Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting

Article abstract of DOI:10.1021/jacs.6b03697

The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to

Full text of DOI:10.1021/jacs.6b03697

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Article
Integrating Display and Delivery Functionality with a Cell Penetrating
Peptide Mimic as a Scaffold for Intracellular Multivalent Multi-targeting
Yugang Bai, Lien T. Nguyen, Ziyuan Song, Shaohong Peng, JuYeon Lee, Nan Zheng, Iti Kapoor,
Lauren D. Hagler, Kaimin Cai, Jianjun Cheng, H. Y. Edwin Chan, and Steven C. Zimmerman
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03697 • Publication Date (Web): 29 Jun 2016
Downloaded from http://pubs.acs.org on June 30, 2016
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Integrating Display and Delivery Functionality with a Cell Penetrat-
ing Peptide Mimic as a Scaffold for Intracellular Multivalent Multi-
targeting
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Yugang Bai,†,§ Lien Nguyen,†,§ Ziyuan Song,‡,§ Shaohong Peng,⫽ JuYeon Lee,† Nan Zheng,‡ Iti Kapoor,†
Lauren D. Hagler,† Kaimin Cai,‡ Jianjun Cheng,‡ H. Y. Edwin Chan,⫽ and Steven C. Zimmerman†,*
†Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States
‡Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States
⫽Laboratory of Drosophila Research and School of Life Sciences, The Chinese University of Hong Kong, Shatin N.T., Hong Kong
SAR, China
ABSTRACT: The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets
with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a
series of multivalent ligand constructs that bind to the expanded dCTG (CTGexp) and rCUG nucleotide repeats (CUGexp) known to cause
myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are
solubilized, delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of
splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of
(CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a
Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for
intracellular multivalent targeting. PLACE HOLDER PLACE HOLDER PLACE HOLDER PLACE HOLDER
Introduction
Oligovalent or polyvalent (multivalent) ligands are synthetic
constructs that present multiple copies of receptor-binding
moieties. Compared with their monovalent analogs, these con-
structs offer both thermo-dynamic and kinetic advantages originat-
ing from a range of mechanisms including the chelate effect, subsite
binding, steric stabilization, receptor clustering, and statistical and
local concentration effects.1 Not surprisingly, the affinity of
multivalent ligands toward their biological targets is highly
dependent on the molecular structure and conformation of ligands
in aqueous solution. Well-designed multivalent constructs may
afford significant enhancements in affinity and selectivity toward
their targets.2 Indeed, the use of multivalency is now an established,
effective strategy for developing potent inhibitors of a range of
biological processes.3
The majority of reported multivalent ligands function at the cell
surface. Although multivalent ligands for intracellular targets have
been reported,4 an obvious limitation is the need for large
structures to pass the cell membrane. Polyvalent constructs have
been developed as targeted delivery vehicles,5 some of which enter
the cell by endocytosis and deliver their cargo.6 Such approaches
are especially important for cancer therapy allowing significant
Figure 1. Schematic illustration of the polymeric approach for the
quantities of hydrophobic cytotoxic agents to be delivered selectivi-
fabrication of multivalent construct with synthetic polypeptide having
dual function as scaffold and transporter. The stability of the complex
ty to cells within the target tissue.2f,2g,5 By encapsulating large quan-
tities of the drug in a nanoscale vehicle that enhances selective lo-
calization through the display of multiple copies of a targeting
formed between the multivalent ligand and the target RNA are
improved both kinetically and thermodynamically.
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Figure 2. Proposed mechanism for how CTG expansion in DMPK gene leads to DM1, and a simple illustration of how acridine-melamine small
molecule targets CUGexp, selectively inhibiting formation of MBNL1-CUGexp RNA complexes.
moiety, the polyvalent construct becomes an essential part of the
therapeutic formulation, increasing local drug concentration and
reducing unwanted, off-site cytotoxicity.
linking two or more of these monomeric ligands can produce a
multivalent effect, resulting in enhanced selectivity and affinity.4b,10
Indeed, compounds with nanomolar Ki values for inhibiting
CUGexp-MBNL1 complex are now known, but these often show
partial rescue of pre-mRNA mis-splicing in the DM1 model cells or
require relatively high concentrations (10−100 µM) for full
rescue.10 These findings may indicate that cell permeability limits
the compound activity. Herein, we report the use of a synthetic
cell-penetrating peptide (CPP) mimic11 to function as both a
delivery vehicle for transmembrane transport and as a scaffold to
display multiple copies of a CUGexp-selective ligand (Figure 1). By
combining these two functions, a series of tunable polymeric,
multivalent DM1 ligands are readily available that exhibit
significantly improved efficacy in a DM1 cell model, showing low
nanomolar Ki values and high splicing rescue of insulin receptor
(IR) pre-mRNA at low concentrations. The polymers were tested
in vivo using a DM1 Drosophila larval crawling assay and a signifi-
cant improvement in the pathogenic phenotype was observed.
