(Received for publication, October 13, 1995; and in revised form, December 18, 1995)
From the
A strategy has been developed for the identification of inhibitors of toxins or regulatory proteins. This approach is based on blocking the access of such proteins to their biological targets during their solution transport. This approach uses the strength of nonsupport-bound synthetic combinatorial libraries (SCLs) for the study of acceptor-ligand interactions. A non-receptor assisted toxin, melittin, was selected for the present study to illustrate this application of the SCL approach. Hexapeptide SCLs were assayed for their ability to inhibit the cytolytic activity of melittin toward bacterial and erythrocyte cells. Over 20 inhibitory hexapeptides were identified following the screening and deconvolution processes from millions of sequences. The identified inhibitory peptides appeared to interact directly with melittin. These interactions appear to decrease melittin's ability to undergo lipid- and/or polysaccharide-induced conformational changes, and are demonstrated by fluorescence and circular dichroism spectroscopy.
The mechanism of action of the majority of biologically active
peptides involves an initial interaction between the peptide and a
specific, singular acceptor system (e.g. receptor, antibody,
enzyme, etc.). However, a number of toxins have been reported to exert
their biological activity directly on cell membranes not by binding to
a specific single binding site but through a cascade of different
possible mechanisms (reviewed in (1) ). Due to the potential
difficulty in targeting a specific mechanism of action, an effective
means to inhibit the activity of such toxins can be envisioned to
involve inhibition of the toxins' access to cell membranes during
the solution transport of the toxins. We have developed an approach
involving the use of synthetic combinatorial libraries (SCLs) ()to identify short peptide sequences able to bind to the
toxins and, in turn, to inhibit their biological effects. The SCLs
generated in this laboratory are composed of mixtures of
nonsupport-bound compounds that can be used in virtually any bioassay
system. Recently, highly specific, short bioactive peptides have been
discovered from pools of millions of other related peptides using SCLs
based on their binding affinity to specific receptors of interest (i.e. antibodies(2, 3, 4, 5) ,
enzymes(6, 7) , and opioid
receptors(8, 9) ). The use of SCLs for the study of
nonspecific interactions was only applied to the identification of
antimicrobial, antifungal, and antiviral
compounds(2, 10, 11, 12, 13) .
Although potent compounds were discovered in this manner, their
mechanisms of action have yet to be determined. These successful
applications suggest that SCLs may be a useful means for the rapid
identification of inhibitors of nonreceptor-assisted, biologically
active proteins such as toxins and regulatory proteins.
In a first study of the ability to use SCLs for inhibiting protein activities through peptide-protein interactions, inhibitors of the cytolytic activity of melittin, a potent toxin isolated from bee venom (14) , were identified. Besides its implication in the toxic effect of a bee sting, melittin is useful model system to study nonreceptor-assisted toxicity, because it is known to bind spontaneously to biological and synthetic model membranes (reviewed in (15) ). Although the mechanism of melittin's cytolytic activity remains unclear, binding to a specific receptor has not been reported. One of the proposed mechanisms of melittin's toxicity toward erythrocyte cells involves the accumulation of peptide molecules on the outer leaflet of the bilayer, which causes a perturbation in the arrangement of the membrane structure. This, in turn, results in an increased membrane permeability and, ultimately, cell lysis (15, 16, 17) .
Using an SCL composed of 52 million hexapeptides, we have developed an inhibitory assay in which each peptide mixture making up an SCL can be tested for its ability to inhibit melittin's lytic activity on erythrocyte or bacterial cells. Several series of individual hexapeptides were identified that were found to inhibit melittin's hemolytic and antimicrobial activity. As demonstrated by CD and fluorescence spectroscopy, this inhibition of melittin's toxicity was found to result from the formation of a complex between melittin and these peptides.
