Rationally designed PMAP-23 derivatives with enhanced bactericidal and anticancer activity based on the molecular mechanism of peptide–membrane interactions

Antimicrobial peptides (AMPs) are a crucial component of the natural defense system that the host employs to protect itself against invading pathogens. PMAP-23, a cathelicidin-derived AMP, has potent and broad-spectrum antimicrobial activity. Our earlier studies led us to hypothesize that PMAP-23 adopts a dynamic helix–hinge–helix structure, initially attaching to membrane surfaces through the N-helix and subsequently inserting the C-helix into the lipid bilayer. Here, we rationally designed PMAP-NC with increased amphipathicity and hydrophobicity in the N- and C-helix, respectively, based on the hypothesis of the interaction of PMAP-23 with membranes. Compared to the parental PMAP-23, PMAP-NC showed two–eightfold improved bactericidal activity against both Gram-positive and Gram-negative strains with fast killing kinetics. Fluorescence studies demonstrated that PMAP-NC largely disrupted membrane integrity, indicating that efficiency and kinetics of bacterial killing are associated with the membrane permeabilization. Interestingly, PMAP-NC exhibited much better anticancer activity against tumor cells than PMAP-23 but displayed low hemolytic activity against human erythrocytes. Collectively, our findings suggest that PMAP-NC, with the structural arrangement of an amphipathic helix–hinge–hydrophobic helix that plays a critical role in rapid and efficient membrane permeabilization, can be an attractive candidate for novel antimicrobial and/or anticancer drugs.


Introduction
Antimicrobial peptides (AMPs) produced by a variety of animals and plants have been recognized as critical components in host innate immune defenses against invading pathogens (Haney et al. 2019;Hilchie et al. 2013;Zhang and Gallo 2016).AMPs have broad-spectrum bacteriostatic and/or bactericidal effects against Gram-positive and Gram-negative species.Importantly, because of the low risk of developing antimicrobial resistance, AMPs are considered as a promising alternative antibiotic group to overcome the emergence of widespread antibiotic resistance (Pfalzgraff et al. 2018;Mahlapuu et al. 2016;Lohner 2016;Biswaro et al. 2018;de Breij et al. 2018;Kumar et al. 2018;Browne et al. 2020).It is commonly thought that the primary mode of action for AMPs is their interaction with the cytoplasmic membrane of target pathogens, which causes membrane permeabilization and ultimately leads to cell death (Stone et al. 2019;Guha et al. 2019;Sani and Separovic 2016;Matsuzaki 2019).AMPs share common structural features of being cationic and amphipathic, allowing them to bind to and permeabilize the negatively charged bacterial phospholipids, such as phosphatidylglycerol (PG).In addition, several AMPs have been reported to exhibit selective anticancer activity because the outer leaflet of the plasma membrane in cancer cells contains higher levels of negatively charged phosphatidylserine (PS) than in normal cells (Bevers and Williamson 2016;Gaspar et al. 2013).Therefore, the interaction studies of AMPs with bacterial and cancer cell membranes are essential to clarify the mode of antibacterial and anticancer action (Tossi 2011;Fjell et al. 2011;Scott et al. 2008;Yang et al. 2006d;Lee et al. 2019).Particularly, since the membrane permeabilization is closely related to effective antimicrobial and anticancer activity, understanding the factors regulating membrane permeabilization caused by AMPs may lead to new antimicrobial and anticancer drugs with improved potency and selectivity.
The predominant family of AMPs found in mammals is derived from cathelicidins (Kosciuczuk et al. 2012;Zanetti 2005).A porcine cathelicidin, PMAP-23 exerts antimicrobial activity against a broad range of bacteria and certain fungi but does not cause cytotoxicity against human erythrocytes (Liu et al. 2020(Liu et al. , 2021;;Veldhuizen et al. 2017).Earlier nuclear magnetic resonance and circular dichroism studies have shown that PMAP-23 shows an unordered structure in aqueous buffer solution but forms a helix-hinge-helix conformation in membrane-mimetic environments (Park et al. 2002).Previous studies by our research group have demonstrated that the PXXP motif located in central region is crucial for conferring structural flexibility and amphipathicity to PMAP-23, which plays a crucial role in its antimicrobial potency and selectivity by preferential interaction with negatively charged membranes (Yang et al. 2006a).The central PXXP motif is inferred to assist the N-terminal helix for electrostatic bonding to the membrane surface and the C-terminal helix for hydrophobic insertion into the interior of lipid bilayers (Yang et al. 2006a).We further investigated the physicochemical properties of the central PXXP motif by substituting two Pro residues by the α-helix former Ala or α-helix breaker Gly and demonstrated that the PXXP motif is essential for PMAP-23 translocation across artificial lipid membranes (Yang et al. 2021).Although the antimicrobial efficacy of PMAP-23 is related to a complex mode of action beyond simply membrane permeabilization, understanding PMAP-23 interactions with lipid bilayers at the molecular level is of great importance for antimicrobial and anticancer drug development.
In this study, based on how PMAP-23 interacts with lipid bilayers at the molecular level, we designed PMAP-NC with a more amphipathic N-terminus and a more hydrophobic C-terminus while retaining the central PXXP hinge and investigated antimicrobial and anticancer activities.In addition, we characterized the hemolytic activity of the peptide to assess its cytotoxicity against human erythrocytes.We found that compared to parental PMAP-23, PMAP-NC had much higher bactericidal and anticancer activity without significant cytotoxicity against human erythrocytes.Physicochemical studies of peptide-lipid interactions revealed that PMAP-NC acted primarily by loss of transmembrane potential and/or disruption of the cytoplasmic membrane.Importantly, the membrane permeability of PMAP-NC was highly dependent on the content of PS, which explains its selectivity between cancer cells and human erythrocytes.We propose that understanding the structural and physicochemical properties of PMAP-NC for rapid and efficient membrane permeabilization can greatly aid in the development of novel therapeutics with antibacterial and/or anticancer activity.

