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The Use of Tethered Bilayer Lipid Membranes to Identify the Mechanisms of Antimicrobial Peptide Interactions with Lipid Bilayers


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This review identifies the ways in which tethered bilayer lipid membranes (tBLMs) can be used for the identification of the actions of antimicrobials against lipid bilayers. Much of the new research in this area has originated, or included researchers from, the southern hemisphere, Australia and New Zealand in particular. More and more, tBLMs are replacing liposome release assays, black lipid membranes and patch-clamp electrophysiological techniques because they use fewer reagents, are able to obtain results far more quickly and can provide a uniformity of responses with fewer artefacts. In this work, we describe how tBLM technology can and has been used to identify the actions of numerous antimicrobial agents.
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The Use of Tethered Bilayer Lipid Membranes to
Identify the Mechanisms of Antimicrobial Peptide
Interactions with Lipid Bilayers
Amani Alghalayini, Alvaro Garcia, Thomas Berry and Charles G. Cranfield *
School of Life Science, University of Technology Sydney, Ultimo, NSW 2007, Australia; (A.A.); (A.G.); (T.B.)
Received: 8 January 2019; Accepted: 29 January 2019; Published: 30 January 2019
This review identifies the ways in which tethered bilayer lipid membranes (tBLMs) can
be used for the identification of the actions of antimicrobials against lipid bilayers. Much of the
new research in this area has originated, or included researchers from, the southern hemisphere,
Australia and New Zealand in particular. More and more, tBLMs are replacing liposome release
assays, black lipid membranes and patch-clamp electrophysiological techniques because they use
fewer reagents, are able to obtain results far more quickly and can provide a uniformity of responses
with fewer artefacts. In this work, we describe how tBLM technology can and has been used to
identify the actions of numerous antimicrobial agents.
antimicrobial peptides; tethered bilayer lipid membranes; electrical impedance spectroscopy
1. Introduction
Antimicrobial peptides (AMPs) are of increasing interest as potential lead candidates for treating
infection because they are thought to be more impervious to antibacterial resistance mechanisms [
One of the most rapid and increasingly effective methods for testing the actions of antimicrobials that
target microbial membranes is to use tethered bilayer lipid membranes (tBLMs) in association with
electrical impedance spectroscopy (EIS). In this review, we outline the advantages of this technique
compared to other lipid membrane techniques and describe how tBLMs, in conjunction with EIS,
have been used to rapidly and reliably identify the actions of many AMPs.
The desire to study membranes and determine changes in their physical and electrical properties
encouraged the development of artificial lipid bilayer systems. The first of these was developed by
Mueller et al. [
] and was subsequently named black lipid membranes. These model membranes
consisted of a painted phospholipid bilayer across a small aperture, usually 1 mm in diameter,
between two chambers that held aqueous solutions. This system provided a planar lipid bilayer
that could be probed with electrical or basic optical techniques. The name black lipid membrane was
derived from the optical technique used to determine when the bilayer had formed. Once the film
had been painted on, the mixture would begin to thin and become a single spanning bilayer, this
would then cause interference of reflected light and render the membrane opaque [
]. The fact that
no underlying support existed meant that the membrane-associated proteins were able to function
without interference. However, non-polar solvents are required in the manufacture of these membranes
which alter the integral properties of the lipid bilayer [4].
Attempts to find alternatives, which abolished the solvent artefact in the membrane, led to the
production of phospholipid bilayers supported by solid substrates. Tamm and McConnell (1985)
Antibiotics 2019,8, 12; doi:10.3390/antibiotics8010012
Antibiotics 2019,8, 12 2 of 13
were the first to describe this technique, which enabled them to create extremely stable bilayers
supported by a number of different substrates (silicon oxide wafers, glass coverslips and quartz
slides). The ability to adhere to these surfaces was believed to originate from the silicious materials,
which have deprotonated free silanols at the surface at neutral pH [
]. Even though they had been
using zwitterionic phospholipids, an apparent interfacial potential was established allowing the
surface of the bilayer to be attracted to the surface of the substrate. However, as acknowledged by
Tamm and McConnell themselves, the fact that the substrate was adjacent to the bilayer meant that
a very thin water layer existed between the phospholipid and the solid support [
]. This meant that
the incorporation of transmembrane proteins was hampered by the high probability of the protein
interacting with the substrate surface [
]. This could cause both immobilization of the protein with
inhibition of function or cause denaturation of the protein in the vicinity of the contact point between
protein and substrate [
]. This has particularly been noted in the inability of integral proteins to diffuse
freely through solid-supported bilayer systems [
]. Not only is the inclusion of integral membrane
proteins difficult in solid-supported bilayer systems, but the study of the electrical properties of
these membranes is difficult to perform when the aqueous reservoir between the membrane and
substrate is so small [
]. Thus, a supported lipid bilayer with an appropriate reservoir space to allow
accommodation of transmembrane proteins and a free diffusion of ions was pursued.
Attempts at constructing novel solid-supported membranes that contained a larger reservoir
were made by laying phospholipid bilayers onto water swellable polymer cushions sitting between
the solid substrate and bilayer [
]. The silicious materials used in these studies enabled the
construction of polymer cushions that adhered directly to either the surface itself or a functionalized
surface, through silinisation, using either acrylamide, dextran or agarose substrates. Silinisation
could be used on metal solid substrates which could be incorporated into impedance spectroscopy
systems to determine the electrical properties of the membrane [
]. Further modification of the
polymer cushion substrates themselves allowed adherence to substrates, such as gold, through sulphur
coordination [18].
It was noted that applying a phospholipid bilayer onto a polymer cushion did not mitigate
inherent problems associated with membrane stability [
]. The issue of stability was circumvented
with a new functionalized polymer cushion that created anchor points for customized lipids to attach
and, in turn, tether the membrane to the cushion through hydrophobic forces [
]. This improvement
in stability did not abrogate the continuing issue of a reservoir that restricted ion diffusion between the
solid substrate and the phospholipid bilayer. A new membrane technology from Australia was reported
by Cornell et al., (1997), consisting of a membrane separated from the surface of the substrate through
the use of double-length reservoir half-membrane spanning diphytanyl (DLP) ethylene glycol tethers.
