 (Table 1). Oxime mismatch polyamide 42 mini-
mally affected cell viability, while 43 and 44 had estimated
IC50 values for cell viability of g30 μM. The IC50 values for
gene regulation for 31 and 33 are 20- and 10-fold lower than
28, respectively. In the case of the 4-fluorooxime-linked
compound 33, the IC50 values for growth inhibition and gene
regulation are within error of each other. The IC50 for growth
inhibition of 28 and 31 is ∼5-fold higher than the IC50 for gene
regulation, but both these values are ∼20-fold lower for 31
than for 28, indicating that 31 is ∼20-fold more potent than 28
and more specific than 33.
IC50 (μM)
(growth inhibition
IC50 (μM))/(gene
regulation IC50
growth
inhibition
gene
regulation
compd
(μM))
28
29
30
31
32
33
>30
>30
6.0 ( 2.6
g5
>30
1.7 ( 0.3
0.6 ( 0.1
0.4 ( 0.0
0.3 ( 0.1
0.6 ( 0.3
6.1
0.7
a Data are shown in Figure 5. IC50 for gene regulation was measured
only for 28, 31, and 33. For 28, 31, and 33, the (IC50 for growth
inhibition)/(IC50 for gene regulation) ratio was calculated.
These results indicate that the oxime linkage can effect a
significant increase in the cell uptake and gene regulation
activity of polyamides. For unconjugated hydroxylamine
polyamides 27 and 38, a ∼50% and 10% down-regulation
was measured, respectively, of induced PSA mRNA expres-
sion in LNCaP cells at 5 μM, and 27 and 38 were significantly
less active than the oxime conjugates. Although previous
studies have rigorously verified the stability of oxime-linked
conjugates in vivo, a control experiment was performed where
polyamide-lacking compounds 45-48 were synthesized and
their cytotoxicity measured.19 It was rationalized that cyto-
toxicity equivalent to that of oxime polyamide conjugates
would serve as an indirect measure of bond lability, whereas a
lack to toxicity would reinforce the robustness of this chemical
bond. The growth inhibitory effects of these compounds were
studied in U251 and LNCaP cells (parts A and B of Figure
SI2, respectively). Treatment with 45-48 in both cells lines
failed to cause dramatic cell death at any concentrations used
(10-0.002 μM in U251, 50-0.02 μM in LNCaP), indicating
that the oxime linkage did not decompose and invoke cell
death. These results suggest that the oxime linkage is promis-
ing for future work aimed at increasing polyamide potency.
Although not the focus of the current study, the mechan-
isms of polyamide cell entry, nuclear localization, and cell exit
are active areas of investigation.11 Our current working
hypothesis for polyamide biological activity is that passive
or nonspecific mechanisms of polyamide cell uptake and
competing efflux pathways influence polyamide accumula-
tion in the cell nucleus, andstructuralmodifications thatresult
in improved polyamide biological activity may reflect a
decrease in efflux. Polyamides are believed in part to undergo
active efflux by p-glycoprotein pumps, which have more
pronounced activity in immortalized cancer cells such as those
employed in the current study.20,21 The chemical agent ver-
apamil, which inhibits p-glycoprotein pump activity, has been
found to result in nuclear localization for some fluorophore-
polyamide conjugates.10 The mechanistic basis for the im-
proved activity observed upon introduction of structural
features such as the IPA tail is unknown, but we speculate
that it is partially due to reduced polyamide efflux.
A similar result was seen for the C7- and C3-linker, amide-
linked IPA conjugate pairs 7 and 18, and 8 and 19, in U251
cells. Thus, although there does appear to be tolerance of
linker variations, the greatest difference in biological activity
within an HRE match-mismatch pair in U251 cells is the
triamine-linked IPA compounds 6 and 17.
