Full Sequence Amino Acid Scanning of θ-Defensin RTD-1 Yields a Potent Anthrax Lethal Factor Protease Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.6b01689

θ-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC₅₀ = 390 ± 20 nM, K_i = 365 ± 20 nM) and a weak inhibitor of other mammalian metalloproteases such as TNFα converting enzyme (TACE) (K_i

Full text of DOI:10.1021/acs.jmedchem.6b01689

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Article
Full sequence amino acid scanning of #-defensin RTD-1
yields a potent anthrax lethal factor protease inhibitor
Yilong Li, Andrew Gould, Teshome Aboye, Tao Bi, Leonard
M. Breindel, Alexander Shekhtman, and Julio A. A Camarero
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01689 • Publication Date (Web): 02 Feb 2017
Downloaded from http://pubs.acs.org on February 7, 2017
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Full sequence amino acid scanning of θ-defensin RTD-1 yields a potent anthrax lethal
factor protease inhibitor
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Yilong Li , Andrew Gould , Teshome Aboye , Tao Bi , Leonard Breindel , Alexander Shekhtman
1,2*
and Julio A. Camarero
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Department of Pharmacology and Pharmaceutical Sciences and Department of Chemistry,
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University of Southern California, Los Angeles, CA 90089-9121, USA; Department of Chemistry,
State University of New York, Albany, NY 12222, USA
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ABSTRACT. θ-Defensin RTD-1 is a non-competitive inhibitor of anthrax lethal factor (LF) protease
(
IC = 390 ± 20 nM, K = 365 ± 20 nM) and a weak inhibitor of other mammalian
50 i
metalloproteases such as TNFα converting enzyme (TACE) (K = 4.45 ± 0.48 µM). Using full
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sequence amino acid scanning in combination with a highly efficient ‘one-pot’ cyclization-folding
approach we obtained an RTD-1 based peptide that was around 10-times more active than wild
type RTD-1 in inhibiting LF protease (IC = 43 ± 3 nM, K = 18 ± 1 nM). The most active peptide
50
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was completely symmetrical, rich in Arg and Trp residues, and able to adopt a native RTD-1-like
structure. These results show the power of optimized chemical peptide synthesis approaches for
the efficient production of libraries of disulfide-rich backbone-cyclized peptides to quickly
perform structure-activity relationship studies for optimizing protease inhibitors.
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INTRODUCTION
Defensins are cysteine-rich antimicrobial peptides that are important in the innate immune
1-3
defense of mammals. Defensins are classically known for their antimicrobial activities, but
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they have been also shown to be involved in other defense mechanisms including immune
3, 4
modulation, neutralization of endotoxins, and anti-cancer properties. Mammalian defensins
are positively charged peptides containing mostly β-sheet structures and six conserved cysteines
forming three intramolecular disulfides. They can be classified into α-, β- and θ-defensins
depending on their overall structure.
The overall fold of α and β-defensins is very similar except for the disulfide connectivities, and
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the presence of an N-terminal α-helix segment in β-defensins that is missing in α-defensins. In
contrast, θ-defensins are backbone cyclized peptides formed by the head-to-tail covalent
1
assembly of two polypeptides derived from α-defensin related precursors. Although the
mechanism for RTD-1 processing still remains unclear, it is regarded to involve two
1
dodecapeptides, each containing a three residue C-terminal pro-sequence. θ-Defensins are to
1
date the only known cyclic polypeptides expressed in animals.
Rhesus θ-defensin-1 (RTD-1) was the first θ-defensin to be discovered from an extract of Rhesus
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macaques leukocytes. Defensin RTD-1, unlike α- and β-defensins, possesses a β-hairpin-like
structure with two anti-parallel β-strands stabilized by three disulfides in a ladder configuration
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(
Fig. 1). Other less abundant RTD variants have been named RTD-2 to RTD-6, have also been
7-9
found in Rhesus macaques. Circular θ-defensins have also been found in other primate
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species.
Intriguingly, although humans possess genes encoding θ-defensins, the ability to
produce these cyclic defensins have been lost due to a stop codon mutation within the signal
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sequence, which prevents subsequent translation.
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θ-Defensins have antimicrobial activity against both Gram-positive and Gram-negative bacteria,
The antibacterial activity, however, has been shown to strongly depend on the experimental
conditions, mostly buffer composition and presence of serum proteins, used in the antibacterial
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assays. θ-Defensins also have anti-fungal and anti-HIV
activities. Chemically-synthesized
human θ-defensins (also called retrocyclins), which are derived from the human pseudogene
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sequences, have been shown to protect human cells from infection by HIV-1 and have been
16-18
evaluated as a topical anti-HIV agent.
θ-Defensins have also been shown to inactivate
germinating anthrax spores and act as a non-competitive inhibitor of anthrax lethal factor (LF)
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protease and other metalloproteases like TNFα converting enzyme (TACE). Due to its network
of disulfide bonds and backbone-cyclized topology θ-defensins present high resistance to
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proteolytic degradation in serum and plasma. θ-Defensins have been shown to possess anti-
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inflammatory properties in animal models. Altogether, these data suggest that the θ-defensin
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is an ideal molecular framework for the development of novel peptide-based therapeutics.
We have recently developed a method for the rapid parallel production of disulfide-rich
backbone cyclized polypeptides using a ‘tea-bag’ approach in combination with high efficient
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cyclization-folding protocols.
This approach has been recently used for the production of
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cyclotide-based amino acid scanning libraries. We decided to use this approach for the rapid
production of a full sequence amino acid scanning library on defensin RTD-1 in order to improve
+
2
its inhibitory activity against the protease LF that was used as a model. LF is a Zn -dependent
metalloprotease that cleaves several mitogen-activated protein kinases and is responsible for
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the lethality of anthrax lethal toxin and therefore is a validated molecular target for the
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development of more efficient therapeutics to treat anthrax
.