This enabled the formation of a lipid bilayer around the end of the DLP through the half-membrane
spanning component of the DLP chain [
]. The other end of the DLP was firmly attached to a gold
substrate surface through sulphur–gold coordination chemistry. This system took the concept of
tethering or anchoring the membrane to the solid substrate surface described by Beyer et al., (1996) and
removed the polymer cushion, thus creating a much larger reservoir space for transmembrane protein
insertion and free ion diffusion (Figure 1). This optimized reservoir space was enhanced by mixing
in polar spacer molecules, which prevented the formation of a monolayer beneath the membrane by
providing lateral separation of the tethers [
]. This system provides an enormous amount of flexibility
in the composition of the phospholipid bilayer, as any mixture of phospholipids able to create a bilayer
could be studied. Since then, several studies have investigated and described alternative tethering
components [
] and substrates such as liquid mercury [
] to create stable supported bilayers.
Southern hemisphere researchers of note in the development of new tethered bilayer architectures
are researchers from the McGuillivray group at the University of Aukland, and the Köper group at
Flinders University, in South Australia.
The variety of novel approaches to the design of tethering systems to maintain stable and dynamic
bilayer systems provides a large toolkit to create explicit solutions for particular biological problems
Antibiotics 2019,8, 12 3 of 13
such as identifying how antimicrobials interact with lipid bilayers. This review does not seek to
identify all the research into AMPs using tBLMs, rather, it seeks to detail what tBLM technology can
do to identify the actions of AMPs.
Figure 1.
A basic tethered bilayer lipid membrane architecture. The use of membrane-tethering
molecules and spacer molecules creates a reservoir between the membrane and the substrate to provide
space for the transport of ions and the insertion of extended membrane-bound peptides or proteins.
2. Models of AMP–Lipid Membrane Interactions
In studying the interactions between lipid bilayers and antimicrobial peptides, a variety of models
have been proposed to identify the exact mechanism of action associated with these interactions.
Each family of peptides appears to interact with lipid bilayers in a unique manner and most
current interactions have been identified as either pore forming, intrinsic pore modulating or having
surfactant-like properties that induce membrane rupture or lysis [
]. Individual types of peptides
may not conform to a particular mode of action or have the qualities of each model, with some
peptides not fitting within any of these models [
]. The proposed mechanisms of action of
AMPs are briefly summarized here, along with a subsequent explanation of how tBLMs can be used
in conjunction with swept frequency electrical impedance spectroscopy (EIS) to distinguish between
these mechanisms.
2.1. Barrel-Stave Model
This model describes the formation of an ionic conductive pore through the membrane resembling
a barrel with individual peptides forming the staves. [33,47,48]. The process of pore formation involves
the insertion of peptides through the lipid bilayer, along with a successive aggregation to form a
transmembrane channel (Figure 2). An amphipathic peptide structure is required, in which the
hydrophobic regions are aligned with the hydrophobic inner domain of the lipid bilayer and the
hydrophilic regions face the pore lumen. This creates a hydrophilic channel through the lipid bilayer
allowing the free passage of ions and solutes through the membrane [
]. The diameter of the pore
lumen is intrinsically linked to the number peptides recruited, with larger groupings of peptides forming
larger pores, resulting in increased leakage of cell contents and potentially leading to cell death [35].
Figure 2.
Schematic of how individual peptides might form the barrel-stave pore configuration in a
cell membrane.
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2.2. Interdigitated Peptide Toroidal Pore Model
The barrel-stave model has evolved into different forms, with a number of antimicrobial
peptides not conforming to the rudimentary barrel-stave model, namely magainins, protegrins and
melittin [4852]
. An alternate method was put forth by Matsuzaki et al. [
]. The toroidal model differs
from the barrel-stave model in that the lipid headgroups of the bilayer participate in the formation
of the pore. The structure of this pore demands that the inner and outer leaflets bend in such a way
as to form a pore composed of interdigitated peptide and phospholipid headgroups (See Figure 3).
In contrast to the barrel-stave model, the peptides remain associated with the headgroups of the lipids
and do not permeate through the hydrophobic chain regions [33].
Figure 3.
The toroidal pore model has the antimicrobial peptides (AMPs) interdigitated between lipid
head groups as part of a pore within the membrane.
2.3. Carpet Model
This model describes the gathering of peptides at the lipid–water interface of the lipid bilayer,
attracted there through electrostatic forces. It was first theorized to describe the interactions of the
peptide dermaseptin [
]. This model has also been used to describe the interactions of peptides
such as ovispirin and melittin [
]. As the concentration of peptides increases at the lipid–water
interface, they form a ‘carpet’ across the surface of the bilayer (Figure 4). The peptides are reported
to permeabilize the membrane by disrupting phospholipid packing and are suggested to have
surfactant-like qualities in high concentrations, leading to the removal of small segments of the
bilayer through micellization [40].
Figure 4.
The carpet model of cell membrane disruption has the accumulation of amphipathic AMPs as
a ‘carpet’ across the membrane, eventually promoting to the micellization of individual lipids creating
membrane defects.
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2.4. Intrinsic Pore Modulation Model by Changing the Critical Packing Paremeter (CPP)
A more recent model of peptide–lipid interactions, suggested by Australian researchers amongst
others, describes the process in terms of altering the critical packing parameter (CPP) of the bilayer
and, thereby, altering the size of membrane pores already present in the membrane [36,37,5660].