An important class of intracellular therapeutic targets, well
suited to a multivalent ligand approach, is found in a set of rare
diseases, known as trinucleotide repeat expansion diseases
(TREDS). TREDS are associated with unstable microsatellites, for
example, Huntington’s disease, myotonic dystrophy type 1 (DM1),
fragile X syndrome, and various spinocerebellar ataxias being
associated with CAGexp, CTGexp, CCGexp, and CNGexp repeats,
respectively.7 In the case of DM1, the CTG expansion located in
the 3’-untranslated region of the dystrophia myotonica protein
kinase (DMPK) gene produces a toxic CUGexp transcript. Thus, as
outlined in Figure 2, a deleterious, RNA gain-of-function model for
DM1 involves the CUGexp transcript sequestering the alternative
splicing regulator, muscleblind-like 1 (MBNL1) into nuclear foci,
which leads to splicing defects in >100 pre-mRNAs.8
One therapeutic strategy has focused on cell-permeable small
molecules that selectively inhibit the transcription of CTGexp or
bind CUGexp and inhibit MBNL sequestration.9 As described
above, the repeating nature of the DNA and RNA target means that
Experimental Section
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Materials and Methods. All reagents were purchased from
glutamate, were prepared by acid-catalyzed esterification of 3-
chloro-1-propanol and the corresponding glutamic acid (L-, D-, or
DL-isomer). Treatment with phosgene in THF afforded the corre-
sponding γ-(3-chloropropyl) L-, D- and DL-glutamic acid N-
carboxyanhydrides (L-, D- and DL-Glu-NCA). For the ligand, we
selected acridine-melamine conjugate 2, which is an alkyne-
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Acros Organics, Fisher Scientific, AK Scientific, TCI America, or
Sigma-Aldrich, and used without further purification unless
otherwise noted. Water was obtained from a Milli-Q purification
system. Instrument setup and synthetic procedures for small
molecules, polymers and proteins as well as methods used to
determine the bioactivity of the polymeric ligands can be found in
the Supporting Information.
containing derivative of a ligand reported by Baranger and
Zimmerman in 2009 to selectively bind CUGexp with high
nanomolar affinity.9c
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The polymerization of 1 was effected using hexamethyl-
disilazane (HMDS) as the initiator16 to give the corresponding
polypeptides, namely poly[γ-(3-chloro-propyl)-L-glutamate]
(PCPLG), poly[γ-(3-chloropropyl)-D-glutamate] (PCPDG), and
poly[γ-(3-chloropropyl)-DL-glutamate] (PCPDLG). The degree
of polymerization (DP) of polypeptide is controlled by monomer-
to-initiator (M/I) feed ratio. As seen in Figure 3a, the GPC traces
for PCPLGs prepared with 1:25, 1:50, and 1:75 initiator to monomer
ratios gave symmetrical peaks with decreasing retention times. In each
case, the polydispersity index (PDI) was <1.10. The stereochemistry
of the amino acid residues on each polypeptide (L-, D-, or DL-)
were determined by the NCA monomer used. The side-chain
chloro groups were converted to azido groups by treatment with
NaN3 in DMF giving polyazide 3. In each case, the chloride to
azide conversion appeared to be quantitative based on the 1H
Results and Discussion
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Synthesis of the Polypeptide-based DM1 Polymeric
Ligands. In selecting a membrane-penetrating polymer to serve
as a scaffold for multi-ligand display, there were many options. The
guanidinium-rich polypeptide system recently developed by Cheng
and coworkers12 was selected because of its accessibility and
defined structure. In particular, these α-helical polymers can be
synthesized using a facile and controlled living polymerization of
N-carboxyanhydride (NCA) 1. The product polyazide has the
potential for multiple post-functionalization reactions using the
copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC).13 The
synthesis of 1 generally followed the reported approach.14,15 Thus,
the functionalized amino acids, γ-(3-chloropropyl)-L-glutamate, γ-
(3-chloropropyl)-D-glutamate
and
γ-(3-chloropropyl)-DL-
Figure 3. (a) GPC curve overlay of parent polymers, poly[γ-(3-chloropropyl)-L-glutamate] (PCPLGs), of three different molecular weights.