Materials and microorganisms
PMAP-23 and its derivatives were prepared by solid-phase peptide synthesis methods using fluoren-9-yl-methoxycarbonyl (Fmoc) chemistry and validated with reversed-phase high-performance liquid chromatography, as described previously (Yang et al. 2006b(Yang et al. , 2021)).The level of purity of the peptides used in the study was > 98%.N-α-Fmoc amino acids were purchased from Novabiochem (Läufelfingen, Switzerland) and other reagents for peptide synthesis were purchased from Applied Biosystems (Foster City, CA, USA).Fluorescent probes and phospholipids including phosphatidylglycerol (PG), phosphatidylserine (PS) and phosphatidylcholine (PC) were purchased from Invitrogen (Carlsbad, CA, USA) and Avanti Polar Lipids (Alabaster, AL, USA), respectively.Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacterial strains were obtained from the KCTC (Daejeon, South Korea).

Circular dichroism (CD) spectroscopy
The CD spectra of peptides were measured in a Jasco J-810 spectropolarimeter as described previously (Yang et al. 2019).Briefly, peptides dissolved in sodium phosphate buffer and in 30 mM sodium dodecyl sulfate (SDS) micelle were loaded into a 0.1 cm quartz cell and wavelengths were scanned from 190 to 250 nm with 50 nm/min speed, 0.1 nm step resolution, and 1 nm bandwidth.The spectra are presented as the mean residue ellipticity [θ], measured in units of deg⋅cm 2 ⋅dmol −1 as a function of wavelength.