In general, all the positions in RTD-1 were amenable to mutation with exception of the Arg
residues located in the β-sheet region of the peptide. Replacement of these residues by non-
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charged residues yielded cyclic peptides that were unable to fold correctly. The anti-LF activity
of all the folded cyclic peptides was evaluated in vitro using a fluorogenic reporter. We found
that the native Arg residues in RTD-1 are key for anti-LF activity. Mutation of these residues to
negatively charged amino acids significantly decreased the anti-LF activity of the resulting
peptides. Mutation of the non-hydrophobic native residues located in the loops of RTD-1 to Arg
also resulted in peptides with improved biological activity. In addition, we observed that the
replacement of hydrophobic residues by Trp also increased the anti-LF activity. Combining this
information we were able to produce a natively folded RTD-1-based backbone cyclized
polypeptide that was about 10 times more active than native RTD-1. These results highlight the
power of optimized chemical cyclization/folding techniques for the rapid production of
positional scanning libraries, which can be quickly used to perform structure activity relationship
studies for improving the biological activity of bioactive peptides.
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RESULTS
Peptide library design
Every residue in the sequence of RTD-1, except for the cysteines that are required for disulfide
formation and oxidative folding, was systematically replaced by a set of amino acids containing
small (Ala), hydrophobic (Leu, Ile or Val), aromatic (Tyr or Trp), positively (Arg, Lys or His) or
negatively (Asp or Glu) charged residues to provide a total of 12 sub-libraries with the objective
to evaluate the importance of every native residue for the anti-LF activity of RTD-1 (Fig. 1). Each
amino acid position was scanned with 9 residues (except position 2 that was scanned with only
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residues) from the different groups described above. Therefore, positions in RTD-1 were
replaced by a set of amino acids containing Ala, Asp, Glu, Leu, Val, Lys, Arg, Tyr or Trp. When the
native the residue on the RTD-1 sequence was Arg, Leu, or Val, the corresponding amino acid in
the set indicated above was replaced by His (Arg) or Ile (Val and Leu). The residue Leu was not
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included in the Phe2 sub-library therefore this sub-library contained only 8 members.
Accordingly, a total of 107 (=11 x 9 + 1 x 8) different f RTD-1 variants containing a single
mutation were chemically prepared (Table S1). Wild-type RTD-1 was also included in the study
as a positive control. In addition, the disulfide-free cyclic form of the θ-defensin, RTD-1 C3S, C5S,
C12S, C14S, C16S (RTD-1-C*S) (Table S1), was also used to evaluate the importance of the
disulfide network for the anti-LF activity of defensin RTD-1.
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Peptide synthesis
All the cyclic single mutant variants of defensin RTD-1 (Table S1) were chemically synthesized
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employing Fmoc-based solid-phase peptide synthesis on a sulfonamide resin
and a ‘tea-bag’
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approach (Fig. 2). We used the peptide bond between residues Leu6 and Cys7 as the
cyclization site for most of the positional scanning libraries except for the R4 and L6 sub-
libraries, where the peptide bond between residues Phe2 and Cys3 was used instead. Both
cyclization sites have shown to provide very good cyclization yields in the synthesis of RTD-1.
The synthesis of the common C-terminal sequence for all the cyclic RTD-1 sub-libraries was
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performed at 0.1 mmol scale on an automated peptide synthesizer ABI433A. When the position
to be scanned was reached for a particular sub-library, the resin was divided in 9 equimolar
aliquots containing around 11 µmol each (Fig. 2). Each resin was manually coupled in parallel
with the corresponding activated Fmoc-amino acid derivative. Once the couplings were
complete, each resin was placed in an individual sealed poly-propylene ‘tea-bag’ (measuring
around 30 x 20 mm), properly tagged for identification and the synthesis was continued on the
synthesizer using the ABI433A 0.25 mmol scale reaction vessel, which has a capacity of 42 mL
(
Fig. 2). This reaction vessel was able to accommodate up to 9 ‘tea-bags’ with the dimensions
described above. The number of ‘tea-bags’, however, could likely be easily increased by using
smaller ‘tea-bags’ to increase the number of amino acids used in particular position of the
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sequence. Once the synthesis of all the RTD-1 linear precursor analogs was complete, activation
of the corresponding peptide sulfonamide-resins was performed in parallel with
iodoacetonitrile, followed by cleavage with ethyl mercaptoacetate and acidolytic deprotection
provided the fully deprotected linear peptide α-thioester precursors (Fig. 2). The peptide crudes
were characterized by HPLC and electrospray mass spectrometry (ES-MS) and in all the cases the
major product was the corresponding linear precursor thioester (Fig. 3).
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Peptide cyclization/folding and purification
The corresponding peptide thioester precursors were directly cyclized and folded in parallel in a
‘
one-pot’ reaction using sodium phosphate buffer at pH 7.2 in the presence of 1 mM glutathione
(
GSH). Under these conditions defensin RTD-1 has been shown to cyclize and adopt a native θ-
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defensin fold as determined by NMR. The cyclization/folding reactions were followed by HPLC
and shown to be complete in 24–48 h. As previously reported the cyclization of RTD-1 under
these conditions provided a very clean reaction crude where the natively folded defensin was
the major product (≈90% by HPLC) (Fig. 3). The control peptide 1 (RTD-1 C*S), which has all
native Cys residues mutated to Ser except Cys7, also showed an efficient backbone cyclization
(Fig. S8). This result suggests that the presence of disulfides is not required for the effective
chemical cyclization of the RTD-1 linear precursors when performed in vitro using an
intramolecular version of native chemical ligation and therefore that the backbone cyclization in
RTD-1 is likely not driven by the formation of the native disulfide network. It is important to
note, however, that as the mechanism for the biosynthesis of RTD-1 remains unclear, the in vivo
processing of θ-defensins may still rely on prior formation of the cysteine ladder.