Taking into account the large number of individual peptides present in a peptide mixture, one must optimize an assay system in order to maximize the signal detection from the background level, permitting the differentiation of the activities between the peptide mixtures. In a competitive assay, the intensity of the signal depends on the concentration of the compound being competed against or, in other words, on its inhibited level of activity at the concentration used. Melittin was therefore initially used at the minimum concentration necessary to lyse 100% of the RBC suspension (i.e. 7.5 µg/ml, 2.6 µM). Each peptide mixture was screened at a concentration of 1.25 mg/ml. Thus, if one assumes an average molecular mass of 720 g/mol for the hexapeptides making up the library, then at a 1.25 mg/ml peptide mixture solution, each individual hexapeptide is present at 13 nM. Although this results in a molar ratio between each individual peptide and melittin of 1:200, one expects the presence of analogs having similar activity in the same peptide mixture, and therefore, a substantially higher ``effective'' molar concentration.
As shown in Fig. 1, a small number of the 400 peptide mixtures assayed from
the SCL were found to have significant inhibitory activity at 1.25
mg/ml. In addition, at 1.25 mg/ml, none of the peptide mixtures
affected cell membranes, as shown by the absence of significant
hemolytic activity. In order to determine the relative activities of
the most active peptide mixtures found through this initial screening,
IC values were calculated for those mixtures inhibiting
more than 50% lysis by melittin at this concentration. The IC
values are based on the percentage of inhibition at 2-fold serial
concentrations in peptide mixtures, thereby providing a more accurate
relative evaluation of the peptide mixture activities than a single
concentration screening. Fifteen out of the 400 peptide mixtures showed
IC
values varying from 350 to 1300 µg/ml. Upon
screening an equivalent non-N-acetylated hexapeptide SCL,
weaker inhibitory activities were observed (IC
>2000
µg/ml), which indicates the value of having a blocked N-terminal
amino group for the desired inhibitory activity. Although several of
the 15 active mixtures have been selected for the iterative process,
Ac-IVXXXX-NH
is used here to illustrate these
studies.
Figure 1:
Screening of
Ac-OOXXXX-NH for inhibition of melittin's
hemolytic activity. Each peptide mixture was screened at 1.25 mg/ml
against 7.5 µg/ml melittin and 0.25% RBCs. Each panel represents a set of 20 peptide mixtures having a common first
residue, and each bar represents the percentage of inhibition
of each peptide mixture, with the x axis corresponding to the
second residue defining the mixture.
A 5-fold increase in
activity was obtained upon defining the fourth position of
Ac-IVIXXX-NH (i.e. AC-IVIOXX-NH
; Table 1). Ten peptide
mixtures had inhibitory activity ranging from 20 to 40 µg/ml,
indicating a lower specificity for the fourth position. As was found
for the third position, the 10 most active mixtures were defined by an
hydrophobic residue at the fourth position. Although the iterative
process is illustrated in Table 1for the peptide mixture
Ac-IVILXX-NH
, the mixtures
Ac-IVIVXX-NH
, Ac-IVICXX-NH
,
Ac-IVIWXX-NH
, and Ac-IVIFXX-NH
were also selected as leading sequences for parallel iterative
processes. Upon defining the fifth position (as illustrated in Table 1), increases of up to 2-fold were observed. In the final
iterative step, a number of potent defined inhibitors of the hemolytic
activity of melittin were identified (IC
values ranging
from 5 to 10 µg/ml; Table 1). Other inhibitors identified
through parallel iterative processes are shown in Table 2. All of
the inhibitors identified can be described as having a strong
hydrophobic character and by the presence of a negatively charged
residue or a polar amino acid at the C terminus.
The sequences of the inhibitors identified
from the PS-SCL are very similar to those obtained using the iterative
process described above in that they are strongly hydrophobic and
contain a glutamic acid at the C terminus. It should be noted that
whereas the three N-terminal positions were highly specific in the
screening of the PS-SCL, the last three positions were found to be
redundant with at least 10 of the 20 peptide mixtures showing similar
activity(23) . These results may explain the differences in the
final sequences obtained from the two libraries, which appear primarily
in the last three residues of the identified sequences. They are also
in agreement with the large number of peptide mixtures having similar
activity found upon defining the last three positions. As shown in Table 3, the three N-terminal residues identified were among
those defining the mixtures having the three greatest activities of
each set of the positional SCLs. For instance, isoleucine represented
the only peptide mixture having activity from the single position SCL,
Ac-XXOXXX-NH and was found to characterize the
most active peptide mixtures upon defining the third position during
the separate iterative processes. On the other hand, the peptide
mixtures corresponding to the two N-terminal residues of the individual
peptide inhibitors derived from the screening of the PS-SCL,
Ac-FIXXXX-NH
and
Ac-IIXXXX-NH
, were among the 15 most active
peptide mixtures of the dual defined position SCL (Table 3).