Polydispersity indices (PDI) < 1.10 for all three. These results are typical for the polymers studied. (b) HPLC elution curve of the alkyne-bearing
DM1 ligand using C-18 reverse-phase column and gradient elution from solvent A/B = 20/80 to 80/20; solvent A = CH3CN with 0.1 % TFA,
solvent B = H2O. (c) Synthesis of the polymeric ligand in schematic format. The guanidinium and acridine moieties on the side-chains are placed on
the exterior of the α-helical polypeptide rod, as visualized by the top-down view.
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Figure 4. (a) CD spectra of PLG50-1/5, PDG50-1/5 and PDLG50-1/5 (pH = 7.0 aqueous solution). The L- and D-polymers showed characteristic
double minimum or maximum for helical polypeptides at 208 and 222 nm. On the contrary, the DL-polymers did not show any helicity. (b)
Measurement of dissociation constant of GST-MBNL1N-(CUG)16 complex by fluorescence anisotropy and curve-fitting. (c) Representitive
fluorescence anisotropy titration curve of the polymeric ligand (PDLG50-1/2) and the corresponding IC50 and Ki calculated from curve-fitting. Study
of other polymeric ligands were performed analogously. (d) Summary of measured Ki of all polymeric ligands. ‘*’ indicates P ≤ 0.05; ‘**’ indicates P ≤
0.01; and ‘***’ indicates P ≤ 0.001 as determined by a two-tailed Student’s t-test.
NMR characterization (Figure S4 and S5 in Supporting
Information) giving poly[γ-(3-azidopropyl)-L-glutamate]
(PAPLG), poly[γ-(3-azidopropyl)-D-glutamate] (PAPDG), and
poly[γ-(3-azido-propyl)-DL-glutamate] (PAPDLG).
Homochiral Polymers are Charged Helical Rods.
The α-helical structure of multivalent ligand 4 is an essential design
feature by: (1) incorporating cell membrane permeability and (2)
maintaining the ligands on the exterior of the helical rod and
available for binding. The CD spectra of PLG50-1/5, PDG50-1/5,
and PDLG50-1/5 are shown in Figure 4a. Note that the naming
convention of the polymeric agents is outlined in Figure 3, with P
referring to polymer, D, L or DL the chirality of the monomeric
units, G the glutamate monomer unit, the subscript the degree of
polymerization, and the ratio of ligand to guanidinium side-chain.
For PLG50-1/5, a characteristic double minimum at 208 and 222
nm was observed, indicating that the ligand-loaded polypeptide
The azido-rich polypeptides served as the parent polymers for
the conjugation of N-propargylguanidine, ligand 2 (Figure 3b),
and an RNAzyme-mimicking pentapeptide (vide infra).17
A
schematic illustration of the synthesis of polymeric, multivalent
ligands 4 is shown in Figure 3c. The Cu(I) catalyzed alkyne-azide
cycloaddition is both a highly chemoselective and high yielding
reaction, so the conjugation was considered likely to be
quantitative and the ratio of side-chains to be equal to the reagent
stoichiometry. Support for this assumption came from several
observations, including a characteristic triazole peak in the 1H
NMR (Figure S6). The acridine resonances were not observed
possibly because packing of the hydrophobic chromophores within
the helix leads to their reduced mobility (slow tumbling) and poor
solvation.18,19 The ligand incorporation into the polymers was
established by the acridine fluorescence observed for each polymer,
and particularly by UV studies that showed an approximately linear
increase in absorbance with L/G feed ratio (Figure S3). The
molecular weight of each polymer 4 was calculated based on the
molecular weight of parent PCPLGs determined by multi-angle
laser light scattering (MALLS) and the alkyne loading
stoichiometry (Table S1).
adopts
a standard helical structure. Using Equation 1 (see
Experimental section in SI), the helicity % was estimated to be
approximately 50%. For PDG50-1/5, prepared from the D- mono-
mers, positive bands at 208 and 222 nm were observed in the CD
spectrum of PDG50-1/5, closely resembling a mirror image of the
spectrum of PLG50-1/5. In contrast, the random D-/L-copolymer,
PDLG50-1/5, showed a flat curve near the baseline, indicating a
random coil structure with no Cotton effect.