MIC and MBC determination
The antimicrobial activity of the peptides was determined against Gram-positive S. aureus and B. subtilis, and Gramnegative E. coli, and P. aeruginosa by the standard microdilution methods of the Clinical and Laboratory Standard Institute (CLSI) (Shin et al. 2021).Briefly, a colony of bacteria was incubated in Mueller Hinton (MH) broth in a 37 °C shaking incubator for 24 h.An aliquot (50 μL) of the culture was transferred to fresh 10 mL MH broth, grown to reach the mid-logarithmic phase, and diluted with MHB to a bacterial count of 2 × 10 6 CFU ml −1 .A total of 100 μL of bacteria were added to 96-well microtiter plates in the presence of serial dilutions of peptides and incubated overnight at 37 °C.The minimal inhibitory concentration (MIC) was expressed as the lowest concentration of peptides at which the bacteria did not grow by more than 90%.To determine minimal bactericidal concentrations (MBC) of peptides, the culture medium with a concentration lower than MIC was plated onto an MH agar plate and incubated overnight at 37 °C.The MBC refers to the minimum concentration that completely prevented bacterial growth in MH broth.Experiments were conducted in triplicate and repeated thrice.

Time-kill kinetics assay
The time-kill experiments were performed according to CLSI guidelines, as described previously (Lee et al. 2020).Briefly, Gram-positive S. aureus and Gram-negative E. coli grown to the mid-logarithmic phase were diluted in LB broth to obtain a cell density of approximately 2 × 10 6 CFU/mL.PMAP-23 and its derivatives (8 μM) were added to the bacterial cells and incubated at 37 °C.Aliquots (100 μL) were drawn at different time intervals, plated after dilution on LB agar plates, and incubated at 37 °C for 24 h to allow full colony development for calculating the viability of cells.The killing rate was plotted as the log CFU/mL over time.Experiments were repeated thrice and their results were averaged.

Membrane depolarization
The measurement of membrane potential was conducted utilizing the voltage-sensitive fluorescent dye diSC3(5), as described previously (Yang et al. 2006d;Zhang et al. 2000).Briefly, S. aureus was grown to the exponential phase at 37 °C and resuspended at 1 × 10 8 CFU/mL in 20 mM HEPES buffer (100 mM KCl, pH 7.2).Fluorescence intensity was monitored at 670 nm (excited at 622 nm) on an RF-5301 spectrofluorometer (Shimadzu, Tokyo, Japan).The membrane potential of S. aureus was completely dissipated by adding gramicidin D. Experiments were repeated thrice and their results were averaged.

Peptide-induced dye leakage from liposomes
Membrane disruption was investigated using the calcein leakage assay, as described previously (Yang et al. 2019).Briefly, the lipid film composed of PC/PG(1:1), PC/PS(9:1) or PC was hydrated in a solution containing 70 mM calcein in 20 mM HEPES buffer.After extruding the suspension through 100 nm polycarbonate filter, unencapsulated calcein was removed by passing liposomes through a Sephadex G-50 column.To evaluate the effect of peptides on membrane disruption, the release of calcein from the liposomes was measured by examining the fluorescence intensity at 520 nm (excited at 490 nm) using a Shimadzu RF-5301 spectrofluorometer.To determine the percentage of calcein release caused by the peptides, the following equation was used: Leakage (%) = 100 × (F − F 0 )/(F t − F 0 ), where F 0 is the initial fluorescence intensity in the buffer, F and F t are the fluorescence intensities after exposure to the peptide and 0.1% Triton X-100, respectively.Experiments were repeated thrice and their results were averaged.

Peptide translocation
The translocation ability of the peptides across lipid membranes was evaluated using fluorescence transfer from Trp to a dansyl group, as described previously (Lee et al. 2019;Kobayashi et al. 2000).Briefly, to assess the peptides' ability to translocate across lipid bilayers, liposomes (PC/PG/DNS-PE, 50:45:5) containing chymotrypsin were prepared by extrusion, and a trypsin-chymotrypsin inhibitor was added to inactivate extraliposomal enzymes.Using a spectrofluorometer, the transfer of fluorescence from Trp residues of the peptides to a dansyl group in DNS-PE was observed after the peptides were added to the liposome suspension.The Trp residues were excited at 280 nm, leading to fluorescence transfer to the dansyl group in DNS-PE, and the emission was recorded at 510 nm.If the internalized peptide was digested by the enzyme within the liposomes, reduced fluorescence would be observed after peptide addition.

