RTD-1 mimic containing γPNA scaffold exhibits broad-spectrum antibacterial activities

Article abstract of DOI:10.1021/ja211867j

Macrocyclic peptides with multiple disulfide cross-linkages, such as those produced by plants and those found in nonhuman primates, as components of the innate immunity, hold great promise for molecular therapy because of their broad biological activities

Full text of DOI:10.1021/ja211867j

Communication
pubs.acs.org/JACS
RTD-1Mimic Containing γPNA Scaffold Exhibits Broad-Spectrum
Antibacterial Activities
Srinivas Rapireddy,† Linda Nhon,† Robert E. Meehan,†,∥ Jonathan Franks,‡ Donna Beer Stolz,‡ Dat Tran,§
Michael E. Selsted,§ and Danith H. Ly*,†
†Department of Chemistry and Center for Nucleic Acids Science and Technology (CNAST), Carnegie Mellon University, 4400 Fifth
Avenue, Pittsburgh, Pennsylvania 15213, United States
‡Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S362 BST, 3500 Terrace Street,
Pittsburgh, Pennsylvania 15261, United States
§Department of Pathology and Laboratory Medicine, Keck School of Medicine, University of Southern California, Los Angeles,
California 90089, United States
S
* Supporting Information
peptide with three disulfide bonds recently isolated from the
leukocytes of rhesus macaques9 and baboons10 (Chart 1A).
ABSTRACT: Macrocyclic peptides with multiple disul-
fide cross-linkages, such as those produced by plants and
those found in nonhuman primates, as components of the
innate immunity, hold great promise for molecular therapy
because of their broad biological activities and high
chemical, thermal, and enzymatic stability. However, for
some, because of their intricate spatial arrangement and
elaborate interstrand cross-linkages, they are difficult to
prepare de novo in large quantities and high purity, due to
the nonselective nature of disulfide-bond formation. We
show that the disulfide bridges of RTD-1, a member of the
θ-defensin subfamily, could be replaced with noncovalent
Watson−Crick hydrogen bonds without significantly
affecting its biological activities. The work provides a
general strategy for engineering conformationally rigid,
cyclic peptides without the need for disulfide-bond
reinforcement.
Though humans do not naturally produce this peptide, due to a
premature stop-codon occurrence which took place ∼9 million
years ago, a chemically synthesized version of this peptide
called “retrocyclin”11 has been made and, like the rhesus RTD-1
counterpart, has been shown to exhibit a broad spectrum of
antibacterial,9,12 antifungal,9 and antiviral activities.11,13,14 The
discovery of these biologically active, cyclic peptides has
spawned considerable interest in their development as
therapeutics for treating fungal, bacterial, and viral infections
as well as a number of chronic conditions including neuropathic
pain.15 While a number of these cyclic peptides have been
chemically synthesized, they are generally difficult to prepare in
large quantities and high purity needed for most clinical
applications, especially those containing multiple and elaborate
disulfide cross-linkages, due to the nonselective nature of
disulfide-bond formation. As a proof-of-concept, we show that
the disulfide bridges of RTD-1 could be replaced with
noncovalent, Watson−Crick hydrogen bonds without signifi-
cantly affecting its biological activities. The newly developed
RTD-1 mimic could be easily prepared in large scale and high
purity and is effective in killing both Gram-positive and
-negative bacterial strains.
eptides and peptidomimetics could potentially be a rich
Psource of pharmacophores for drug discovery because of
the ease and flexibility of their synthesis and diverse sequence
space that can be generated from a limited number of chemical
building blocks; however, the main drawback is conformational
flexibility. Because of their diminutive size and high degree of
conformational freedom, they neither bind with high affinity
nor selectivity, which has been the Achilles’ heel of peptide
therapeutics. Plants and certain organisms, including nonhu-
man primates, however, have devised a creative solution to this
problem by stitching linear peptides into cyclic1 and knotted2−5
structures. Such architectural transformations not only confer
conformational rigidity but also chemical, thermal, and
enzymatic stability to otherwise highly flexible and enzymati-
cally labile molecular entities.6,7 These post-translationally
modified peptides are commonly referred to as “cyclotides”,7
for those produced by plants, and as “defensins”,8 for those
found in primates as part of an innate immunity. The latter
class is further divided into three subfamilies, α, β, and θ, based
on the spacing and pairing of the six disulfide-bonded cysteines.