Computational studies20 have shown this class of polymers to
have hindered, hydrophobic backbones that reside in a water-
depletion zone and, indeed they have been shown to be
significantly more stable to enzymatic cleavage by peptidases.21 It
was anticipated that the esters would be similarly protected from
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fluorescence anisotropy upon titration of TAMRA-r(CUG)16 with
1
2
3
GST-MBNL1N. Curve fitting gave a dissociation constant (Kd) for
the protein-RNA complex, Kd = 11.3 nM, which is close to that
reported previously.22
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To measure the inhibition potential of the polymeric ligands,
the [GST-MBNL1N]·[TAMRA-r(CUG)16] complex (abbreviated
as MBNL1⋅r(CUG)16) was titrated with the polymeric ligand
(Figure 4c). Curve fitting the plot of fluorescence anisotropy
against the polymeric ligand concentration afforded IC50 and Ki
values that were in the low nanomolar range. The comparative
performance of the various polymeric ligands is shown graphically
in Figure 4d and Figure S10. Similar to the trend reported by
Kiessling and coworkers for glycopolymers,24 a higher ligand
loading generally led to lower Ki values. PLG50-1/5 and PDG50-1/5
showed similar Ki values, indicating that the chirality of α-helix
plays a minor role in the peptide-RNA binding. Interestingly,
PDLG50-1/5 showed improved IC50 and Ki, indicating that the
recognition units are still very much available for binding. The
increased ability of PDLG50-1/5 to inhibit the MBNL1⋅r(CUG)16
complex likely results from the random-coil conformation of the
polypeptide allowing for a flexible fit between macromolecules.
However, as shown in the confocal studies (vide infra), PDLG50-
1/5 was not as cell permeable as the helical polymers.
As noted above, the binding affinity and inhibition potency of
the polymeric ligands were seen to increase with the acridine-
melamine loading on the polymer. Although further improvements
in inhibition potency may be observed by further increasing ligand
loading, solubility or aggregation of the resulting polymeric
constructs is likely to be an issue. Thus, decreased aqueous
solubility was observed for PLG50-1/2 compared to PLG50-1/10
and PLG50-1/5, although PLG50-1/2 was still soluble enough for all
studies. Increasing the polymer length is another approach to in-
crease the multivalency, but that approach raises concerns about
the synthetic accessibility and ultimately the issue of body
clearance.
Figure 5. HeLa cellular uptake studies with PLG50-1/5, PDG50-1/5
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and PDLG50-1/5 monitored by confocal microscopy of live cells.
Hoechst dye was used as a nuclear stain (blue). The acridine moieties
on the polypeptide showed inherent fluorescence (green), allowing
the location of the polymer-ligand conjugates to be determined. In-
cubation time was 4 h with a ligand concentration of 500 nM. Scale
bar = 20 µM.
cytosolic esterases and, indeed, porcine liver esterase (PLE), which
has a broad substrate scope showed no cleavage of PLG50-1/5 over
a 72 h period (Figure S8 in Supporting Information). With this set
of stable polymers containing different chirality, it was possible to
determine the importance of the helical structure to be established.
Polymers are Potent in Vitro Inhibitors of the
MBNL1⋅r(CUG)16 Interaction Exhibiting Nanomolar
Ki Values. To assess the ability of the polymeric ligands to inhibit
the MBNL protein sequestration by CUGexp
, fluorescence
anisotropy inhibition experiments were performed using GST-
MBNL1N protein22 and TAMRA-labeled r(CUG)16 (see
Supporting Information).23 Figure 4b shows the change in
Figure 6. Reduction of nuclear foci in the DM1 model cells were observed by treating the cells with 500 nM polymeric ligand (PLG50-1/5). Con-
trol polymer (without ligand conjugated) showed no efficacy.
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Confocal Microscopy Shows Polymeric Ligands are transfecting HeLa cells with plasmids containing (CTG)960
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Taken up by HeLa Cells. Confocal microscope images of live
HeLa cells treated with the DM1 polymeric ligands 4 provided
semi-qualitative evidence of the ability of the polymeric ligand to
pass the cell membrane and at least partially enter the nucleus. The
results for PLG50-1/5, PDG50-1/5, and PDLG50-1/5 are shown in
Figure 5. The polymers containing D- or L-monomers exclusively
showed better cellular uptake relative to the DL-polymer consistent
with cell penetration mediated by the helical structure.12b The
helical polypeptides were observed in the endosome, cytoplasm
and some in the nucleus of cells. It was reported that hydrophobic
groups such as alkyl chains on PLG are necessary for better cellular
uptake, and the uptake process is governed by both endocytosis
and direct penetration.12b In this work, the acridine-melamine
ligands likely provide the hydrophobic moieties, although they led
to more uptake by the endocytosis pathway (Figure S11).