In LNCaP cells (Figure 5B), the HRE-oxime series 9-11
were more active than the amide-linked series 6-8 and in fact
reduced VEGF mRNA expression levels to below baseline
levels. In this case, introduction of fluorine on the tail restored
the match-mismatch activity difference to ∼2-fold (i.e., 9 and
20, or 10 and 21, or 11 and 22). The Tm data for the oxime-
linked polyamides and their parent unconjugated polyamides
do not indicate that differences in binding affinity play any
role in the differences in activity; the Tm values measured for
the amide series, both match and mismatch cores, are compar-
able to those measured for the oxime series.
Polyamide regulation of VEGF expression in LNCaP had
not been studied, but it was known that polyamide-effected
regulation of PSA in LNCaP cells can be achieved with 10 and
5 μM 28. A 10-fold higher concentration of HRE polyamides
was required in LNCaP cells compared to U251 cells to effect
comparable VEGF gene regulation. Although the effects on
VEGF expression in LNCaP and U251 cells of the HRE
polyamide series vary somewhat, it is encouraging that there
are no drastic differences in biological activity measured for
any of the compounds tested between the two cell lines. This
suggests thata structuralmodification thatresultsinincreased
gene regulation in one cell line may have a similar effect for the
same core in other cell lines.
The data for the oxime-linked ARE series (31-33, 42-44)
in LNCaP cells (Figure 5C) indicate that the oxime linkage
greatly increases the gene regulation activity of the ARE
polyamides. PSA mRNA expression was negligible in cells
treated with 31-33 at 5 μM, but there was evidence of cell
death upon visual inspection (vide infra) and levels of total
mRNA were very low. The average gene expression titration
curve generated by quantitative real-time RT-PCR for 28, 31,
and 33 is shown in Figure 6B. These data illustrate the impact
of the oxime linkage on the biological activity, as the activity
curves of the oxime compounds 31 and 33 are shifted down by
a factor of g10-fold from the amide-linked triamine IPA
parent polyamide 28 (IC50 values in Table 1).
The tolerance of modifications to the linker group suggests
that further modifications to the linker group may lead to
improved polyamide biological activity. Comparison of the
data gathered for the HRE match and mismatch polyamides
in U251 and LNCaP cells indicate that while the concentra-
tions required to achieve acceptable levels of gene regulation
activity will vary between cell lines, the effect of structural
modifications will most likely be comparable between cell
lines; i.e., the trends in activity will translate between cell lines.
In addition, the use of an oxime linkage in place of an amide
To quantitate the effect of 28-33 and 39-44 on cell growth
in LNCaP cells, cells were treated with compounds and
viability was measured (Figure 6A (28-33) and Figure SI1
(39-44)). Cells treated with the amide-linked compounds at

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7387
linkage between the tail and linker groups was found to
increase the gene regulation activity of polyamides. This
increase was most noticeable for the ARE oxime-linked series
31-33 in LNCaP cells. The dramatic increase in activity
measured for 31 compared to the previously published results
for 28 makes this compound and related derivatives useful for
future ARE studies. In addition, these results suggest that the
oxime linkage may be a useful structural feature to increase
the biological activity of polyamides in other biological
studies. Although this study did not uncover a set of predictive
structural requirements for polyamide cell uptake, the dis-
covery of the utility of the oxime linkage for gene regulation
studies is a welcome addition to the known set of structural
modifications to increase polyamide cell uptake.
108.3 mg (93% yield) of the 3-((3-acetamidopropoxyimino)-
methyl)benzoic acid product 46. 1H NMR (DMSO-d6):
δ 13.14 (s, 1H), 8.33 (s, 1H), 8.18 (t, 1.8 Hz, 1H), 7.95 (app dt,
7.5, 1.5 Hz, 1H), 7.87 (br t, 4.8 Hz, 1H), 7.83 (app dt, 8.3, 1.5 Hz,
1H), 7.54 (app t, 7.8 Hz, 1H), 4.14 (t, 6.5 Hz, 2H), 3.12 (m, 2H),
1.79 (s, 3H), 1.78 (m, 2H). 13C NMR (DMSO-d6): δ 169.1, 166.8,
148.0, 132.5, 131.3, 130.9, 130.4, 129.2, 127.4, 71.5, 35.4, 28.9,
22.6. Exact mass (M þ Hþ): calcd, 265.1188; found, 265.1199.
Synthesis of 47. The synthesis and purification of 47 pro-
ceeded as described for 45 but with 4-fluorobenzaldehyde.