One such example of the last subfamily is RTD-1, a cyclic
The structure of RTD-1 has been determined by NMR,16
comprised of an extended β-hairpin reinforced by three
disulfide bonds. Though its mechanism of action is not yet
fully understood, studies of related members of this peptide
family suggested that they confer their biological effects by
interacting with the membrane of bacteria or pathogens.18,19
The interaction causes perturbation in the membrane dynamics
or potential, resulting in cell death or rendering the pathogens
incapable of infecting the host cells. Based on this general
mechanism of action, we surmised that it might be feasible to
develop a synthetically simpler version of RTD-1, by replacing
the disulfide bridges with noncovalent Watson−Crick hydrogen
bonds, without significantly affecting its biological activities.
Received: December 20, 2011
Published: February 14, 2012
© 2012 American Chemical Society
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a
Chart 1RTD-1
Figure 1. CD spectra of RTD-1, RTD-1M, and RTD-1M-P at 5 μM
each in 10 mM sodium phosphate buffer (pH 7.4), recorded at 37 °C.
Inset: UV-melting curves of the corresponding RTD-1M and RTD-
1M-P in the same buffer.
coupling patterns in the 200−230 nm regions. This spectral
regime has been previously assigned to the n−π* transition of
the amide in the peptide backbone.24 The CD spectrum in the
230−300 nm range is an indication of RTD-1M adopting a
right-handed helix.20 Since the duplex is relatively short, three
base-pairs in length, it is expected to adopt an extended, β-
sheet-like conformation but with a slight twist, as shown in
Chart 1B. The UV-melting data revealed a significant increase
in the thermal stability of the cyclic as compared to the hairpin
precursor, with a Tm of ∼80 °C for RTD-1M as compared to 51
°C for RTD-1M-P, corresponding to a net ΔTm of +29 °C.
These results together show that despite its small size, RTD-
1M maintains a stable, β-sheet-like conformation at physio-
logical temperatures.
a
(A) Chemical composition of RTD-1, cyclic RTD-1M, and acyclic
RTD-1M-P. Note that in RTD-1M and RTD-1M-P, the disulfide
bonds are replaced with C-G base pairs. (B) NMR structure of RTD-1
as determined by Craik and coworkers16 and energy minimized
structure of RTD-1M using γPNA-γPNA duplex as a starting point.17.
Next, we assessed the antimicrobial activities of RTD-1,
RTD-1M, and RTD-1M-P against Escherichia coli. The result
showed that while RTD-1M and RTD-1M-P are progressively
less potent, as compared to the native RTD-1; it is only by a
small margin (Figure 2A). Like RTD-1, RTD-1M exhibits
To test this hypothesis, we synthesized a cyclic RTD-1 mimic
(RTD-1M) containing a mixture of natural L-amino acids and
γ-peptide nucleic acid (γPNA)20 building blockschiral
derivatives of PNA with an amino acid side-chain installed at
the γ-backbone position [Chart 1A and Chart S1, Supporting
Information (SI)].21 To preserve the chemical functionalities of
the native RTD-1, we grafted the side chains of a selected set of
amino acids onto the γ-backbone positions of PNA and
replaced the cysteine side chain with cytosine (C) or guanine
(G) nucleobase. The rest of the amino acid residues in the loop
were kept intact except the glycine residues depicted in circles,
which were inserted to provide an optimal loop-size for
intramolecular hybridization (Chart 2S and Figures S1 and S2,
SI). The acyclic precursor, RTD-1M-P, was prepared on solid-
support according to a standard procedure20 and cyclized
postcleavage via intramolecular, template-directed ligation to
give the desired RTD-1M (Figures S3−S6, SI).