Most of the RTD-1 single mutants were able to cyclize and fold with yields similar to that of wild-
type RTD-1 (≈80-90%), and only the non-charged mutants in the R4 and R13 sub-libraries failed
to fold correctly under the conditions described above providing in all cases a complex
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cyclization/folding crude (Fig. S1). Complete chemical reduction of these crudes showed a major
product that was identified by ES-MS as the corresponding fully reduced backbone cyclized
polypeptide (Fig. S2). These results indicate that these mutations, while allowing cyclization,
were unable to produce a major folded product. Intriguingly, replacement of these Arg residues
by either positively (Lys or His) or negatively (Asp or Glu) charged residues provided very clean
cyclization/folding crudes in all the cases (Fig. S1).
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Given the relatively high purity of majority of the cyclization/folding crudes, most of the RTD-1
single mutants were desalted using a C18 cartridge solid phase extraction (SPE) to remove the
buffer components. Only mutants R8L and V11E were purified by C18 reverse-phase HPLC (Fig.
S3).
Biological activity of single RTD-1 mutants against lethal factor protease
To evaluate the effect of the different mutations on the biological activity of the corresponding
θ-defensin analogs, we tested the ability of all the single mutant RTD-1-based peptides to inhibit
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the metalloprotease lethal factor using a FRET-based fluorogenic substrate. This FRET-based
reporter was designed to contain a LF recognition sequence flanked by the FRET pair formed by
CyPet and YPet fluorescent proteins (Figs. 4 and S4). The cyclic defensin RTD-1 was able to
inhibit metalloprotease LF in a dose dependent manner with an IC value of 390 ± 20 nM (K =
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65 ± 20 nM) and Hill-slope of -1.1 ± 0.1 (Fig. 4). On the other hand, the disulfide free form of
RTD-1 (peptide 1) did not show any activity up to 10 µM, therefore indicating that the folded
structure of RTD-1 is required for LF inhibitory activity (Fig. 4A). All the RTD-1 mutants tested
were also able to inhibit LF in a dose dependent manner with IC values ranging from 6.4 ± 0.7
50
µM (RTD-1 R4D) to 198 ± 26 nM (RTD-1 V11W, peptide 7) and an average Hill slope of -1.0 ± 0.2
(Figs. 4 and S4). These data suggest that these RTD-1 analogs were correctly folded and that the
molecular stoichiometry of the inhibitory reaction is 1:1.
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The relative activities of all the single mutants that were able to provide a single folded product
are summarized in Fig. 4B. θ-Defensin RTD-1 is highly rich in positively charged (≈30% of
residues are Arg) and hydrophobic/aromatic (≈ 22% of residues are Phe, Leu, Val or Ile) residues.
In general, the introduction of negatively charged residues (Asp or Glu) reduced the inhibitory
activity of resulting RTD-1 analogs. This effect was more apparent when the mutations were
affecting native Arg residues on the sequence of RTD-1. For example, mutations R4E, R8E, R9E
and R13E mutants showed only from 8% to 29% of the original activity of RTD-1. Similar
reduction in activity was observed when Arg residues were mutated to Asp. Introduction of
negatively charged residues in other positions also had a negative effect on the biological
activity although the results were not that pronounced with the biological activity ranging from
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≈
14% (F2 position) to ≈57% (average for G1 and G10 positions) of that of RTD-1 (Fig. 4B).
Replacing Arg residues by positively charged residues (Lys or His) also produced less active RTD-
1 analogs, with activities ranging from ≈41% (average for positions R4 and R13) to ≈75%
(
average for R9 and R18 positions), hence highlighting the importance of the Arg residues for
anti-LF activity. Introduction of positive charged (Lys or Arg) residues in non-hydrophobic
positions had in general a positive effect on the activity (Fig. 4B). Introduction of Arg in positions
G1, G10 and T17 gave RTD-1 analogs with activities ranging from 114% (T17 position) to ≈138%
(
average for G1 and G10 positions) of that of wild-type RTD-1. The effect was slightly less
marked when these positions were mutated to Lys.
In general, the mutation of the native hydrophobic/aromatic residues (F2, L6 and I15 positions)
present in the sequence of RTD-1 by positive or negatively charged residues had a detrimental
effect on the activity of the resulting mutants (Fig. 4B). Intriguingly, replacement of the V11
position by either Lys or Arg residues had practically no effect on the biological activity of the
resulting mutants. The introduction of Trp in the L6, V11 and I15 positions, however, yielded
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mutants with significant better activity than that of wild-type RTD-1. For example, RTD-1 V11W
(
peptide 7) and RTD-1 I15W had both around 183% of RTD-1 anti-LF activity, while mutant RTD-
1 L6W (peptide 2) had around 176% of the RTD-1 activity. Replacement of Phe at position 2 of
RTD-1 by any amino acid besides Trp was detrimental for the biological activity, while the RTD-1
F2W mutant had basically the same activity than RTD-1 (Fig. 4B).
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Since mutants R8L and RTD-1 V11E were purified by HPLC, we decided to compare the anti-LF
activity of the corresponding desalted cyclization/folding crudes with the purified peptides. The
biological activities in both cases were almost identical within the experimental error (Fig. S3).
For peptide RTD-1 R8L, the IC values of the desalted crude and purified peptide were 505 ± 59
50
nM and 558 ± 55 nM, respectively.
Design of RTD-1 analogs with multiple mutations
Encouraged by these results we decided to explore if the effects seen with individual mutations
were additive. Accordingly, we designed a total of six RTD-1 analogs containing multiple
mutations that were shown to be positive for the anti-LF biological activity (Fig. 5A). The six
RTD-1 analogs (peptides 8 through 13, Fig. 5A) were chemically synthesized as described
previously. The ‘one-pot’ backbone cyclization/folding was also very efficient in all the cases.