The cell uptake results underline the importance of using a
suitable vector for ligands to be transported into the cells to find
their target. Although CPP-like functionality have not yet found
use in the clinic, CPP-conjugated oligonucleotides are being inves-
tigated as potential treatments of various muscular dystrophies.25 In
this study that target is the expanded rCUG transcript located in
the nucleus. The random-coiled DL-polymer showed less uptake
by the HeLa cells. Thus, despite the better inhibition potency ob-
served in the in vitro fluorescence anisotropy studies, these ligands
are less attractive as agents for treating DM1. Moreover, helicity is
not only important for cell-penetrating capability, but also affecting
the ultimate fate of the polymer. Although beyond the scope of this
study the D-polypeptides are expected to resist enzymatic
degradation. It is likely that long-term toxicity and clearance rates
will depend on the chirality. Beyond whether D- or L-monomers
are used, the synthesis outlined in Figure 3c is sufficiently flexible
to allow other functionality to be added and the many different
structural parameters to be varied in a systematic way.
(DT960) and the IR minigene. The splicing of IR pre-mRNA was
chosen for study because it is relatively difficult to be rescued.28 As
seen in Figure 7a, the IR pre-mRNA undergoes splicing to form
two isoforms, A and B, the former without and the latter with exon
11 included.
The DM1 model cells were treated with polymeric ligands at
concentrations where high levels of foci dispersion and cell viability
were observed. The relative amounts of two IR isoforms were
measured for treated and untreated cells. The splicing of IR pre-
mRNA in untreated HeLa cells containing the IR minigene but
lacking the DT960 minigene, produced ca. 47% of isoform B,
whereas the DM1 model cell produced only 27% (Figure 7b).
These differences in the levels of the IR isoforms generally
reproduces that observed in normal and DM1 patient cells.29
Treatment of the DM1 model cells with the polymeric ligands
reversed the splicing defect of IR pre-mRNA to within
experimental error of normal cells (Figure 7b and Figure S12).
Ligand PLG50-1/2 appeared to be most effective giving full rescue
at ≤225 nM but all ligands studied corrected the splicing defect at
concentrations from 0.1 to 1 µM (Figure S12). In comparison to
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other small molecules that target CUGexp 22
exhibited significantly higher activity.
, the polymeric ligands
Polymeric Ligands Exhibit Acceptable Cytotoxicity.
Minimal cell death was observed during the splicing recovery and
foci dispersion experiments described above and at a level that was
indistinguishable from the control using no compound. Indeed, the
parent CPP-mimicking polypeptides used here were reported to
have relatively low toxicity.12b Nonetheless, the cytotoxicity of
polymer 4 at their respective working concentrations was evaluated
using multiple cell lines, including DM1 patient cells (see Figure
S13). The results showed that 4 exhibited no or relatively low
cytoxicity at their working concentrations of 225 nM for PLG50-
Nuclear Foci Formation is Inhibited in Model DM1
Cells by Polymeric Ligands. One of the hallmarks of DM1 is
the formation of nuclear foci, resulting from MBNL sequestration
by CUGexp 26
These foci can be readily visualized by confocal
.
microscopy. In this study, DM1 model cells were constructed by
transfecting HeLa cells with a GFP-DT960 plasmids27 containing
(CTG)960 in a truncated DMPK gene and a sequence encoding
GFP protein to assess the transfection efficiency.
PLG50-1/5 was used in the initial study along with PLG50-0/1, a
control polymer lacking acridine ligands. In the untreated cells, the
co-localization of Cy3-(CAG)10 and an anti-MBNL antibody led to
the visualization of nuclear foci (Figure 6). In contrast, cells treated
with 500 nM PLG50-1/5 for 2 d, showed fewer foci and their size
were noticeably smaller (Figure 6, row 3). No significant change in
the foci number was observed for the cells treated with the control
polymer (PLG50-0/1) under the same conditions (Figure 6, row 2).
These results revealed the importance of the CUGexp-binding
moieties on the polypeptides. In addition, the working
concentration of the polymeric ligand PLG50-1/5 (500 nM) was
two orders of magnitude lower than that of the monomeric small
molecule ligand (50 to 100 µM),19,22 showing
a significant
improvement in efficacy for this rationally designed polymeric
ligand system.