Since there have been few reports of nucleic acid systems
with three or fewer base pairs22,23 and all of which contained
entirely different backbone skeletons, it was not clear whether
such a relatively short cyclic RTD-1M can maintain a stable, β-
sheet-like conformation at physiological temperatures. To
address this question, we performed circular dichroism (CD)
and UV−vis spectroscopic analyses of RTD-1M and RTD-1M-
P. Our result showed that both the cyclic and acyclic forms
adopt a similar backbone conformation as that of the native
RTD-1 (Figure 1), evidenced from the overlapping exciton
Figure 2. Microbicidal activities of (A) RTD-1M, RTD-1M-P, and
RTD-1 against E. coli and (B) RTD-1M against a panel of Gram-
positive and -negative bacterial strains. EC: Escherichia coli, LM:
Listeria monocytogenes, SA: Staphylococcus aureus, and ST: Salmonella
typhimurium. Each test organism was incubated with different
concentrations of antimicrobial agents in 10 mM PIPES buffer (pH
7.4) containing 5 mM glucose and 0.03% TSB for 4 h at 37 °C in
accordance with our published report.9 The limit of detection was 1
colony per plate, corresponding to 1 × 102 colony forming units
(CFU) per mL.
broad spectrum antimicrobial activities against both Gram-
positive (Staphylococcus aureus and Listeria monocytogenes) and
Gram-negative (E. coli ML 35 and Salmonella typhimurium)
bacteria strains (Figure 2B).9 The viability of E. coli, S. aureus,
and L. monocytogenes was reduced by more than 99% after 4 h
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incubation with 2 μM RTD-1M, whereas it is slightly less for S.
typhimurium.
the former, the membrane is dramatically distorted and broken-
up, with cytoplasmic material leaking out; whereas in the latter,
the outer membrane appeared to be swollen (or enlarged),
coincided with aggregation and shedding of the extracellular
membrane materials.
Consistent with the proposed mechanism of action that θ-
defensins confer their bactericidal activity by interacting with
and perturbing the membranes of the pathogens, treatment of
bacteria with RTD-1M followed by brief incubation with
SYTO-9 and propidium iodide revealed an intense red stain
(Figure 3). SYTO-9 is a cell-permeable, green fluorescent probe
Consistent with the previous observations with cyclotides7
and defensins,25 we noted that cyclic RTD-1M is significantly
more stable in human serum than the linearized RTD-1, with a
half-life of ∼24 h as compared to ∼2 min for the acyclic RTD-1
with Cys→Ser substitutions (Figures S2 and S8, SI). Hemolysis
assay further revealed that RTD-1M is relatively nonlytic, as
compared to most antimicrobial peptides found in nature.26 At
250 μM, the highest concentration examined, less than 3.4%
lytic activity was observed against human red blood cells (Table
S1, SI). This result shows that like RTD-1, RTD-1M is
cytoselective.