Due to the low number of analogs, we decided to purify them by C18-reversed phase HPLC to
provide high purity peptides for biological and structural assays (Fig. S5). The different RTD-1
analogs were tested for anti-LF activity as described before. In all cases the peptides inhibited LF
in a dose dependent manner, showed Hill-slopes close to -1 and showed better activities than
the best of the single mutation RTD-1 analogs, mutant V11W (peptide 7, IC =197 ± 26 nM) (Fig.
50
5
). Analog 8, which contains mutations G10R and V11W located in one of the loops of RTD-1,
was around three times more potent than RTD-1 (IC = 125 ± 15 nM). Peptide 8 was more
50
active than the single mutants G10R (peptide 4, IC = 262 ± 24 nM) and V11W (peptide 7, IC
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=
197 ± 26 nM) indicating some degree of cooperativity between these two mutations. Analog 9
included a third mutation (L6W) located in the β-sheet region of RTD-1. This defensin was
around four times more potent than wild-type RTD-1 with an IC value of 92 ± 10 nM. Peptide
50
10
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12
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0 added a fourth mutation (L6W), also located in the β-sheet region of RTD-1. The activity of
this peptide (IC = 100 ± 10 nM) was similar to that of analog 9.
50
At this point and given the pseudo-symmetry of RTD-1 we decided to explore adding similar
mutations to the other loop of RTD-1. θ-Defensin 11 contained the additional mutation F2W
providing an analog that was about five times more potent than RTD-1 (IC = 83 ± 7 nM).
50
Adding mutations G1R (peptide 12) and G1R,T17R (peptide 13) further improved the biological
activity of the resulting defensins by around six and ten times, respectively, when compared to
that of the wild-type RTD-1 peptide (IC = 61 ± 6 and 43 ± 3 nM, respectively) (Fig. 5). The K
50
i
value for peptide 13 against LF protease was estimated to be 18 ± 1 nM.
Inhibitory activity of RTD-1 mutants against metalloprotease TNFα converting enzyme (TACE)
We decided to test the selectivity of the most active RTD-1 based defensins against TACE. TACE
is a member of the ADAM (a disintegrin and metalloproteinase) family, which is also known as
30, 31
ADAM17.
TACE is the proteolytic enzyme that processes the membrane-bound TNFα
precursor into soluble TNFα. Since TNFα is a pro-inflammatory cytokine that plays an important
role in inflammatory process in many immune-mediated disorders and cancer, TACE has
32, 33
become a molecular target for anti-inflammatory therapies.
We used a fluorogenic peptide substrate to test the activity of the different RTD-1 mutants.
Initial studies were done using the catalytic domain of TACE, fragment 223-474. Wild-type RTD-
1, however, failed to inhibit the proteolytic activity of TACE (223-474). In contrast, RTD-1 was
able to inhibit in a dose-dependent fashion and a Hill-slope of -1.0 ± 0.1 the whole extracellular
domain of TACE (fragment 223-671), although with a relatively modest activity (IC = 4.5 ± 0.5
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µM) (Table 1 and Fig. S6). These data suggest that RTD-1 is a modest non-competitive inhibitor
of TACE. Under the conditions used in the inhibitory assay the K value was estimated to be
i
similar to the IC .
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Next we investigated the activity of some the single mutant RTD-1 analogs. Again, all the
mutants analyzed were able to inhibit TACE in a in a dose-dependent fashion and a Hill-slope
around -1.0 (Table 1 and Fig. S6). For example, introduction of mutations L6D and G10D in RTD-
1
, peptides 3 and 5 (Table 1), were able to improve around four times the biological activity
against TACE, while the same mutations had a significant detrimental effect on the activity
against LF, around 3-4 times less active than wild-type RTD-1 (Table 1 and Fig. S6). Intriguingly,
introduction of mutation V11W (peptide 7) produced a significant increase in anti-TACE activity
of the corresponding mutant, about 25 times more potent than RTD-1 (IC = 175 ± 19 nM),
50
while the same mutation had only a modest effect on anti-LF activity (Table 1 and Fig. S7). In
contrast with LF protease, the addition of additional mutations had a detrimental effect on the
anti-TACE activity. For example analogs 8 and 9, both containing the additional mutations G10R
and G10R/L6W, respectively, were about 4 times less potent than the single mutant V11W
peptide 7 (Table 1). Furthermore, peptide 13, which contains multiple mutations and is the most
active RTD-1 analog against LF (K = 18 ± 1 nM), was only a modest inhibitor for TACE (K =1.20 ±
i
i
0
.15 µM) being around 7 times less potent than the single mutant V11W (peptide 7). Altogether,
these data indicate that θ-defensin analog 13 is a selective and more potent LF inhibitor than
RTD-1.
P      13 is a specific inhibitor of LF
To eliminate the possibility of non-specific inhibition, the most active RTD-1 analog (peptide 13)
was also tested in the presence of 1 mg/mL bovine serum albumin (BSA) (≈ 15 µM) (Table 1).
The use of BSA at this concentration is widely used in high throughput screening assays to avoid
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the selection on non-specific inhibitors.
Under these conditions, θ-defensins RTD-1 and 13
were able to inhibit LF in a dose-dependent fashion with a IC values of 1.06 ± 0.14 and 250 ±
50
39 nM, respectively (Table 1 and Fig. S7). In contrast, this peptide did not show any activity
against TACE under these conditions (IC ≥ 10 µM) (Table 1 and Fig. S7). The single mutant
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V11W (peptide 7) was also able to inhibit the enzyme TACE in the presence of BSA in a dose-
dependent. However, it was only a modest inhibitor under these conditions with an IC value of
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3
.08 ± 0.50 µM (Table 1 and Fig. S7). These results strongly indicate that peptide 13 is a specific
inhibitor for LF protease.