The IR pre-mRNA Mis-splicing is Fully Corrected
by the Polymeric Ligands in DM1 Model Cells. Given
the ability of the polymers to inhibit foci formation and
sequestration of MBNL1, the downstream effect on splicing mis-
regulation was examined in DM1 model cells constructed by co-
Figure 7. Reversal of IR mis-splicing with DM1 polymeric ligand
(a) Schematic illustration of the splicing of IR pre-mRNA. (b) Results
from three concentrations of PLG50-1/5 after 72 h incubation. Error
bars represent standard error of the mean of the three independent
experiments. “*” P< 0.05 (two-tailed Student’s t-test)
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those that simply inhibit MBNL1 sequestration. We recently
1/2, 500 nM for PLG50-1/5, and 1 µM for PLG50-1/10. The
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cytotoxicity of 4 with HEK-293 cells at higher polymer
concentrations was also examined in an effort to understand the
relationship between the polymer structure and its toxicity (Figure
S14). The more hydrophobic polymer, PLG50-1/2, showed higher
toxicity at higher concentrations compared to other polymers. This
is possibly resulted from the higher loading of the hydrophobic
ligands on the polymer backbone, which may lead to more
aggressive cell membrane disruption that compromises membrane
integrity, leading to cell death.12b,30 Nonetheless, the higher inhibi-
tory power of PLG50-1/2 allows for lower dosing, and it showed
excellent activity in the foci and splicing assays along with PLG50-
1/5 and PLG50-1/10 without significantly affecting model cell via-
bility.
Polymeric Ligands Suppress Cellular Levels of
CUGexp RNA Transcript. The DM1 disease pathobiology is
complex and the CUGexp may produce additional toxicity beyond
the sequestration of MBNL. For example, CUGexp undergoes
repeat-associated non-ATG (RAN) translation producing homo-
peptides, some of which are known to be toxic.31 The CUGexp
transcript also disrupts the translation of MEF2 protein, leading to
microRNA dysregulation in DM1 heart tissue.27b,32 For these
reasons, ligands that suppress CUGexp levels may be superior to
reported several small molecules that target DM1 simultaneously
through three separate pathways.33 In particular, these multi-
targeting agents were shown in vitro to (1) inhibit the transcription
of the expanded dCTG DNA, (2) slowly and selectively cleave
CUGexp, and (3) inhibit the formation of the CUGexp-MBNL
complex. One of both of the first two capabilities likely led to the
reduced CUGexp levels observed in DM1 model cells.
Taking advantage of the ease in synthesis, a potential RNA-
cleaving pentapeptide (propargyl-glycine-His-Gly-His-Lys) was
incorporated into the polymeric ligand (Figure 8a). This
pentapeptide is a clickable derivative of a tetra-peptide, HGHK,
whose acridine conjugate was reported to cleave tRNA under
physiological conditions.34 The new polymeric ligand, PLG50-1/5-
2P, was prepared using the same synthetic approach outlined in
Figure 3a. PLG50-1/5-2P had an acridine-melamine to guanidine
ratio of 1:5, and an average of two pentapeptides per polymer
chain. CD spectroscopy was used to show that the attached
pentapeptides did not disrupt the helical structure of the polypep-
tide (Figure S15a). Cell uptake studies using PLG50-1/5-2P re-
vealed an uptake profile similar to that of PLG50-1/5 (Figure S15b).
To examine the potential for these ligands to regulate the
cellular levels of CUGexp, the two polymeric ligands, PLG50-1/5 and
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Figure 8. (a) Structure of alkyne-containing pentapeptide that, along with 2 and N-propargyl guanidine was conjugated to 3 to produce a poten-
tial RNA-cleaving 4. (b) Change in CUGexp levels in DM1 model cells after 3 d treatment of polymeric ligands PLG50-1/5 and PLG50-1/5-2P. The
error bar represent standard error of mean from at least three independent experiments, “*” P < 0.05, “**” P < 0.01, “***” P < 0.001 (two tailed t-test).
(c) In vitro transcription experiments using a linearized plasmid containing (CTG·CAG)74 and control plasmids in presence of DM1 polymers. (d).
Dose-dependent rescue (PLG50-1/2) and the effect of polymer chirality (PLG50-1/5, PDG50-1/5 and PDLG50-1/5) on the rescue of locomotion
defect in DM1 larvae at low concentration in the larvae food. See text, Supporting Information, and reference 9h for additional details.