1
Yield: 66.8 mg (74%). H NMR (DMSO-d6): δ 8.27 (s, 1H),
7.86 (br s, 3H), 7.66 (m, 2H), 7.26 (m, 2H), 4.17 (t, 6.3 Hz, 2H),
2.90 (m, 2H), 1.94 (m, 2H). 13C NMR (DMSO-d6): δ 164.0,
162.0, 129.0 (J=28 Hz), 128.5 (J=11 Hz), 115.9 (J=86 Hz),
70.4, 36.2, 27.0. Exact mass (M þ Hþ): calcd, 197.1090; found,
197.1091.
Synthesis of 48. The synthesis and purification of 48 pro-
ceeded as described for 46 but with 4-fluorobenzaldehyde.
Yield: 103.3 mg (89%). H NMR (DMSO-d6): δ 8.24 (s, 1H),
Materials and Methods
1
Synthesis of Polyamides. Polyamides were synthesized by
solid-phase methods on Kaiser oxime resin (NovaBiochem) or
Boc-β-alanine-PAM resin (Peptides International), were
cleaved from resin with 3,30-diamino-N-methyl-dipropylamine,
N,N0-dimethylpropane-1,3-diamine (Dp), heptane-1,7-diamine,
propane-1,3-diamine, or tert-butyl 3-aminopropoxycarbamate,
and purified by reverse-phase HPLC.16,22-24 Synthesis of IPA
conjugates and oxime conjugates was as previously described.8,16
Reaction progress was monitored by analytical HPLC at
310 nm. Turn deprotection was done through addition of
1 mL of 1:1 TFA/DCM at ambient temperature, and purificat-
ion by reverse-phase preparative HPLC was performed imme-
diately following successful deprotection. Reverse-phase HPLC
solvent systems were 0.1% TFA (aqueous) and acetonitrile.
Polyamide purity and identity were assessed by analytical
HPLC (310 nm) and MALDI-ToF MS, and polyamides were
quantitated by UV-vis at 310 nm (ε= 69 360 M-1 cm-1 for
eight-ring hairpin polyamides). Purity of all compounds was
g95% by analytical HPLC. Supporting Information Table SI 1
provides expected and observed (MþHþ) (MALDI-ToF MS)
7.87 (br s, 1H), 7.65 (m, 2H), 7.24 (m, 2H), 4.10 (t, 6.5 Hz, 2H),
3.11 (m, 2H), 1.78 (s, 3H), 1.76 (m, 2H). 13C NMR (DMSO-d6):
δ 169.1, 164.0, 162.0, 129.0 (J=33 Hz), 128.6 (J=13 Hz), 115.8
(J=87.5 Hz), 71.3, 35.5, 28.9, 22.6. Exact mass (M þ Hþ): calcd,
239.1196; found, 239.1189.
Determination of DNA Melting Temperature (Tm) Values.
Melting temperatures were measured according to a previously
published methodology on a Varian Cary 100 spectrophoto-
meter equipped with a thermocontrolled cuvette holder and
quartz cuvettes (1 cm path length).18 The buffer was an aqueous
solution of 10 mM sodium cacodylate, 10 mM KCl, 10 mM
MgCl2, and 5 mM CaCl2 at pH 7.0. Oligonucleotide sequences
were 50-GTGCATACGTGGGC-30 (HRE 14-mer top), 50-
GCCCACGTATGCAC-30 (HRE 14-mer bottom), 50-TTGCA-
GAACAGCAA-30 (ARE 14-mer top), and 50-TTGCTGTT-
CTGCAA-30 (ARE 14-mer bottom).