The CPP concept was first introduced by Jacob Israelachvili during his time at Australian National
University [
]. This model aims to predict the morphology of lipids based on the ratio of the
lipid head groups’ surface area (a
) and the hydrophobic lipid chain lengths (l) with the overall
volume of the individual lipids (v), such that CPP = v/a
l. Within a planar bilayer, the overall CPP
= 1. An overall CCP = 1/3 describes a micelle but also describes the CPP of the lipids that make
up the curved regions of a toroidal pore (Figure 5) [
]. The CPP-pore modulation model suggests
the interaction of peptides is influencing the size of pre-existing pores or defects found within the
lipid bilayer [
]. Upon interacting with the lipid bilayer, the peptides disrupt the packing of the
surrounding lipids causing changes in the overall CPP of the bilayer. This interaction of lipids and
peptides can cause a change in the effective head group surface area (a
) compared to the lipid chain
length (l), either increasing or decreasing the CPP depending on the geometry of the peptide in the
bilayer. When the CPP of an individual lipid drops below unity within a lipid bilayer, it lacks sufficient
laxity for lateral movement beyond the existing boundaries. This model suggests that the need for
space may be resolved through movement of lipids into or out of existing toroidal pores where the
CPP = 1/3, and/or with an alteration in chain length to compensate for the change in the surface area
in the planar bilayer sections where the CPP has to remain equal to one.
Figure 5.
The Critical Packing Parameter (CPP) model of toroidal pore modulation by antimicrobial
agents. This model predicts that the shape of peptides influences the lipid packing arrangement leading
to either an increase in intrinsic membrane pore radius and an overall slight thinning of the membrane,
as more lipids diffuse into pore regions, or a decrease in the intrinsic membrane pore radius with a
thickening of the membrane as more lipids diffuse out of the pore regions.
2.5. Identifying Mechanisms of Membrane Interaction Using EIS Techniques
Swept frequency electrical impedance spectroscopy is the method most commonly used to
identify the ionic conduction across a tethered bilayer lipid membrane. It also characterizes the
membrane capacitance, which identifies changes in membrane thickness and/or water content. These
changes in membrane thickness and/or water content are derived from the geometric properties of
a capacitor. In its simplest form, a lipid bilayer can be modelled as a parallel-plate capacitor, itself
in parallel with a resistor. The membrane thickness pertains to the distance between the two plates
of the capacitor and the water content determines the relative permittivity (
). Thus, as membrane
capacitance increases, the membrane thickness (distance between the plates) decreases and/or the
water content (relative permittivity between the plates) increases. The opposite is true when the
Antibiotics 2019,8, 12 6 of 13
membrane capacitance decreases. When studying the interactions of antimicrobial peptides with lipid
bilayers, the ability to identify changes in either or both of these values can assist in isolating the
mechanisms of antimicrobial interactions.
A large increase in membrane conduction, with limited or only a small change in membrane
capacitance, indicates the formation of ion channels within the membrane, particularly if the
conduction effect does not readily disappear with subsequent wash steps. This type of response
is typical of pore forming AMPs where it would be expected that insertion and aggregation of the
peptides in the membrane would disrupt the packing of adjacent lipids, with a reduced influence on
packing structure at further distances from the annular ring [
]. A classic example of this would be
channels formed by the antibiotic
-hemolysin (Figure 6A,B) [
]. We can see a significant increase
in conduction across the membrane with an associated small increase in membrane capacitance
(Figure 6A). The small membrane capacitance change in this case can be attributed to the localized
disruption of phospholipid packing in the annular ring around the
-hemolysin as determined by
neutron reflectometry [65].
A large increase in membrane conduction that is also associated with a large increase in membrane
capacitance is suggestive of antimicrobials having a lytic or surfactant-like effect. It is expected that
these effects would be concentration dependent and that reaching a particular concentration threshold
would enable sequestration of lipids from the bilayer through micellization. These effects are typically
irreversible with subsequent wash steps and often washing will compound the effects. The increase in
membrane capacitance in this case is suggestive of the membrane getting thinner, probably due to the
removal of lipids from the bilayer as a result of the surfactant-like activity of the AMPs. An example of
this response is the human cathelicidin AMP LL-37 [
] (Figure 6C,D). The initial interaction of this
peptide can be described by the “carpet model”, with disruption of the membrane due to intercalation
of the peptide with lipid headgroups [
] and an associated decrease in membrane capacitance.
However, as has been noted [
], peptides that follow the “carpet model” can possess a threshold
concentration at which significant disruption of the phospholipid bilayer is observed. This would then
manifest itself as a large increase in bilayer capacitance as the surfactant-like effect of removing lipids
thins the bilayer.
In many cases, AMPs can be added that are known to be too small to traverse the membrane yet
can still induce increases or decreases in overall membrane conduction. In these sorts of responses,
there are typically only very small changes in membrane capacitance, if any. Typically, to some degree,
these AMPs can be readily washed out of the membrane (Figure 6E,F). There have been numerous
reports of AMPs or peptidomimetics that induce these responses [
]. The change in
membrane conduction in these cases has been assigned to a modulation of intrinsic membrane pores
via a rearrangement of the packing of the lipids according the CPP model (Figure 5).
A difficulty arises in comparing the concentration dependence of membrane disruption in
tBLM systems to the minimal inhibitory concentrations derived from
in vitro
bacterial growth
experiments [
]. Given that EIS measurements are limited to the antimicrobial peptides’ ability
to modulate the physical structure of the lipid membrane, the fact that these peptides have other
purported antimicrobial properties unrelated to membrane disruption must be considered [
Other considerations in the use of tBLMs include the ratio of tethered lipids to freely diffusing lipids
and the relative volume of the reservoir region between the substrate and the bilayer. In principle, it is
better to have as few tethered lipids, compared to freely diffusing lipids, as possible, and to have as
large a reservoir region as possible to enable the free passage of ions [75].
Antibiotics 2019,8, 12 7 of 13
Figure 6.
The tethered bilayer lipid membrane (tBLM) responses presented here are actual data
obtained in the laboratory of the authors. (
) Membrane conduction and capacitance responses to
the membrane ion channel forming antibiotic
-hemolysin. (
) Phase versus frequency (Bode Plot)
before and after addition of the antibiotic
-hemolysin. The phase minima typically shift to higher
frequencies with little increase in the phase angle. (
) Membrane conduction and capacitance responses
to the human defensin peptide, LL-37, that causes membrane lysis. The responses are particularly
evident after the membrane undergoes a wash step which induces mild sheer stress at the membrane.