Figure 2:
Effect of preincubation on the inhibitory
activity of Ac-IIIYFE-NH and Ac-FIIWFE-NH
. A, melittin (7.5 µg/ml) was preincubated with
Ac-IIIYFE-NH
(
, 14 µg/ml) or Ac-FIIWFE-NH
(
, 6 µg/ml) at 37 °C for up to 30 min, prior to
the addition of RBCs (0.25%). B, melittin (7.5 µg/ml) was
preincubated with RBCs (0.25%) at 22 °C for up to 60 min, prior to
the addition of Ac-IIIYFE-NH
(
, 14 µg/ml) or
Ac-FIIWFE-NH
(
, 6 µg/ml). In both cases, the
percentage of inhibition is plotted as a function of preincubation
time.
Although the lysis mechanism of RBCs relative to that of bacterial cells by melittin may differ, it is logical to assume that it involves the initial accumulation of melittin on the respective cell membranes. If the mechanism of inhibition involves a strong interaction between melittin and the peptide inhibitors rather than an interaction between the cells and the peptide inhibitors, as indicated above, then similar inhibitory activities would be expected with bacterial cells. The four peptides tested were found to inhibit melittin against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria (Table 5).
Figure 3:
Fluorescence studies of
Ac-IIIYFE-NH-melittin interactions. A, the
fluorescence spectra of melittin (7 µM) in the absence (solid line) or presence of Ac-IIIYFE-NH
(85
µM, dotted line). B, stoichiometric
titration of melittin (7 µM) in the presence of up to 85
µM Ac-IIIYFE-NH
. The tryptophan fluorescence
of melittin at 340 nm after excitation at 294 nm was measured. The
increases in tryptophan fluorescence at 340 nm relative to the spectra
of melittin in the absence of the inhibitor
(
I
) are plotted as a function of the
inhibitor concentration.
The occurrence of conformational changes
in melittin upon adding an increasing concentration of
Ac-IIIYFE-NH was studied by CD spectroscopy. Melittin was
found to have a random conformation at 22 µM in
phosphate-buffered saline ([
]
=
-5000 degrees cm
dmol
) with no
significant change in ellipticity at 222 nm upon the addition of
Ac-IIIYFE-NH
, even at an Ac-IIIYFE-NH
to
melittin ratio of 7:1. These results indicate that the binding of the
inhibitory peptides to melittin does not induce significant changes in
the overall conformation of melittin in buffer. Furthermore, these
results support the lack of an inhibitor-induced tetramerization of
melittin, which would result in an induced
-helical conformation (30, 31, 32) , and, in turn, agree with the
formation of a melittin/inhibitor complex evidenced earlier by
fluorescence spectroscopy. The occurrence of conformational changes of
melittin in the presence of the inhibitors was further evaluated when
melittin binds to two model biological membrane systems: naturally
occurring polymers of N-acetyl neuraminic acid (referred to as
polysialic acids), which are present on the surface of cell membranes
and lysophosphatydylcholine micelles as representative of the lipid
bilayer of the cell membranes. As shown in earlier
studies(32) , melittin was readily induced into an
-helical conformation upon binding to colominic acid
(poly-2,8-N-acetyl neuraminic acid; Fig. 4). After
subtracting the contribution of the inhibitor from the overall spectra,
the ellipticity at 222 nm of melittin CD spectra was found to decrease
dramatically in the presence of Ac-IIIYFE-NH
(Fig. 4). This was found to be independent of the mixing
order of the three components (melittin, Ac-IIIYFE-NH
, and
colominic acid; Fig. 4). In a similar manner, lower ellipticity
at 222 nm was observed at several melittin:lysophosphatydylcholine
ratios (R
varying from 0 to 30) in the presence
of Ac-IIIYFE-NH
(Fig. 5).