Cell Culture Experiments. For cell culture experiments, a
polyamide stock solution in DMSO and DNase/RNase-free
water was prepared by dissolving 5-30 nmol of polyamide in
DMSO (1-3 μL), followed by addition of DNase/RNase-free
water to a target concentration of 50 M. Suspensions were
centrifugated to remove undissolved material, and solution
concentration was determined by UV-vis measurement of a
100ꢀ diluted solution at 310 nm (ε=69 360 M-1 cm-1 for eight-
ring hairpin polyamides). The DMSO concentration of cell
media solutions was kept below 0.1%, and a DMSO control
was included in quantitative real-time RT-PCR (see below).17
Isotherms were generated using the following modified Hill
equation: Y=m1 þ (m2 - m1)/(1 þ (m0/m3)), where m1=100,
1
and purity by analytical HPLC for 1-44. H and 13C NMR
spectra were obtained on a 500 MHz spectrometer.
Synthesis of 45. An amount of 1 equiv (84.3 mg, 0.44 mmol)
of tert-butyl 3-(aminooxy)propylcarbamate was dissolved in
300 μL of DMF, and 1.2 equiv of 3-formylbenzoic acid added
as a 50 mM solution in DMF.25 Reaction progress was mon-
itored by analytical HPLC at 254 nm for starting material
consumption and production of a mixture of the E- and Z-3-
(10,10-dimethyl-8-oxo-3,9-dioxa-2,7-diazaundec-1-enyl)benzoic acid
intermediate. Upon consumption of starting material, the Boc
protecting group was removed by addition of 4 mL of 1:1 TFA/
DCM. After 15 min, the mixture was concentrated by rotovap
and the product purified by reverse phase preparatory HPLC.
The E and Z products were separable by preparative HPLC but
interconvert at room temperature on the time-scale of the
experiments and so were recombined to yield 43.3 mg (44%
yield) of 3-((3-aminopropoxyimino)methyl)benzoic acid (45).
1H NMR (DMSO-d6): δ 13.23 (s, 1H), 8.36 (s, 1H), 8.20 (app t,
J=1.8 Hz, 1H), 7.96 (app dt, 7.8, 1.8, 1 Hz, 1H), 7.88 (br s, 3H),
7.84 (app dt, 8.0, 1.5 Hz, 1H), 7.55 (app t, 7.8 Hz, 1H), 4.20 (t, 6.3
Hz, 2H), 2.91 (t, 7.3 Hz, 2H), 1.96 (m, 2H). 13C NMR (DMSO-
d6): δ 166.8, 148.6, 132.3, 131.4, 131.0, 130.6, 129.2, 127.4, 70.6,
36.2, 27.0. Exact mass (M þ Hþ): calcd, 223.1083; found,
223.1078.
m2=5000, m3=5 e-7
.
Measurement of Cell Viability in U251 and LNCaP Cells.
U251 and LNCaP cells were maintained as previously described.8,14
For the growth inhibition assay, cells were plated in 96-well
plates in 0.2 mL at (10-15)ꢀ102 cells per well ((15-20)ꢀ103
cells/mL). After 24 h (U251) or 48 h (LNCaP), 150 μL of
medium was removed and replaced with 50 μL of 2ꢀ (polyamide
concentration) cell medium solutions. After 48 h, the medium
was replaced with 100 μL of fresh medium, and cells were
allowed to recover for 24 h before addition of 10 μL of WST-1
reagent (Roche) to each well. Cells were incubated for 30 min,
and absorbance at 450 nm was measured. Untreated controls
and cell-free, media-only controls were included on each plate,
and each well was corrected for average background absorption
of media-only controls before normalization of the average
absorption for each concentration to untreated controls.