) Phase versus frequency (Bode Plot) response of LL-37. The phase minima typically shift to higher
frequencies with very large increase in the phase angle. This phase signature is evidence of the tBLM
undergoing irrevocable disruption. (
) Membrane conduction and capacitance responses to the AMP
cys-Melimine [
]. A mild increase in membrane conduction is evident at relatively high concentrations
of the AMP with a small change in membrane capacitance. The responses are partially reversed after
washing. Note that the concentration of the AMP is 10 times larger than that for the ion channel
-hemolysin (Figure 6A). (
) Phase versus frequency (Bode Plot) response of the AMP cys-Melimine.
The phase minima typically shift to higher frequencies with only a small increase in the phase angle.
3. Antimicrobial–Lipid Membrane Interactions Investigated Using tBLMs
3.1. Testing the Lipid Specificity of AMPs
There have been numerous studies that have shown how antimicrobials have a preference for
negatively charged lipids over zwitterionic lipids [
]. The relative high number of positively
charged amino acids reported in AMPs confer this preference through electrostatic attraction. However,
there is not always a correlation between a peptide’s charge and its affinity for negatively charged
lipids. The bespoke antimicrobial peptide chimera of melittin and protamine, melimine, and its analogs
have been investigated by Australian researchers using tBLMs and their actions have been described
Antibiotics 2019,8, 12 8 of 13
according to the CPP model as mentioned above [
]. Despite the presence of multiple positively
charged amino acid residues in these peptides, there was little correlation with their interactions in
tBLMs comprised of a high percentage of negatively charged lipids. Instead, a correlation was made
according to the number and location of hydrophobic peptide residues.
Researchers in Sydney, Australia, studied a group of bespoke biphenyl peptidomimetics using
tBLMs to identify their mechanism of action. Zwitterionic and negatively charged lipids were
employed to determine how headgroup charges may modulate the electrostatic interactions of these
peptidomimetics [
]. For each synthetic peptidomimic, their capacity to disrupt tBLMs was compared
to their minimum inhibitory concentration (MIC) against bacteria and was found to not consistently
correlate. This was used as evidence that the antimicrobial mechanisms of these peptidomimetics were
not necessarily as a result of their actions on negatively charged bacterial membranes.
Other small molecular antimicrobial peptidomimics, N-naphthoyl-phenylglyoxamide-based and
N-sulfonylphenylglyoxamide-based antimicrobial peptides, were investigated to determine whether
the mechanism of action was related to their capacity to disrupt phospholipid membranes [
In each case, their membrane interactions were described using the CPP pore modulation model and
only partially correlated with the MIC observed in bacterial experiments.
In the case of the naturally occurring Kalata B1 and Kalata B2 cyclic antimicrobial and insecticidal
peptides (of which researchers from the David Craik laboratory at the University of Queensland are
recognized world leaders [
]), their specificity for membranes that contain phosphotidyl ethanolamine
(PE) lipids was confirmed using tBLMs [
]. This specificity was derived from a binding pocket within
the peptide containing both negatively and positively charged amino acid residues which specifically
targeted PE lipids and had little affinity for negatively charged lipid headgroups [
]. Electrical
impedance spectroscopy identified large changes in the membrane capacitance and membrane
conductance suggesting activity of these cyclotides was due to a surfactant-like mechanism rather
than the previously reported pore forming mechanism [79].
These results demonstrate the capacity of tBLMs in conjunction with electrical impedance
spectroscopy to elucidate the membrane disruptive properties of novel peptidomimetics and provides
valuable information regarding affinities for particular lipid compositions. Further, tBLM technology
used in this way permits a rapid characterization of peptide/membrane interactions and provides a
basis for implementing an iterative development of synthetic peptides.
3.2. Voltametric Techniques to Explore Antimicrobial Interations
As well as electrical impedance spectroscopy, other electrical techniques, such as ramped or pulsed
amperometry can be employed to identify the actions of various antimicrobial agents. The amphipathic
antimicrobial peptide trichogin GA IV (TCG) was investigated using applied potential steps at 50 mV
increments [
]. Using this technique, the researchers determined the required membrane potentials
for incorporation of the peptide. The researchers also employed cyclic voltammetry and identified that
TCG has a voltage-gated behavior similar to the fungal peptaibol peptide alamethicin.
The use of potential steps and ramped amperometry has also been employed to identify how
peptides can make use of membrane defects to incorporate into membranes. Led by Australian
researchers, Cranfield et al. (2014) showed that by rapidly increasing the potential across the tBLM,
they could induce a detectable electroporation effect. They were then able to show that defects caused
by electroporation induced an increase in the activity of the African clawed frog antimicrobial peptide
PGLa [64].
3.3. Bacterial Surface tBLM Mimics
Significant effort has gone into creating tBLMs that better mimic the actual surface of bacteria.
There have been efforts to incorporate commercially supplied lipids from E. coli sources in tBLMs
with some success [
], but these membranes do not have the lipopolysaccharide (LPS) layer that is
associated with bacterial membranes. Recently, however, Andersson et al. (2018) from the Köper group
Antibiotics 2019,8, 12 9 of 13
at Flinders University in South Australia in collaboration with researchers from the Australian Nuclear
Science and Technology Organisation (ANSTO) were able to fuse liposomes of a lipopolysaccharide
purified from E. coli onto a monolayer of tethering lipids [
]. They were then able to test their
LPS–tBLM using the antimicrobial colistin sulfate and were able to elicit a change in the membrane
structure as evidenced by neutron scattering and EIS measures.
Spencelayh et al., (2006) were able to form tBLMs that incorporated Lipid I and Lipid II, which are
precursors to the peptidoglycan layer of bacterial cell walls. They werethen able to test the glycopeptide
antibiotics vancomycin and ramoplanin against these tBLM architectures. These types of antibiotics
interfere with the formation of the peptidoglycan coatings that protect Gram-positive bacteria from
lysis. Surface plasmon resonance and EIS were employed to measure changes in membrane thickness
as a result of adding these antibiotics. Significantly, purified inner E. coli membranes were used to
form these tBLMs [82].