P      13 is not toxic to mammalian cells
Due to the high content of Arg and Trp residues in peptide 13, we decided to evaluate the
cytotoxicity of this peptide against mammalian cells. Trp and Arg residues possess some key
chemical properties that make them ideal components for peptides able to interact and
interfere with the biological activity of cellular membranes. For example, Arg residues provide
the peptides with cationic charges and hydrogen bonding properties necessary for interaction
with the abundant anionic components of cellular membranes, while Trp has a distinct
preference for interacting with the interfacial region of lipid bilayers. In addition, Arg and Trp
residues can interact through cation–π interactions, thereby facilitating enhanced peptide–
membrane interactions.
The cytotoxicity of peptides 13 and RTD-1 (used as control) was tested against HeLa cells using
an MTT assay. Under these conditions none of the peptides show any cellular toxicity at
concentrations up to 100 µM (Fig. S13)
Peptide 13 adopts a native θ-defensin fold
Cyclic peptide 13 was fully characterized by NMR (Figs. 6 and S10, and Table S2). The NMR
15
spectrum of 13 was monodispersed showing a single conformation in solution. The N-HSQC
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spectrum of uniformly N labeled [U- N] 13 was relatively simple and well dispersed in the
amide proton region, indicating a folded structure and a high degree of structural symmetry
(Fig. 6A, see also Fig. S10). The indole and backbone NH cross-peaks for all the Trp residues and
backbone and guanidino NH cross-peaks for residues Arg4, Arg8, Arg12, and Arg17 were highly
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clustered around 10 ppm ( H)/129 ppm ( N), 9.2 ppm ( H)/125 ppm ( N), 8.6 ppm ( H)/122.5
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ppm ( N), and 7.1 ppm ( H)/116.5 ppm ( N) , respectively (Fig. 6A). The backbone NH cross-
peaks for residues Arg1, Arg9, Arg10, and Arg18 were completely broadened, possibly, due to
the chemical exchange of the amide protons with solvent. In the homology model of 13 (Fig.
6
B), which is based on the wt RTD-1, the amides of Arg1, Arg9, Arg10, and Arg18 are located in
the loops and are solvent exposed; these two conditions facilitate amide-solvent proton
chemical exchange.
Based on the assigned cysteine Cβ chemical shifts (Table S2), all cysteine residues in peptide 13
form disulfide bonds. Although clearly visible, the corresponding Cys NH cross-peaks were
broadened in the nitrogen dimension when compared to those from the Trp or Arg residues.
Broadening of the Cys NH cross-peaks has been attributed to the isomerization of disulfide
3
6, 37
bonds in other Cys-rich polypeptides
.
Cys amides were clustered in two distinct groups,
suggesting two different types of similar chemical environments. Interestingly the backbone
cross-peaks for the Cys residues C5 and C14 were identical indicating the presence of a
symmetry element between these two residues.
Backbone, HN, Hα, N, Cα, and side chain Cβ chemical shifts are exquisitely sensitive to the
protein conformation and used to establish secondary structure fold. We used the chemical
38
shift index (CSI), which is calculated based on the difference between protein chemical shifts
and that of random coil, to identify the secondary structure elements within the sequence of
p       13. The CSI values per residue of peptide 7 were consistent with segments Phe2-Arg8
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and Val11-Thr17 adopting an antiparallel β-sheet structure, and with residues Arg1, Arg9, Arg10,
and Arg18 located in the corresponding connecting loops (Figs. S11 and S12). In addition, we
were able to identify seventeen interresidual NOEs for peptide 13 (Table S3). The number of
unambiguous NOEs was much lower that what is required for the determination of a high-
resolution structure, usually 5 NOEs/residue. This was due to the highly symmetrical nature of
peptide 13 primary structure, which resulted in the extremely overlapped NMR spectra.
Nevertheless, these experimental data, altogether, are fully consistent with peptide 13 adopting
a native θ-defensin fold.
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DISCUSSION AND CONCLUSIONS
We report the first full sequence amino acid scanning of backbone-cyclized θ-defensin to
improve its biological activity against the anthrax metalloprotease LF. LF protease is a
component of the lethal factor toxin produced by the bacterium Bacillus anthracis, which is key
24
to the pathogenesis of anthrax, and therefore a therapeutic target for the development of
25
novel potential treatments for anthrax disease. The LF catalyzes the hydrolysis of mitogen-
activated protein kinase kinase 1 (MAPKK1) and MAPKK2 rendering them inactive and hence
39, 40
incapable of activating the mitogen-activated protein protein kinase (MAPK) pathway.
Cleavage of the MAPKK proteins leads to inhibition of a wide variety of immune cell functions,
24
cellular necrosis and apoptosis.
Wild-type RTD-1 is a moderate non-competitive inhibitor of LF protease (K = 365 ± 20 nM) and a
i
weak inhibitor of other metalloproteases such as TACE (K = 4.45 ± 0.48 µM). We have used a full
i
sequence amino acid scanning approach to explore the contribution of every amino acid, except
the Cys residues required for folding, to the anti-LF activity and whether this could be improved
by replacing the original residue with a set of different amino acids including hydrophobic,
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aromatic, positively and negatively charged residues. This required the synthesis of over 100
individual θ-defensin analogs. Defensin analogs were rapidly produced in high purity by using a
novel approach for the rapid parallel production of disulfide-rich backbone cyclized polypeptides
using a ‘tea-bag’ approach in combination with high efficient cyclization-folding protocols that
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require minimal purification.