Synthesis of 46. An amount of 1 equiv (83.8 mg, 0.44 mmol) of
tert-butyl 3-(aminooxy)propylcarbamate was used to synthesize
45, as above. Following Boc deprotection and prior to purifica-
tion, 2 equiv of (Ac)2O (83.2 μL, 0.88 mmol) and 2.5 equiv of
DIEA (191.6 μL, 1.1 mmol) were added. Reaction progress was
monitored by analytical HPLC at 254 nm, and the product was
purified by preparative reverse phase HPLC. As above, the E
and Z enantiomers were separable but were recombined to yield
Measurement of Induced Gene Expression in U251 and
LNCaP Cells. Measurement of hypoxia-induced VEGF expres-
sion in U251 cells and DHT-induced PSA expression in LNCaP
cells was as previously described.8,14 Measurement of hypoxia-
induced VEGF expression in LNCaP cells followed the protocol
for DHT-induced PSA expression in LNCaP cells but with the

7388 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Jacobs and Dervan
following changes: gene induction was by DFO, and FBS (not
charcoal stripped) was used. Primer sequences were as follows:
(10) Crowley, K. S.; Phillion, D. P.; Woodard, S. S.; Schweitzer, B. A.;
Singh, M.; Shabany, H.; Burnette, B.; Hippenmeyer, P.; Heitmeier,
M.; Bashkin, J. K. Controlling the intracellular localization of
fluorescent polyamide analogues in cultured cells. Bioorg. Med.
Chem. Lett. 2003, 13, 1565–1570.
(11) Best, T. P.; Edelson, B. S.; Nickols, N. G.; Dervan, P. B. Nuclear
localization of pyrrole-imidazole polyamide-fluorescein conju-
gates in cell culture. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12063–
12068.
VEGF,
L
50-AGGGCAGAATCATCACGAAG-30,
R
50-
GGGTACTCCTGGAAGATGTCC-30; PSA,
L
50-TCTG-
CGGCGGTGTTCTG-30, R 50-GCCGACCCAGCAAGATC-
A-30; β-glucuronidase, L 50-CTCATTTGGAATTTTGCCG-
ATT-30, R 50-CCGAGTGAAGATCCCCTTTTTA-30.
(12) Edelson, B. S.; Best, T. P.; Olenyuk, B.; Nickols, N. G.; Doss,
R. M.; Foister, S.; Heckel, A.; Dervan, P. B. Influence of structural
variation on nuclear localization of DNA-binding polyamide-
fluorophore conjugates. Nucleic Acids Res. 2004, 32, 2802–2818.
(13) Olenyuk, B. Z.; Zhang, G. J.; Klco, J. M.; Nickols, N. G.; Kaelin,
W. G.Jr.; Dervan, P. B. Inhibition of vascular endothelial growth
factor with a sequence-specific hypoxia response element antago-
nist. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16768–16773.
(14) Nickols, N. G.; Dervan, P. B. Suppression of androgen receptor-
mediated gene expression by a sequence-specific DNA-binding
polyamide. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10418–10423.
(15) Nickols, N. G.; Jacobs, C. S.; Farkas, M. E.; Dervan, P. B.
Modulating hypoxia-inducible transcription by disrupting the
HIF-1-DNA interface. ACS Chem. Biol. 2007, 2, 561–571.
(16) Harki, D. A.; Satyamurthy, N.; Stout, D. B.; Phelps, M. E.;
Dervan, P. B. In vivo imaging of pyrrole-imidazole polyamides
with positron emission tomography. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 13039–13044.
Acknowledgment. We thank the National Institutes of
Health for support (Grant GM051747), Dr. Giovanni Melillo
for U251 cells, and Dr. Daniel Harki for providing tert-butyl
3-aminopropoxycarbamate, tert-butyl 3-(aminooxy)propyl-
carbamate, and 11 and for help with the manuscript.
Supporting Information Available: MALDI-ToF and purity
by analytical HPLC data for 1-44, Tm data for 1-44, cell
viability data for 39-44 in LNCaP cells, and cell viability data
for 45-48 in LNCaP and U251 cells. This material is available
free of charge via the Internet at http://pubs.acs.org.
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