Tryptophan (Trp) fluorescence
Trp fluorescence measurements were used to estimate the binding of peptides to membranes, as described previously (Yang et al. 2006c).Briefly, small unilamellar vesicles (SUVs) were generated by sonication and the peptides were added to the vesicles at a peptide/lipid molar ratio of 1:100.The mixture was then incubated for 10 min to allow for interaction.The Trp emission spectra of peptides were recorded at 300-400 nm, while acrylamide was introduced to quench the Trp fluorescence.The Stern-Volmer constant (K SV ) was determined using the formula where [Q] represents the concentration of acrylamide, F 0 and F are Trp fluorescence measured in the absence and presence of acrylamide, respectively.Experiments were repeated thrice and their results were averaged.

Cell viability assay
Cell viability was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously (Yang et al. 2019(Yang et al. , 2009)).Briefly, the A549 (human lung cancer) and MDA361 (human breast cancer) cell lines were cultured in RPMI-1640 medium with antibiotics and 10% FBS.These cells were seeded in 96-well microplates at a density of 2 × 10 4 cells per well in DMEM with 10% FBS and incubated for 24 h under 5% CO 2 at 37 °C.After that, 20 µL of peptide solution (diluted serially twofold in DMEM) was added to each well and the plates were incubated for 2 days.Then, approximately, 20 µL of MTT solution (5 mg MTT/mL in PBS) was added to each well, followed by a 4-h incubation at 37 °C.The MTT formazan precipitate was dissolved in 50 µL of 20% SDS containing 10 mM HCl, and the percent cell viability was determined by calculating the ratio of the absorbance at 570 nm of cells treated with peptide to untreated cells.Experiments were conducted thrice and their results were averaged.

Cytotoxicity against human erythrocytes
To determine the hemolytic activity of the peptides against human erythrocytes, the release of hemoglobin was measured after incubation with various peptide concentrations, as described previously (Yang et al. 2006c).Briefly, fresh human erythrocytes were collected by centrifugation (1500 × g) for 10 min, washed twice with PBS (pH 7.2), and diluted to a concentration of 4% in the buffer.Erythrocytes were introduced to a 96-well plate in the presence of peptides and incubated at 37 °C for 1 h.The plates were then centrifuged at 1000 × g for 5 min, and the resulting supernatant was transferred to a new 96-well plate.The hemolytic activity of the peptides was evaluated using an ELISA microplate reader to measure the absorbance of the supernatant at 414 nm, indicative of hemoglobin release.A zero hemolysis control was established using PBS, while 0.1% Triton X-100 resulted in complete (100%) hemolysis.Experiments were repeated thrice and their results were averaged.

Peptide design
Previous studies on the structural characterization of PMAP-23 have shown that PMAP-23 presents a random coil structure in aqueous buffer, whereas it forms a helix-hinge-helix structure in membrane-mimetic environments.Upon interaction of PMAP-23 with lipid bilayers at the molecular level, the N-terminal helix first binds to negatively charged membrane surface and then inserts the C-terminal helix into the hydrophobic membrane core (Fig. 1).In this study, based on the interaction of PMAP-23 with lipid bilayers, we designed derivatives to make the N-helix more amphiphilic for efficient electrostatic binding to negatively charged membranes and the C-helix more hydrophobic for insertion into hydrophobic interiors.PMAP-N was designed to increase amphiphilicity at the N-terminal helix by exchanging the amino acids between Leu5 and Arg10.To increase the hydrophobic property at the C-terminal helix (PMAP-C), the hydrophilic amino acids (Thr19 and Arg23) were substituted with Leu residues.Leu residues are typically buried deeper within the hydrophobic core of lipid bilayers, while Trp residues are often primarily located at the lipid-water interface.The substitution of Leu residues instead of Trp residues facilitates stronger hydrophobic interactions with the lipid bilayer, which is advantageous for promoting membrane permeabilization.PMAP-NC was designed to have both increased amphiphilicity at the N-terminus and increased hydrophobicity at the C-terminus.Since the central hinge plays an important role in the interaction of PMAP-23 with lipid bilayers and antimicrobial potency and selectivity [11,13], we retained the central PXXP motif.As anticipated, PMAP-23 and the designed peptides exhibited similar CD spectra (Fig. S1) and α-helicities (Fig. 1), indicating their adoption of similar secondary structures.The sequences and helical wheel diagrams of PMAP-23 and the designed peptides are shown in Fig. 1.