Outer membrane protein F (OmpF) is one of the porin transmembrane proteins found in E. coli
outer membranes and is a target for antibiotics such as colicin N [
]. Stora et al. (1999) were able to
incorporate OmpF into tBLMs and demonstrate that colicin N was able to reduce overall membrane
conduction as a result [
]. The same group were later able to self-assemble tBLMs containing cysteine
mutants of the OmpF protein which itself anchors onto the gold substrate via coordination of the
cysteine thiol group [85].
4. Conclusions
Australia and New Zealand, in particular, are home to some of the world’s leading researchers
into the use of tethered bilayer lipid membranes for antimicrobial research. Australia is also the home
of the world’s only commercial supplier of tethered bilayer lipid membranes. In this work, we have
reviewed how this technology has been used to assist in identifying how antimicrobial agents interact
with lipid bilayers and, where appropriate, highlighted the works of the southern hemisphere research
groups who are the leaders in this field of research.
Author Contributions:
Writing—original draft preparation, A.A, A.G., T.B., and C.C.; Writing—review and
editing, A.G., and C.C.
This research was funded by Australian Research Council (ARC) Discovery Program (DP160101664),
the ARC Research Hub for Integrated Device for End-user Analysis at Low-levels (IDEAL) (IH150100028) and the
UTS Chancellor’s Postdoctoral Research Fellowship Scheme.
We wish to acknowledge the contribution of Bruce Cornell (SDX Tethered Membranes Pty.
Ltd.) for helpful comments concerning the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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... All measurements involving the SLB and intact vesicle platforms were conducted using the quartz crystal microbalance-dissipation (QCM-D) technique, which is widely used to track membrane-peptide interactions at solid-liquid interfaces in a label-free format and its time-resolved resonance frequency (∆f) and energy dissipation (∆D) signals are sensitive to the acoustic mass and viscoelastic properties of the model membrane adlayers, respectively [34][35][36][37]. Additionally, tBLM platform experiments were conducted using the electrochemical impedance spectroscopy (EIS) technique, which tracks membrane-peptide interactions by evaluating changes in the electrical conductance (G m ) and capacitance (C m ) properties of the lipid bilayer membrane [38][39][40][41]. ...
... The EIS technique tracked changes in the conductance (G m ) and capacitance (C m ) signal of the tBLM platform. When a peptide causes membrane disruption, the G m signal increases due to greater ion flow across the more permeable membrane, while an increase in the C m signal indicates membrane thinning of the tethered lipid bilayer, which provides insight into membrane structural integrity [38,57,58]. Furthermore, changes in the frequency and the phase value of phase minima of the EIS spectra were evaluated using Bode plot analysis, which provides insight into the state of ion leakage that corresponds to the G m signal and membrane thinning corresponding to the C m signal, respectively [59]. ...
... Overall, across all tested lipid compositions, LTX-315 peptide addition and subsequent buffer washing caused increases in both the G m and C m signals, which is consistent with increased membrane permeability and a thinning effect [38]. Notably, the EIS results demonstrated that the G m and C m shifts persisted even after buffer washing, further supporting that peptide-mediated membrane disruption is irreversible. ...
Full-text available
LTX-315 is a clinical-stage, anticancer peptide therapeutic that disrupts cancer cell membranes. Existing mechanistic knowledge about LTX-315 has been obtained from cell-based biological assays, and there is an outstanding need to directly characterize the corresponding membrane-peptide interactions from a biophysical perspective. Herein, we investigated the membrane-disruptive properties of the LTX-315 peptide using three cell-membrane-mimicking membrane platforms on solid supports, namely the supported lipid bilayer, intact vesicle adlayer, and tethered lipid bilayer, in combination with quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. The results showed that the cationic LTX-315 peptide selectively disrupted negatively charged phospholipid membranes to a greater extent than zwitterionic or positively charged phospholipid membranes, whereby electrostatic interactions were the main factor to influence peptide attachment and membrane curvature was a secondary factor. Of note, the EIS measurements showed that the LTX-315 peptide extensively and irreversibly permeabilized negatively charged, tethered lipid bilayers that contained high phosphatidylserine lipid levels representative of the outer leaflet of cancer cell membranes, while circular dichroism (CD) spectroscopy experiments indicated that the LTX-315 peptide was structureless and the corresponding membrane-disruptive interactions did not involve peptide conformational changes. Dynamic light scattering (DLS) measurements further verified that the LTX-315 peptide selectively caused irreversible disruption of negatively charged lipid vesicles. Together, our findings demonstrate that the LTX-315 peptide preferentially disrupts negatively charged phospholipid membranes in an irreversible manner, which reinforces its potential as an emerging cancer immunotherapy and offers a biophysical framework to guide future peptide engineering efforts.
... With appropriate tBLM platform design and electrical sealing, the relatively large ionic reservoir between the lower bilayer leaflet and gold surface makes it possible to measure electrical current flow through the membrane [30,31]. Given these capabilities, EIS measurements have been conducted to study the mechanism of action of membrane-disruptive antimicrobial peptides using the tBLM platform, which has proven useful for distinguishing peptides that induce transverse pore formation [32], membrane lysis [33,34], and size modulation of existing membrane pores [35,36]. Hence, there is excellent potential to apply the EIS sensing technique together with the tBLM platform to characterize the membrane-disruptive interactions of antimicrobial lipids and surfactants from a mechanistic perspective. ...
... In the first case, the test compound can intercalate within the tethered lipid bilayer [43], resulting in changes in membrane thickness and fluidity ( Figure 1C). Such changes in turn affect membrane permeability as well; increased permeability is typically associated with G m and C m shift increases along with a prominent increase in frequency and/or modest increase in phase minima in the phase profile [33,34]. On the other hand, in the second case of membrane lysis, the tethered lipid bilayer surface tension is reduced, followed by membrane solubilization whereby lipid molecules leave the tBLM surface and results in Figure 1D). ...
... On the other hand, in the second case of membrane lysis, the tethered lipid bilayer surface tension is reduced, followed by membrane solubilization whereby lipid molecules leave the tBLM surface and results in Figure 1D). Membrane lysis typically induces large increases in the G m and C m values along with a frequency increase and a very large increase in the phase minima in the phase profile [33]. ...