Figure 4:
CD studies of
Ac-IIIYFE-NH-melittin interactions. The CD spectra of
melittin (22 µM) in the presence of colominic acid
(colominic acid to melittin ratio, 2) is shown in the absence (dotted line) or presence (centered line) of
Ac-IIIYFE-NH
(Ac-IIIYFE-NH
to melittin, 5).
Ac-IIIYFE-NH
was mixed with melittin either prior to (centered line) or following (dashed line) the
addition of colominic acid. The CD spectra of melittin in buffer (solid line) is also shown as a
reference.
Figure 5:
Effect of Ac-IIIYFE-NH on
conformational changes induced by melittin-lipid interactions. The mean
residue ellipticity at 222 nm ([
]
) of the
CD spectra of melittin (22 µM) was determined at R
varying from 0 to 30 in the absence (
) or
presence (
) of Ac-IIIYFE-NH
at a Ac-IIIYFE-NH
to melittin ratio of 5.
The soluble SCL technology developed in this laboratory has been proven in a wide variety of assays to allow the rapid identification of potent biologically active peptides (reviewed in (33) ) and, more recently, peptidomimetics(11) . In the present study, the power of the SCL approach was used to develop a new strategy to identify inhibitors of known cytolytic compounds through peptide-protein interactions in solution using melittin as a model system.
Two deconvolution processes have been used for the identification of individual active compounds from an SCL: an iterative process (2) and a positional scanning process(5) . The comparison of these two approaches described here leads to the conclusion that very similar information about the overall physico-chemical properties of the final sequences and the relative importance of an amino acid or building block at a given position can be obtained using either deconvolution format. This is true even though relatively minor differences in sequences are likely to be obtained depending on the level of specificity of a given amino acid within the sequence. Thus, in the present case, the results from either SCL indicate that the peptides must have an overall hydrophobic character for inhibition to occur and that the presence of a negatively charged residue at the C terminus (E or D) is important but not an absolute necessity in the inhibition mechanism. The strong similarities observed for the first three residues of the individual peptide sequences identified indicate that highly specific residues can be readily identified through the use of either library format. On the other hand, families of amino acids having slightly different chemical character are most likely to be identified in the case of less specific positions (i.e. the three C-terminal residues).
Similar inhibitory activity was found when using different cells (Gram-positive and Gram-negative bacteria and erythrocytes), indicating that the mode of inhibitory action occurred through binding to melittin. These results are supported by the greater inhibitory activity observed upon longer incubation times between melittin and inhibitors prior to the addition of the cells. Loss in inhibitory activity was observed when the N terminus of the inhibitory peptides was not blocked with an acetyl group (data not shown). This can also be explained by inhibitor-melittin interactions. Thus, electrostatic repulsions between the positively charged N terminus of this peptide and the basic residues of melittin may lessen or prevent such interactions from occurring and, in turn, eliminate the inhibitory activity. Melittin is known to bind to a number of proteins such as calmodulin(34) , troponin C(35) , and phosphorylase kinase(36) . In particular, it has been reported that melittin binds to calmodulin (34) or troponin C (35) through the exposed hydrophobic surfaces. The hydrophobic character of the inhibitors found in this study suggests the occurrence of hydrophobic-hydrophobic interactions with melittin similar to those reported for proteins.
Using over 50 single substitution analogs of melittin, we have found that the self association of melittin to its transitory tetrameric aggregate is required for penetration of the carbohydrate barrier present in biological membranes and for lysis to occur(32) . In addition, the seven residues of the hydrophobic segment (i.e. proline-14 to isoleucine-20) of melittin have been described as a nucleation center in the folding of monomeric melittin into its self-assembled tetramer(37) . One can envision the mode of actions of the identified inhibitors through hydrophobic interactions with this segment of melittin. This would perturb melittin's ability to undergo lipid- and/or polysaccharide-induced conformational changes as observed by CD spectroscopy. In agreement with this hypothesis, our fluorescence studies showed that the environment of tryptophan-19 of melittin is more hydrophobic in the presence of the inhibitors. In conclusion, we believe that the present studies show that not only can potent inhibitors of a potentially lytic compound be rapidly identified using the SCL approach but also that one can find specific agents that block the access of a peptide and/or protein to its biological target. The application of combinatorial chemistry may also be useful for blocking or modifying the activity of other toxins, as well as regulatory proteins.