Antimicrobial activity
Table 1 displays the antibacterial activity of PMAP-23 and its rationally designed derivatives as minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against Gram-positive bacteria, including S. aureus and B. subtilis, as well as Gram-negative bacteria, including E. coli and P. aeruginosa.PMAP-23 exhibited activity with MIC values in the 4-8 µM range and MBC values in the 16-64 µM range against the tested strains of bacteria.Importantly, PMAP-NC was found to be the most potent among the peptides against both Gram-positive and Gram-negative bacteria, with MIC values in the 2-4 µM range and MBC values in the 4-8 µM range.The MIC values between PMAP-N and PMAP-C were similar or showed only a twofold difference.In MBC values, however, PMAP-C exerted bactericidal activity about 4 times better than PMAP-N, suggesting that the hydrophobic C-terminal helix is crucial for the bactericidal activity.
To further investigate the differences in their lethal effects, we measured the time-dependent behavior of their bactericidal activity against S. aureus and E. coli, which are examples of Gram-positive and Gram-negative bacteria, respectively.The number (CFUs/ml) of viable bacterial cells trended to decrease with time, after treatment with peptides to the bacteria (Fig. 2).At the 2 × MIC, PMAP-23 and PMAP-N had little immediate impact on the number of surviving CFUs which were reduced by less than only 20% after 120 min of incubation.By contrast, it was observed that PMAP-C and PMAP-NC almost completely killed both bacteria within 120 min.Killing kinetics studies of the peptides on S. aureus and E. coli demonstrated that the hydrophobic C-terminal α-helix has a drastic effect on the rapid killing of the peptides.

Membrane depolarization and permeabilization
The bactericidal activity of AMPs is often associated with membrane depolarization and permeabilization.To investigate whether bacterial killing by PMAP-23 and its derivatives is related to membrane depolarization, the ability to depolarize the cell membrane of S. aureus was evaluated (Fig. 3a).All peptides dissipated the membrane potential in a dose-dependent manner.Interestingly, we found that PMAP-C and PMAP-NC were much higher than PMAP-23 and PMAP-N, indicating that the hydrophobic C-terminal helix has a significant effect on their membrane depolarizing activity.The correlation between bactericidal activity and membrane depolarization of the peptides suggests that loss of membrane potential may be a major contributor to the bactericidal effect.We then generated calcein-containing liposomes composed of PG/PC (1:1) and investigated the ability of the peptides to induce membrane permeabilization by measuring the release of entrapped calcein from the liposomes into an aqueous buffer (Fig. 3b).Similar to their ability to depolarize bacterial membranes, PMAP-23 and PMAP-N induced weak calcein release (~ 30%) from liposomes, whereas PMAP-C and PMAP-NC showed strong membrane-lytic activity (70 ~ 80%), which further confirmed that the C-terminal helix plays important roles in efficient membrane permeabilization.PMAP-NC with the most potent bactericidal activity showed the greatest membrane depolarization and permeabilization, suggesting that membrane disruption is the primary event in bacterial killing for PMAP-NC.The potential intracellular targets associated with the antimicrobial activity of PMAP-23 led us to investigate the peptides' ability to translocate across PC/PG (1:1) liposomes.We accomplished this by monitoring resonance energy transfer from the Trp residues of PAMP-23 and its derivatives to the dansyl-PE, which was incorporated into the liposomes (Fig. 3c).We observed that all peptide efficiently translocated across the lipid bilayers but they exhibited only slight variations in their capacity to translocate across the membrane, indicating that the translocation does not play decisive role in the bactericidal efficacy of the peptides.