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There is extensive interest in developing real-time biosensing strategies to characterize the membrane-disruptive properties of antimicrobial lipids and surfactants. Currently used biosensing strategies mainly focus on tracking membrane morphological changes such as budding and tubule formation, while there is an outstanding need to develop a label-free biosensing strategy to directly evaluate the molecular-level mechanistic details by which antimicrobial lipids and surfactants disrupt lipid membranes. Herein, using electrochemical impedance spectroscopy (EIS), we conducted label-free biosensing measurements to track the real-time interactions between three representative compounds—glycerol monolaurate (GML), lauric acid (LA), and sodium dodecyl sulfate (SDS)—and a tethered bilayer lipid membrane (tBLM) platform. The EIS measurements verified that all three compounds are mainly active above their respective critical micelle concentration (CMC) values, while also revealing that GML induces irreversible membrane damage whereas the membrane-disruptive effects of LA are largely reversible. In addition, SDS micelles caused membrane solubilization, while SDS monomers still caused membrane defect formation, shedding light on how antimicrobial lipids and surfactants can be active in, not only micellar form, but also as monomers in some cases. These findings expand our mechanistic knowledge of how antimicrobial lipids and surfactants disrupt lipid membranes and demonstrate the analytical merits of utilizing the EIS sensing approach to comparatively evaluate membrane-disruptive antimicrobial compounds.
... The solid-supported bilayers have a significant limitation in that there is only a very thin layer of water between the lipid bilayer and the solid support [53]. The insufficient space hampers the incorporation of transmembrane proteins and leads to an inability to have the incorporated membrane proteins freely diffuse through the model membrane [54][55][56]. Thus, tethered bilayer lipid membranes have been developed to provide appropriate space between the bilayer and solid support by using anchorlipids to covalently anchor the lipid bilayer to the solid substrate surface [57][58][59]. ...
Full-text available
Increasing antibiotic resistance has provoked the urgent need to investigate the interactions of antimicrobials with bacterial membranes. The reasons for emerging antibiotic resistance and innovations in novel therapeutic approaches are highly relevant to the mechanistic interactions between antibiotics and membranes. Due to the dynamic nature, complex compositions, and small sizes of native bacterial membranes, bacterial membrane mimetics have been developed to allow for the in vitro examination of structures, properties, dynamics, and interactions. In this review, three types of model membranes are discussed: monolayers, supported lipid bilayers, and supported asymmetric bilayers; this review highlights their advantages and constraints. From monolayers to asymmetric bilayers, biomimetic bacterial membranes replicate various properties of real bacterial membranes. The typical synthetic methods for fabricating each model membrane are introduced. Depending on the properties of lipids and their biological relevance, various lipid compositions have been used to mimic bacterial membranes. For example, mixtures of phosphatidylethanolamines (PE), phosphatidylglycerols (PG), and cardiolipins (CL) at various molar ratios have been used, approaching actual lipid compositions of Gram-positive bacterial membranes and inner membranes of Gram-negative bacteria. Asymmetric lipid bilayers can be fabricated on solid supports to emulate Gram-negative bacterial outer membranes. To probe the properties of the model bacterial membranes and interactions with antimicrobials, three common characterization techniques, including quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), and neutron reflectometry (NR) are detailed in this review article. Finally, we provide examples showing that the combination of bacterial membrane models and characterization techniques is capable of providing crucial information in the design of new antimicrobials that combat bacterial resistance.
... As a result, measurements requiring days or weeks are difficult to achieve. Direct bilayersubstrate contact can also create an insufficient amount of space for bilayer-spanning protein incorporation (Castellana and Cremer 2006;Andersson and Köper 2016;Alghalayini et al. 2019;Tamm and McConnell 1985). Protein-substrate contact induces denaturation or impaired function which hinders functional, electrical, or structural studies (Alghalayini et al. 2019; Tanaka and Sackmann 2005). ...
Full-text available
The complex composition of bacterial membranes has a significant impact on the understanding of pathogen function and their development towards antibiotic resistance. In addition to the inherent complexity and biosafety risks of studying biological pathogen membranes, the continual rise of antibiotic resistance and its significant economical and clinical consequences has motivated the development of numerous in vitro model membrane systems with tuneable compositions, geometries, and sizes. Approaches discussed in this review include liposomes, solid-supported bilayers, and computational simulations which have been used to explore various processes including drug-membrane interactions, lipid-protein interactions, host-pathogen interactions, and structure-induced bacterial pathogenesis. The advantages, limitations, and applicable analytical tools of all architectures are summarised with a perspective for future research efforts in architectural improvement and elucidation of resistance development strategies and membrane-targeting antibiotic mechanisms. Supplementary information: The online version contains supplementary material available at 10.1007/s12551-021-00913-7.
... Most channel-forming peptides are "antimicrobial peptides" ("AMPs"), a class of oligopeptides with a widely varying number of amino acid residues and a broad spectrum of targeted organisms. Reviews of AMPs may be found in Teixeira et al, [95] Bechinger and Salnikov, [96] Bahar and Ren, [97] Mahlapuu et al, [98] Lee et al, [99] Le et al, [100] Alghalayini et al, [101] and Mercer et al. [102] In animals, AMPs are particularly present in tissues and organs exposed to airborne pathogens, which they can recognize and kill. In this regard, AMPs represent the first line of the innate immune defense against viruses, fungi, and bacteria. ...