In summary, we have shown that a cyclic RTD-1 mimic
containing natural L-amino acids and γPNA building blocks
exhibits broad spectrum antimicrobial activities. Like the other
members of the θ-defensin family, RTD-1M exerts its biological
effect by interacting with and disrupting the cell membrane of
bacteria. RTD-1M can be readily prepared on solid-support and
cyclized postcleavage via intramolecular, template-directed
ligation. Because of its relatively small size, such a cyclic
molecule could be prepared in large quantities via modular,
solution-phase methodology. Though standard, CDI-mediated
coupling reaction was employed in the ring closure step in this
study as a proof-of-concept, other coupling reactions including
native chemical ligation,27 Huisgen [3 + 2] cycloaddition,28 and
aziridine-mediated (4-component) condensation29 could also
be employed which may be operationally simpler and more
efficient because of the high degree of orthogonality of these
reactions. Likewise, the potency of RTD-1M could be further
improved by modifications of the amino acid side chains, ring
size, or incorporation of chemical moieties that are known to
facilitate cell membrane anchoring, such as the alky tail of
daptomycin,30,31 or disrupting reagents, such as those inherent
in magainin 2 and melittin.26 While a large variety of classes of
antibiotics have been developed and shown to be effective in
combating bacteria,32 almost invariably they succumb to
resistance, rendering them ineffective over a prolonged
treatment due to the rapid rate of genetic mutations of these
pathogens. θ-Defensins and their synthetic analogues, such as
RTD-1M, that confer their biological activities by interacting
with and perturbing the cell membrane of bacteria or pathogens
are less likely to develop resistance, as compared to those that
target the genetic materials, because the cell membrane is under
less selective pressure to evolve. Overall, the work provides a
rational approach to controlling the spatial arrangement of
peptides and construction of conformationally rigid, cyclic
peptides without the need for covalent-bond reinforcement.
Figure 3. Fluorescent images of bacterial strains with and without
RTD-1M treatment and after brief (15 min) staining with SYTO-9 and
propidium iodide. The treatment was done with 10 μM RTD-1M for 4
h in 10 mM PIPES buffer (pH 7.4). The untreated samples were
assayed the same way but without addition of RTD-1M.
that stains both live and dead bacteria, while propidium iodide
is a cell-impermeant, red fluorescent probe that only stains cells
with damaged membranes. The fact that all four bacterial
strains treated with RTD-1M stained red, while the controls did
not in the TRITC channel, indicates that RTD-1M is capable of
disrupting the extracellular membrane of both Gram-positive
and -negative bacterial strains. This finding was further
corroborated by transmission electron microscopy (TEM),
which revealed unusual extracellular morphologies for bacteria
treated with RTD-1M, as compared to the controls. The effect
is more pronounced in E. coli than in S. aureus (Figure 4). In
ASSOCIATED CONTENT
■
S
* Supporting Information
Monomer and oligomer synthesis protocols, HPLC, and
MALDI-TOF spectra of RTD-1M-P and RTD-1M, UV-melting
curves of various RTD-1M analogues with different loop sizes,
results of enzymatic digestion and hemolysis assays, and other
experimental procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 4. TEM images of E. coli and S. aureus with and without RTD-
1M (20 μM for 2 h) treatments at 150 000× magnification.
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(26) Zasloff, M. Nature 2002, 415, 389−395.
AUTHOR INFORMATION
Corresponding Author
dly@andrew.cmu.edu.
■
(27) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776−779.
(28) Kolb, H. C.; Fin, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
Engl. 2001, 40, 2004−2021.
Present Address
∥Yale University, Department of Chemistry, 266 Whitney
Avenue, New Haven, Connecticut 06520.
(29) Hili, R.; Rai, V.; Yudin, A. K. J. Am. Chem. Soc. 2010, 132,
2889−2891.
(30) Raja, A.; LaBonte, J.; Lebbos, J.; Kirkpatrick, P. Nat. Rev. Drug
Discovery 2003, 2, 943−944.
Notes
The authors declare no competing financial interest.
(31) Makovitzki, A.; Avrahami, D.; Shai, Y. Proc. Nat. Acad. Sci. U.S.A.
2006, 103, 15997.
(32) Fischbach, M. A.; Walsh, C. T. Science 2009, 325, 1089−1093.
ACKNOWLEDGMENTS
■
Financial support for this work was provided by the National
Institutes of Health (M.E.S.: R37AI22931 and R01DE015517;
D.H.L.: GM076251), the National Science Foundation
(D.H.L.: CHE-1012467) and the DSF Charitable Foundation
(S.R.). NMR instrumentation at CMU was partially supported
by NSF (CHE-0130903 and CHE-1039870).
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