In general, all the positions in RTD-1 were amenable to mutation with exception of the Arg
residues (Arg4 and Arg13) located in the β-sheet region of the peptide. In our hands,
replacement of these residues by non-charged residues yielded cyclic peptides that could not
fold correctly. Regarding the biological activity of RTD-1, the presence of a θ-defensin fold is
required for biological activity. Replacement of the Cys residues by Ser yielded an inactive
peptide against LF (IC >10 µM). Also, we found that the native Arg residues in RTD-1 are key for
50
anti-LF activity. Replacement of these residues by negatively charged amino acids, Asp or Glu,
decreased the biological activity of the resulting peptides. In addition, introduction of extra Arg
residues in the non-hydrophobic residues located in both loops (Gly1, Gly10 and Thr17) also
gave peptides with improved biological activity. Replacement of the hydrophobic residues in
RTD-1 by Trp was also able to increase the anti-LF activity. Using this information, we were able
to design a series of RTD-1 analogs with increasing activities for inhibiting LF. The most active
compound, peptide 13, was about 10 times more active than native RTD-1, IC value of 43 ± 3
50
nM versus 390 ± 20 nM for RTD-1. P      7 was able to fold adopting a native θ-defensin fold
as determined by NMR, despite the numerous mutations introduced in this peptide.
Intriguingly, peptide 13 has a symmetrical sequence almost entirely composed by Arg and Trp
residues, with the exception of the Cys residues that are required for disulfide formation and
folding. Despite the high content on Arg and Trp residues, θ-defensin 13 was not cytotoxic to
mammalian cells up to a concentration of 100 µM. This peptide was able to retain significant
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anti-LF activity even in the presence of bovine serum albumin, while was not active under the
same conditions against the metalloprotease TACE (IC > 10 µM). The TACE inhibitory activity of
50
different RTD-1 analogs was also analyzed indicating a different mechanism of inhibition in both
proteases. For example, introduction of negative charge in RTD-1 was able to improve the anti-
TACE activity while had a negative effect on the anti-LF activity (Table 1). More intriguing was
the result obtained with the V11W mutant (peptide 7), which increased the TACE inhibitory
activity over 25 times while the same mutant had only a modest effect on anti-LF activity (Table
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1
). All combined, these data suggest that peptide 13 does not inhibit LF through a non-specific
mechanism, although more structural studies may be required to fully understand at the
molecular level the mechanism of inhibition of this peptide.
A recent study has reported that retrocyclins, synthetic peptides corresponding to a putative
13
ancestral human peptide encoded by a θ-defensin pseudogene, are able to destabilize some
41
bacterial toxins causing them to unfold in thermal denaturation assays. Precipitation of
bacterial toxins in the presence of defensins has been also described as a mechanism the
42
inactivation of some bacterial toxins by these types of peptides. In our study, however, we did
not see any evidence of protein precipitation under the conditions used in our assays.
In summary, we report the use of amino acid positional scanning to improve the inhibitory
activity of the θ-defensin RTD-1 against the bacterial metalloprotease LF. Our results clearly
show the high robustness of the θ-defensin scaffold for the generation of molecular diversity,
43
either generated by chemical synthesis (this work) or recombinant expression methods. These
libraries should allow the rapid screening and selection of novel θ-defensin-based sequences
able to inhibit other proteases or modulate specific protein/protein interactions, therefore
providing leads for the development of novel peptide-based therapeutics.
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EXPERIMENTAL SECTION
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Analytical HPLC was performed on a HP1100 series instrument with 220 nm and 280 nm
detection using a Vydac C18 column (5 mm, 4.6 x 150 mm) at a flow rate of 1 mL/min. All runs
used linear gradients of 0.1% aqueous trifluoroacetic acid (TFA, solvent A) vs. 0.1% TFA, 90%
acetonitrile in H O (solvent B). UV-vis spectroscopy was carried out on an Agilent 8453 diode
2
array spectrophotometer, and fluorescence analysis on a Jobin Yvon Flurolog-3
spectroflurometer. Electrospray mass spectrometry (ES-MS) analysis was performed on an
Applied Biosystems API 3000 triple quadrupole electrospray mass spectrometer using software
Analyst 1.4.2. Calculated masses were obtained using Analyst 1.4.2. All chemicals involved in
synthesis or analysis were obtained from Aldrich (Milwaukee, WI) or Novabiochem (San Diego,
CA) unless otherwise indicated. The purity of all the peptides used in this study was estimated to
be ≥95% as determined by HPLC and ES-MS.
Preparation of Fmoc-Aaa-OH. Fmoc-Phe-F and Fmoc-Leu-F were prepared using
2
3
diethylaminosulfur trifluoride (DAST) as previously described and quickly used afterwards.
Briefly, DAST (160 ꢀL, 1.2 mmol) was added drop wise at 25° C under nitrogen current to a
stirred solution of Fmoc-Phe-OH (387.4 mg, 1 mmol) or Fmoc-Leu-OH (353.4 mg, 1 mmol) in 10
mL of dry dichloromethane (DCM), containing dry pyridine (81 µL, 1 mmol). After 20 minutes,
the mixture was washed with ice-cold water (3 x 20 mL). The organic layer was separated and
dried over anhydrous MgSO . The solvent was removed under reduced pressure to give the
4
corresponding Fmoc-amino acyl fluoride as white solid that was immediately used.
Loading of 4-sulfamylbutyryl AM resin with Fmoc-Aaa-F. Loading of the first residue was
23
accomplished using Fmoc-Phe-F or Fmoc-Leu-F as previously described. . Briefly, 4-
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sulfamylbutyryl AM resin (420 mg, 0.33 mmol) (Novabiochem) was swollen for 20 minutes with
dry DCM and then drained. A solution of Fmoc-Phe-F (≈389 mg, ≈1 mmol) of Fmoc-Leu-OH (≈354
mg, ≈ 1 mmol) in dry DCM (2 mL) and di-isopropylethylamine (DIEA) (180 µL, 1 mmol) was added
to the drained resin and reacted at 25° C for 1 h. The resin was washed with dry DCM (5 x 5 mL),
dried and kept at -20° C until use.
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Chemical synthesis of RTD-1 based peptides. All peptides were synthesized as described in Fig.