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PLG50-1/5-2P, were studied using the same DM1 model cells uninterrupted (CTG)60 or an interrupted (CTG)480 sequence was
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described above containing GFP-DT960. The model cells were
incubated with 500 nM of the polymeric ligands for 3 d. The total
RNA was isolated and the r(CUG)960 mRNA level was determined
by measuring the mRNA levels of exon 15 upstream of CUGexp
using PABP mRNA as an internal standard.33 The results shown in
Figure 8b indicate that the toxic mRNA level was decreased by over
75% for both polymers, with PLG50-1/5-2P outperforming PLG50-
1/5. The possibility that the ligands, if isolated with the RNA,
might interfere with cDNA synthesis and amplification of the target
mRNA was ruled out in control experiments. Thus, the presence of
polymeric ligands in a concentration range of 0.5 – 500 nM showed
no effect on the reverse transcription or RT-PCR reactions (see
Figure S16), ruling out the possibility of a false positive response
resulting from this type of interference.
The two possible mechanisms that account for the observed
suppression of the rCUG transcript levels are: (1) inhibition of
(CTG)960 transcription, and (2) degradation of the r(CUG)960
transcript. To better understand the regulation mechanism, in vitro
transcription inhibition experiments using a (CTG)74-containing
DNA template were performed in the presence of PLG50-1/5 and
PLG50-1/5-2P. Thus, linearized T7 promoter-containing (CTG)74
plasmids35 were incubated for 2 h with T7 polymerase in the
presence of polymeric ligands PLG50-1/5 and PLG50-1/5-2P at
concentrations ranging from 1 nM to 500 nM. The results showed
that PLG50-1/5 and PLG50-1/5-2P significantly inhibited r(CUG)74
formation at a concentration as low as 50 nM (>70% inhibition,
Figure 8 and Figure S17), whereas the control polymer PLG50-0/1
showed little inhibition at all testing conditions. A dose-dependent
effect of the ligands on in vitro transcription was observed. In
contrast, there was no clear trend seen for control polymer PLG50-
0/1, indicating the need for the acridine ligand to selectively
recognizing CTG DNA.9c,9h Consistent with this model, PLG50-1/5
and PLG50-1/5-2P showed no effect on the in vitro transcription of
a control sequence lacking the (CTG·CAG)74 sequence (Figure
8c).
In a parallel experiment, PLG50-1/5-2P was incubated with
r(CUG)74 under the same conditions without the DNA template.
No RNA degradation was observed over a 2 h incubation (Figure
S18 in Supporting Information) suggesting that the decreased level
of r(CUG)74 in the transcription experiments originates primarily
in the inhibition of transcription, not RNA degradation. However,
over longer periods both PLG50-1/5 and PLG50-1/5-2P showed
r(CUG)16 cleavage. Thus, 32P-labeled r(CUG)16 showed smaller
RNA fragments after a 24 h incubation with either PLG50-1/5 or
PLG50-1/5-2P (Figure S19). Given that the cell studies used a 3 d
incubation, it is possible that RNA cleavage contributes to the
suppression in CUG RNA levels. However, it is beyond the scope
of this study to determine the relative importance of transcription
inhibition relative to the RNA cleavage.
Polymer PLG50-1/2 Fully Corrects the Mobility
Defect of DM1 Transgenic Drosophila Larvae. The ex-
pression of expanded CUG repeats in Drosophila was reported to
lead to nuclear accumulation of CUG RNA, splicing misregulation,
and lowered MBNL function, with reduced lifespan and muscle
degeneration.36 A larval crawling assay was initially developed by
Pandey and co-workers for ALS modeling, as motor neuron
expression of mutant FUS/TLS was observed to cause a larval-
crawling defect.37 Based on the above studies, we recently
developed a DM1 Drosophila larval crawling assay and used it to
examine the in vivo activity of two bisamidinium compounds.33
Thus, the locomotion of transgenic larvae expressing either an
measured by placing them on an agarose gel dish over grid paper
and recording the number of lines crossed per minute. The
transgenic larvae expressing (CTG)60 crossed ~12.5 lines/min,
which is considered to be the nonpathogenic control, whereas
those expressing i(CTG)480 exhibited significantly impaired
locomotion, crossing ~9 lines/min (pathogenic state). The same
assay was used to test the in vivo activity of the polymeric ligands
developed herein, the data summarized in Figures 8d and S20.