Full-text available
Ion transport across biomembranes plays a major role in living cells. This fundamental function is normally carried out by molecules with both a hydropho-bic and a hydrophilic side (amphiphilic molecules), which aggregate within the membrane forming a hydrophilic pore (the ion channel) permitting the selective translocation of permeant ions. Countless papers report the conformation of these ion channels in lipid vesicles using several techniques, such as circular dichroism and solid-state NMR spectroscopies. However, the functional activity of ion channels can only be investigated by varying the transmembrane potential. This is also the situation in which ion channels operate in commercialized drugs with intracellular targeting activities, of great interest in pharmaceutical research. A suitable biomimetic membrane must consist of a conducting or semiconducting support, whose "heart" is a lipid bilayer in contact with the aqueous solution of interest on one side. The other side must comprise a hydrophilic region thick enough to completely decouple the lipid bilayer from the support, giving rise to a "tethered bilayer lipid membrane" (tBLM). This review Abbreviations: ϕ, absolute potential difference across the metal|water interface; Φ d , potential difference across the diffuse layer; ϕ m , transmembrane This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
The transient disruption of membranes for the passive permeation of ions or small molecules is a complex process relevant to understanding physiological processes and biotechnology applications. Phenolic compounds are widely studied for their antioxidant and antimicrobial properties, and many biological activities of phenolic compounds are based on their interactions with membranes. Ions are ubiquitous in cells and are known to alter the structure of phospholipid bilayers. Yet, ion-lipid interactions are usually ignored when studying the membrane-altering properties of phenolic compounds. This study aims to assess the role of Ca²⁺ ions on the membrane-disrupting activity of two phenolic acids and to highlight the role of local changes in lipid packing in forming transient defects or pores. Results from tethered lipid membrane (tBLM) electrical impedance spectroscopy (EIS) experiments showed that Ca²⁺ significantly reduces membrane disruption by CAME and CAF. As phenolic acids are known metal chelators, we used UV-vis and fluorescence spectroscopy to exclude the possibility that Ca²⁺ interferes with membrane disruption by binding to the phenolic compound and subsequently preventing membrane binding. Molecular Dynamics simulations showed that Ca²⁺ but not CAME or CAF increases lipid packing in POPC bilayers. The combined data confirm that Ca²⁺ reduces the membrane-disrupting activity of the phenolic compounds and that Ca²⁺-induced changes to lipid packing govern this effect. We discuss our data in the context of ion-induced pores and transient defects and how lipid packing affects membrane disruption by small molecules.
Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.
Connecting molecular interactions to emergent properties is a goal of physical chemistry, self-assembly, and soft matter science. We show that for fatty acid bilayers, vesicle rupture tension, and permeability to water and ions are coupled to pH via alterations to lipid packing. A change in pH of one, for example, can halve the rupture tension of oleic acid membranes, an effect that is comparable to increasing lipid unsaturation in phospholipid systems. We use both experiments and molecular dynamics simulations to reveal that a subtle increase in pH can lead to increased water penetration, ion permeability, pore formation rates, and membrane disorder. For changes in membrane water content, oleic acid membranes appear to be more than a million times more sensitive to protons than to sodium ions. The work has implications for systems in which fatty acids are likely to be found, for example in the primitive cells on early Earth, biological membranes especially during digestion, and other biomaterials.
In this study we developed a methodology for solving an inverse problem to obtain structural information about distribution of nanoscale defects in surface supported, tethered bilayer membranes (tBLMs) using the electrochemical impedance spectroscopy (EIS) technique. We demonstrate that the EIS spectra contain physical information about the electrical and structural parameters of tBLMs as well as information about distribution of density of defects in membranes. Such defects can be naturally occurring collapsed sites of bilayers due to imperfections of solid substrates onto which tBLMs are assembled. Also, the membrane defects can be introduced artificially by insertion of pore-forming toxin proteins into phospholipid bilayers or by other means such as electroporation. The proposed methodology can be used for the development of precision biosensors sensitive to agents impairing integrity of biological membranes, and in general studies of protein membrane interactions that involves damage of phospholipid bilayers.
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Antimicrobial peptides (AMPs) are versatile molecules with broad antimicrobial activity produced by representatives of the three domains of life. Also, there are derivatives of AMPs and artificial short peptides that can inhibit microbial growth. Beyond killing microbes, AMPs at grow sub-inhibitory concentrations also exhibit anti-virulence activity against critical pathogenic bacteria, including ESKAPE pathogens. Anti-virulence therapies are an alternative to antibiotics since they do not directly affect viability and growth, and they are considered less likely to generate resistance. Bacterial biofilms significantly increase antibiotic resistance and are linked to establishing chronic infections. Various AMPs can kill biofilm cells and eradicate infections in animal models. However, some can inhibit biofilm formation and promote dispersal at sub-growth inhibitory concentrations. These examples are discussed here, along with those of peptides that inhibit the expression of traits controlled by quorum sensing, such as the production of exoproteases, phenazines, surfactants, toxins, among others. In addition, specific targets that are determinants of virulence include secretion systems (type II, III, and VI) responsible for releasing effector proteins toxic to eukaryotic cells. This review summarizes the current knowledge on the anti-virulence properties of AMPs and the future directions of their research.
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Antibiotic resistance is projected as one of the greatest threats to human health in the future and hence alternatives are being explored to combat resistance. Antimicrobial peptides (AMPs) have shown great promise, because use of AMPs leads bacteria to develop no or low resistance. In this review, we discuss the diversity, history and the various mechanisms of action of AMPs. Although many AMPs have reached clinical trials, to date not many have been approved by the US Food and Drug Administration (FDA) due to issues with toxicity, protease cleavage and short half-life. Some of the recent strategies developed to improve the activity and biocompatibility of AMPs, such as chemical modifications and the use of delivery systems, are also reviewed in this article.
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Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2) on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores, but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behaviour is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore, but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.
Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilised melimine, and its derivative with a cysteine on N-terminus (cys-melimine), on tethered bilayer lipid membranes (tBLMs) was examined using AC electrical impedance spectroscopy. Addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. Results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the non-cysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability which is then reduced as the melimines aggregate and phase separate within the membrane. The actions of these antimicrobials are modelled in terms of altering the critical packing parameter (CPP) of the membranes. Variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. Results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for Gram positive and Gram negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studies here with their modulation of membrane conductivity.
Through thiolipids a planar lipid bilayer (1) was immobilized on a gold support (2) for use as an electrode. This allows the detection of the ligand-gating function of the natural transmembrane channel protein OmpF (3) reconstituted in the artificial membrane: the binding of a domain (4) of the toxin colicin N, observed by surface plasmon resonance, induces the blocking of the OmpF channel protein, as shown by impedance spectroscopy.