2
and was carried out by solid-phase synthesis on an automatic peptide synthesizer ABI433A
(
Applied Biosystems) using the Fast-Fmoc chemistry with 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU)/diisopropylethylamine (DIEA) activation
protocol at 0.1 mmol scale on a Fmoc-Phe-sulfamylbutyryl AM resin (L6 and R4 position
scanning library) or Fmoc-Leu-sulfamylbutyryl AM resin (the rest of peptides). Side-chain
protection compatible with Fmoc-chemistry was employed as previously described for the
synthesis of peptide α-thioesters by the Fmoc-protocol, except for the N-terminal Cys residue,
which was introduced as Boc-Cys(Trt)-OH. Before coupling of the position to be scanned, the
peptide-resin was manually resuspended in DCM:dimethylformamide (DMF)(1:1) and split into 9
equal aliquots. Each peptide-resin aliquot was placed in a separate 1 mL polypropylene column
(
Qiagen). The resins were manually deprotected with 20% 4-methylpiperidine in DMF (3 x 5 min)
and then individually coupled with each of the different 9 Fmoc-Aaa-OH (Figs. 1 and 2).
Couplings were performed using HBTU and DIEA for 45 min. The resins were then washed,
dried, placed in individual tagged ‘tea-bags’ (30 x 20 mm) and sealed. The synthesis was
continued using the peptide synthesizer 0.25 mmol reaction vessel, which can hold up to 9
different tea-bags of the dimensions reported above. Following chain assembly, the alkylation,
thiolytic cleavage and side chain deprotection were performed for individual peptides in 1 mL
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polypropylene columns as previously described. Briefly, ≈10 mg of protected peptide-resin
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were first alkylated two times with ICH CN (17.4 µL, 0.24 mmol; previously filtered through basic
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alumina) and DIEA (8.2 µL, 0.046 mmol) in N-methylpyrrolidone (NMP) (0.22 mL) for 24 h. The
resin was then washed with NMP (3 x 5 mL) and DCM (3 x 5 mL). The alkylated peptide resin was
cleaved from the resin with HSCH CH CO Et (20 µL, 0.18 mmol) in the presence of a catalytic
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amount of sodium thiophenolate (NaSPh, 0.3 mg, 2.2 µmol) in DMF:DCM (1:2 v/v, 0.12 mL) for
24 h. The resin was then dried at reduced pressure. The side-chain protecting groups were
removed by treating the dried resin with trifluoroacetic acid (TFA):H O:tri-isopropylsilane (TIS)
2
(
95:3:2 v/v, 0.5 mL) for 3-4 h at room temperature. The resin was filtered and the linear peptide
thioester was precipitated in cold Et O. The crude material was dissolved in the minimal amount
2
of H O:MeCN (4:1) containing 0.1% TFA and characterized by HPLC and ES-MS as the
2
corresponding desired linear precursor α-thioester. Cyclization and folding was accomplished by
flash dilution of the linear α-thioester TFA crude to a final concentration of ≈20-50 µM into
freshly degassed 2 mM reduced glutathione (GSH), 0.1 M sodium phosphate buffer at pH 7.2 for
9
6 h. Most RTD-1 analogs were desalted using a C18 cartridge solid phase extraction (SPE) to
remove the buffer components providing the folded peptide with relatively high purity (Fig. S1).
Single mutants R8L and V11E, and peptides 8 through 13 were purified by C18-reversed-phase
HPLC and characterized by ES-MS (Fig. S3 and S5). The complete synthesis of a positional sub-
library took around 1.5 weeks in average, from the start of the synthesis to the lyophilized
purified peptide.
Synthesis of peptide 1. The linear thioester precursor of peptide control 1 was synthesized as
described above. Cyclization was accomplished by flash dilution of the linear α-thioester to a
final concentration of 25 μM into freshly degassed 2% sodium 2-mercaptoethanesulfonate
(
MESNA) in 0.1 M sodium phosphate buffer at pH 7.2 for 1 h. S-alkylation was achieved by
adding a large excess of iodoacetamide into the cyclization mixture for 30 min. The final peptide
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was purified using a linear gradient 15-25% buffer B over 30 min and characterized by analytical
HPLC and ES-MS (Fig. S8).
Peptide quantification. RTD-1 peptide analogs were quantified by HPLC at 214 nm using a
10
11
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13
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relative extinction coefficient calculated as previously described. A solution of pure peptide
RTD-1 of known concentration estimated by amino acid analysis was used as internal control for
HPLC quantification.
Anthrax LF protease inhibition assay. Lethal factor (LF) protease and FRET-based substrate
containing fluorescent proteins CyPet and YPet linked by consensus sequence
(
RRKKVYPYPMEGTIA) containing the linker (GGS) at both sites of the consensus sequence were
6
29, 46
expressed and purified as previously described.
LF inhibition assay was performed in LF
reaction buffer (10 µM CaCl , 10 µM MgCl , 20 µM ZnCl , 20 mM sodium phosphate, and 100
2
2
2
mM NaCl at pH 7.2) in the presence or absence of BSA. Samples of 50 nM LF in LF reaction
buffer (100 µL) were preincubated with different concentrations of RTD-1 ranging from 1 nM to
1
0 µM. After incubation at room temperature for 30 min, residual LF activity was measured by
adding the FRET-based substrate to a final concentration of 10 nM and the decrease in FRET
29
signal was measured every 2 min for 3 h. FRET was measured using an Envision 2103 plate
reader (PerkinElmer) using an excitation wavelength of 405 nm. The relative FRET change was
535
535
calculated using: FRC = It /I0 , where I and I are the fluorescence intensities at time zero and
0
t
at a particular time (t), at 535 nm. The initial velocities for the hydrolysis of substrate Lethal
Factor in the presence of different concentrations of RTD-1 were fitted to a log(inhibitor) versus
response with a variable Hill-slope using the software package Prism (GraphPad Software). Ki
was calculated using the equation for non-competitive tightly bound inhibitors; where K = IC –
i
50
47, 48
E /2 (E = enzyme concentration).