The six polymers studied showed minimal effect on the
mobility of the control (non-pathogenic) transgenic larvae
containing (CTG)60 (Figure 8d and Figure S20). Of the six ligands
investigated, three showed significant improvement in the crawling
defect of the i(CTG)480 larvae, and of these, PLG50-1/2 showed the
largest effect. As seen in Figure 8d, PLG50-1/2 exhibited a dose-
dependent response with larval mobility fully recovered to non-
pathogenic levels at 10 μM. Although this polymer exhibited the
highest cytotoxicity in HEK-293 cells among all the synthesized
polymers, it also showed the highest activity in inhibiting the
MBNL1Ŋr(CUG)16 interaction in vitro. In addition, the three
polymers with similar structure but different chirality, PLG50-1/5,
PDG50-1/5 and PDLG50-1/5, showed a clear trend in locomotive
ability rescue capability: PDG50-1/5 > PDLG50-1/5 > PLG50-1/5,
which correlates with their anticipated resistance to enzymatic
degradation across the longer timeframe of the in vivo experiments:
PDG50-1/5 > PDLG50-1/5 > PLG50-1/5. In fact, no activity was
observed for L-peptides of lower ligand loading at the tested
concentration (10 µM). There are many factors that will determine
the in vivo activity, including the amount of agent consumed in
feeding, as well as its absorption, distribution, and metabolism.
Additional studies would be needed to determine if the D-chirality
is correlated with increased in vivo stability and, in turn, this
accounts for the higher activity.
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Conclusion
Amplifying the affinity and selectivity of ligands that target
trinucleotide repeat diseases can be accomplished logically by
linking two or more of these ligands together with the appropriate
scaffold. However, these multivalent ligands must be taken up by
the cell and the larger the multivalent construct, often the more
difficult it is to achieve suitable uptake. This study reports a general
strategy to avoid this limitation. In particular, we described the first
approach toward DM1 treatment that uses a polymeric delivery
agent, a cell-penetrating peptide mimic, as the scaffold to bring
multiple CTG- and CUG-binding agents to the target DNA and
RNA within the cell. Although the cellular and in vivo fate of these
macromolecular constructs is unknown, and one cannot rule out
the possibility of enzymatic ester or peptide cleavage, the >100 fold
increase in potency, known peptidase resistance of these poly-
mers,21 and demonstrated resistance to esterase cleavage is more
consistent with a multivalent mode of action.
Despite the complexity of the construct, its synthesis was
straightforward using a bottom-up, living polymerization strategy.
Polyazide 3 allowed different loadings of various ligands and other
groups to be easily linked to the scaffold providing considerable
tunability. By preparing a series of DM1 polymeric ligands with
different molecular weights (i.e., chain lengths), ligand loadings
and helicity, the effects of these parameters could be evaluated in
both in vitro and in vivo assays. Further improvements in the
inhibition power and therapeutic efficacy could come from even
more controlled structural variants, such as those biasing the CUG
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ligands to one side of the helical rod, or with precise control over
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Synthetic polypeptide-based ligands (e.g., PLG50-1/5) were
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cantly inhibited the formation of nuclear foci at concentrations of
500 nM, and fully or nearly fully reversed the mis-splicing of the IR
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In a broader sense, the use of a cell penetrating peptide as a
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target that could benefit from a similar polyvalent strategy.
ASSOCIATED CONTENT
Supporting Information. Detailed instrument setup, synthetic
procedures, additional polymer characterization data, additional pro-
tocols for molecular and cell studies, and additional supportive experi-
ments are provided in the support information. This material is availa-
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AUTHOR INFORMATION
Corresponding Author
* sczimmer@illinois.edu
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
Funding from the Muscular Dystrophy Association (MDA grant
295229 to SCZ) and the National Science Foundation (CHE 1153122
to JC) is gratefully acknowledged. The authors thank Auinash Kalsotra
(University of Illinois at Urbana-Champaign, Urbana, IL) for the GFP-
DT0 and GFP-DT960 plasmids, Thomas Cooper (Baylor College of
Medicine, Houston, TX) for the DT960 minigene plasmid, Maurice
Swanson (University of Florida, Gainesville, FL) for the (CTG)74
plasmid, and Nicholas Webster (University of California, San Diego,
CA) for the IR minigene plasmid.
ABBREVIATIONS
DM1, myotonic dystrophy type 1; MBNL1, Muscleblind-like
protein 1; DMPK, dystrophia myotonica-protein kinase; IR, insulin
receptor; SV40, simian virus 40; GFP, green fluorescent protein; CPP,
cell-penetrating peptide; NCA, N-carboxyanhydride; PLG, poly-L-
glutamate.
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