A model membrane system has been developed, which mimics the outer membrane of Gram negative bacteria. The structure is based on a tethered monolayer which has been fused with vesicles containing lipopolysaccharide molecules. The effect of the composition of the monolayer and the lipids in the outer layer on the structural and electrical properties of the membrane has been investigated. By using electrochemical impedance spectroscopy as well as neutron scattering techniques, it could be shown that a relatively high tethering density and a small amount of diluting lipids in the outer membrane leaflet leads to the formation of a stable solid supported membrane. The influence of divalent ions on the membrane stability has been probed as well as the interaction of the bilayer with the antibiotic colistin. A number of different architectures were developed, suited to both the study of bacterial membrane proteins and the screening of antimicrobial activity of potential drug candidates.
Antimicrobial peptides (AMPs) and their synthetic mimics have received recent interest as new alternatives to traditional antibiotics in attempts to overcome the rise of antibiotic resistance in many microbes. AMPs are part of the natural defenses of most living organisms and they also have a unique mechanism of action against bacteria. Herein, a new series of short amphiphilic cationic peptidomimetics were synthesized by incorporating the 3'-amino-[1,1'-biphenyl]-3-carboxylic acid backbone to mimic the essential properties of natural AMPs. By altering hydrophobicity and charge, we identified the most potent analogue 25g that was active against both Gram-positive Staphylococcus aureus (MIC = 15.6 μM) and Gram-negative Escherichia coli (MIC = 7.8 μM) bacteria. Cytoplasmic permeability assay results revealed that 25g acts primarily by depolarization of lipids in cytoplasmic membranes. The active compounds were also investigated for their cytotoxicity to human cells, lysis of lipid bilayers using tethered bilayer lipid membranes (tBLMs) and their activity against established biofilms of S. aureus and E. coli.
Tethered bimolecular lipid membranes are solid supported membrane systems, which provide a versatile model platform for the study of many membrane related processes. Here, such an architecture has been used to study the interaction of the small synthetic antimicrobial peptide, V4, with membranes of various mixed lipid compositions, including membranes containing bacterial lipids. By investigating the binding of the peptide using a range of surface analytical techniques such as surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopy as well as electrochemical impedance spectroscopy, a clear preference of the peptide for negatively charged membranes over zwitterionic ones has been shown. Additionally, the interactions seemed to indicate a cooperative behavior for the peptide binding to a membrane.
Antibiotic resistance is a major global health concern. There is an urgent need for the development of novel antimicrobials. Recently, phenylglyoxamide-based small molecular antimicrobial peptide mimics have been identified as potential new leads to treat bacterial infections. Here, we describe the synthesis of novel phenylglyoxamide derivatives via the ring-opening reaction of N-sulfonylisatins with primary amines, followed by conversion into hydrochloride, quaternary ammonium iodide or gunidinium salts. The antibacterial activity of the compounds against Staphylococcus aureus was evaluated by in vitro assays. Structure-activity relationship studies revealed that 5-bromo-substituent at the phenyl ring, octyl group appended to the ortho sulfonamide group or guanidine hydrochloride salt as the terminal group significantly contributed to potency. The most potent compound, the gunidinium salt 35 d, exhibited a minimum inhibitory concentration value of 12 μM and a therapeutic index of 15. It also demonstrated its potential to act as antimicrobial pore-forming agent. Overall, the results identified 35 d as a new lead antimicrobial compound.
Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms. Although AMPs have been essentially studied and developed as potential alternatives for fighting infectious diseases, their use as anticancer peptides (ACPs) in cancer therapy either alone or in combination with other conventional drugs has been regarded as a therapeutic strategy to explore. As human cancer remains a cause of high morbidity and mortality worldwide, an urgent need of new, selective, and more efficient drugs is evident. Even though ACPs are expected to be selective toward tumor cells without impairing the normal body physiological functions, the development of a selective ACP has been a challenge. It is not yet possible to predict antitumor activity based on ACPs structures. ACPs are unique molecules when compared to the actual chemotherapeutic arsenal available for cancer treatment and display a variety of modes of action which in some types of cancer seem to co-exist. Regardless the debate surrounding the definition of structure-activity relationships for ACPs, great effort has been invested in ACP design and the challenge of improving effective killing of tumor cells remains. As detailed studies on ACPs mechanisms of action are crucial for optimizing drug development, in this review we provide an overview of the literature concerning peptides' structure, modes of action, selectivity, and efficacy and also summarize some of the many ACPs studied and/or developed for targeting different solid and hematologic malignancies with special emphasis on the first group. Strategies described for drug development and for increasing peptide selectivity toward specific cells while reducing toxicity are also discussed.
Antimicrobial resistance in bacteria is becoming increasingly prevalent, posing a critical challenge to global health. Bacterial biofilm formation is a common resistance mechanism that reduces the effectiveness of antibiotics. Thus, the development of compounds that can disrupt bacterial biofilms is a potential strategy to combat antimicrobial resistance. We report herein the synthesis of amphipathic guanidine-embedded glyoxamide-based peptidomimetics via ring-opening reactions of N-naphthoylisatins with amines and amino acids. These compounds were investigated for their antibacterial activity by the determination of minimum inhibitory concentration (MIC) against S. aureus and E. coli. Compounds 35, 36, and 66 exhibited MIC values of 6, 8 and 10 μg mL(-1) against S. aureus, respectively, while compounds 55 and 56 showed MIC values of 17 and 19 μg mL(-1) against E. coli, respectively. Biofilm disruption and inhibition activities were also evaluated against various Gram-positive and Gram-negative bacteria. The most active compound 65 exhibited the greatest disruption of established biofilms by 65% in S. aureus, 61% in P. aeruginosa, and 60% in S. marcescens respectively, at 250 μM concentration, while compound 52 inhibited the formation of biofilms by 72% in S. marcescens at 250 μM. We also report here the in vitro toxicity against MRC-5 human lung fibroblast cells. Finally, the pore forming capability of the three most potent compounds were tested using tethered bilayer lipid membrane (tBLM) technology.