0
0
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6
TACE protease inhibition assay. TACE protease containing the catalytic domain (TACE (233-477),
Enzo Life Sciences) or the whole extracellular domain (TACE (215-671), R&D System), and the
TACE fluorogenic substrate (Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH , Enzo
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Life Sciences, where Mca = (7-methoxycoumarin-4-yl)acetyl and Dpa = N-3-(2,4-dinitrophenyl)-
L-α,β-diaminopropionyl) were used in this work. The proteolytic reaction was performed in TACE
reaction buffer (2.5 μM ZnCl and 25 mM Tris-HCl, at pH 8.0) in the presence or absence of BSA.
2
Samples of 2 nM TACE in TACE reaction buffer (100 μL) were preincubated with different
concentrations of RTD-1 based peptides ranging from 1 nM to 10 μM. After incubation at room
temperature for 30 min, residual TACE activity was measured by adding the fluorogenic
substrate to a final concentration of 3 µM. Fluorescence was measured every 2 min for 3 h in an
Envision 2103 plate reader (PerkinElmer) using an excitation wavelength of 320 nm and an
emission wavelength of 405 nm. The initial velocities for the increase of fluorescent signal in the
presence of different concentrations of the corresponding RTD-1 analog were calculated as
described earlier for LF. The K values were calculated as for LF.
i
1
5
15
13 15
Bacterial expression of N-labeled of θ-defensin 13. Bacterial expression of N- and C, N-
43
labeled 13 was achieved as previously described. The purified peptides were characterized by
analytical HPLC and ES-MS (Fig. S9).
Cytotoxicity Assay
HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS),
penicillin (50 IU/mL), and streptomycin (50 µg/mL) at 37° C with 5% CO . The cellular toxicity of
2
49
3
the RTD-1 peptides was measured using an MTT assay as previously described. Briefly, ≈3 x 10
cells were seeded in 96-well microtiter plates in 100 µL DMEM with 10% FBS and incubated for
24 h. After the incubation period, the cells were washed with PBS and treated with 200 µL/well
DMEM media supplemented with 10% FBS containing the peptides at the indicated
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concentration for 48 h at 37° C in 5% CO and then treated with 20 µL of a solution of 3-(4,5-
2
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL) for 2 h. The medium was
discarded and DMSO (100 µL/well) was added to each well and incubated with gentle shaking
for 20 min at room temperature. The absorbance at 595 nm of the solution was analyzed using a
Tecan Genios Multifunctional Microplate Reader (Tecan System Inc) and the background at 670
nm subtracted.
10
11
12
13
14
15
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Molecular modeling and dynamics of θ-defensin 13. The homology model of θ-defensin 13 was
6
built using the structure of RTD-1 (PDB ID code: 1HVZ) as template. The resulting structure was
subjected to molecular dynamics for 700 ns in a neutral water box containing 0.9% NaCl. The
molecular dynamics was run using the macro md_run.mcr (www.yasara.org/md_run.mcr) using
the Yasara software package (www.yasara.org). The 10 most stable structures were used to
generate a morphed movie using Pymol (www.pymol.org) (Fig. S14).
NMR spectroscopy. NMR samples were prepared by dissolving 13 into 80 mM potassium
2
phosphate pH 6.5, 0.05% trifluoracetic acid, 100 mM d6-DMSO in 90% H O/10% H O (v/v) to a
2
2
15
13
concentration of approximately 0.2 mM for unlabeled 13 and 0.1 mM for [U- N] and [U- C,
15
N] peptide 13. All NMR spectra were acquired on a Bruker Avance III 600 MHz spectrometer
equipped with TCI cryoprobe. The NMR spectra were acquired at 298 K, and 2,2-dimethyl-2-
silapentane-5-sulfonate, DSS, was used as an internal reference. The carrier frequency was
centered on the water signal, and the solvent was suppressed by using either WATERGATE pulse
sequence or a spin lock pulse. Standard triple resonance experiments HNCACB and CBCACONH
1
15
13
15
1
1
as well as HCCH-TOCSY, 3D H, N-NOESY (mixing time 200 ms), 2D C-HSQC, N-HSQC, H, H-
1
15
1
1
NOESY (mixing time 200 ms), 3D H, N-TOCSY (spin lock time 80 ms), and 2D H, H-TOCSY (spin
50
lock time 80 ms) were collected. Spectra were processed using Topspin 3.1 (Bruker).
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15
Assignments for the backbone nitrogen ( N), carbons (Cα and Cβ) and protons (Hα and NH) of
51, 52
peptide 13 (Table S2) were obtained using standard procedures.
Supporting Information Available: Analytical, biological and NMR data on the θ-defensin
peptide analogs produced in this work. The coordinates for the homology model of θ-defensin
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3 are also included. This material is available free of charge via the Internet at
http://pubs.acs.org.
Homology Models: The coordinates for the homology model of θ-defensin 13 are included in
the Supporting Information.
Corresponding Author Information: Julio A. Camarero, Ph.D., Department of Pharmacology and
Pharmaceutical Sciences, Department of Chemistry, University of Southern California, 1985
Zonal Avenue, PSC 616, Los Angeles, CA 90033, Tel: 323-442-1417, Fax: 323-224-7473, E-mail:
jcamarer@usc.edu.
Abbreviations used: ES-MS, electrospray mass spectrometry; GSH, glutathione; LF, lethal factor;
RTD-1, Rhesus θ-defensin; TACE: TNFα converting enzyme
Acknowledgements: We thank Drs. Michael E. Selsted and Andre J. Ouellette (University of
Southern California) for their suggestions and help during this project. This work was supported
by National Institutes of Health Research Grants R01-GM090323 (J.A.C.), R01-GM113363 (J.A.C.)
and R01-GM085006 (A.S.).
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