Cytotoxicity of PMAP-23 and PMAP-NC against human cancer cells and erythrocytes
We evaluated the anticancer potential of PMAP-23 and PMAP-NC on human cancer cells (MDA361 and human A549) using the MTT assay, and their selectivity was assessed using human erythrocytes.While PMAP-23 was almost inactive, PMAP-NC demonstrated robust anticancer activity against MDA361 and A549 cancer cell lines (Fig. 4a, b).The IC50 (50% inhibitory concentration) value of PMAP-NC against MDA361 and A549 cancer cells was 6.4 μM and 7.1 μM, respectively.Even though PMAP-NC at high concentration (64 μM) elicited 20% release from human erythrocytes, the peptide was considerably more toxic to cancer cells than to human erythrocytes (Fig. 4c).
As cancer cells display exposed negatively charged PS on their surface, we further evaluated the potential contributions of PS in peptide-induced membrane permeability.
The peptides exhibited a comparatively low ability to disturb zwitterionic PC liposomes, which is consistent with the results obtained for their hemolytic activity.Interestingly, we found that consistent with the data from cell-based system, PMAP-23 did not have a noticeable impact on the permeabilization of PS-containing liposomes, whereas PMAP-NC exhibited an increase in permeabilization activity with a rise in PS content (Fig. 4d).These results can potentially clarify why PMAP-NC has selective anticancer properties and also indicate that PMAP-NC may serve as a lead compound for developing anticancer peptides.

Partitioning of peptides into lipid bilayers by Trp fluorescence analysis
All peptides presented in Fig. 1   (1:1), PC/PS (9:1), or PC, at a peptide/lipid molar ratio of 1:100.These vesicles serve as models for bacterial, cancer, or normal cell membranes, respectively.The K SV values were determined by conducting a fluorescence quenching experiment in which a water-soluble fluorescence quenching agent called acrylamide was used.The wavelength maxima range of the peptides in aqueous buffer was 352-353 nm, indicating that Trp residues were hydrophilic in nature.In the presence of PC/PG (1:1) vesicles, all peptides caused a blue shift (10-11 nm) in the emission maximum, indicating that they bind to the negatively charged membranes.In contrast, the addition of peptides to PC liposomes only caused a minor shift (1-3 nm) in the Trp emission maxima.This is due to the low exposure to acrylamide when Trp residues are buried in the lipid bilayer, leading to a low K SV value.
In comparison to PC vesicles, the K SV values of PMAP-23 and its derivatives were lower in PC/PG (1:1), which suggests that the Trp residues of peptides penetrate deeper into the hydrophobic core of lipid membranes.These findings explain why the peptides have a selective antimicrobial effect due to the fact that eukaryotic membranes are mostly made up of zwitterionic phospholipids, while prokaryotic membranes are composed of a combination of negatively charged and zwitterionic phospholipids.Interestingly, in the presence of PC/PS (9:1) vesicles, the Trp emission maxima of PMAP-C and PMAP-NC were more shifted than those of PMAP-23 and PMAP-N, suggesting that the increased hydrophobicity at the C-terminus is essential for the strong and deep insertion into the hydrophobic core of the cancermimicking membrane.Importantly, the lower K SV value of PMAP-NC over PMAP-23 in PC/PS (9:1) vesicles further explains the direct correlation between the binding of peptides to cancer-mimicking membranes and their superior anticancer activity.

Discussion
In the design and development of novel AMPs for the treatment of antibiotic-resistant bacteria, there has been considerable interest in understanding the structure-activity relationship of AMPs and their interactions with lipid bilayers.In general, broad-spectrum antibiotic activities are typically the result of the binding of antimicrobial peptides to target cell membranes, as well as the disruption of these membranes.This binding and disruption are primarily due to the electrostatic and hydrophobic interactions that occur between the peptides and the bacterial membranes (Wimley 2010;Lee et al. 2016).The helix-hinge-helix motif is a critical structural element found in potent AMPs, underscoring its importance in their antimicrobial activity and suggesting its potential as a design principle for the development of novel therapeutic agents against microbial infections (Bhunia et al. 2009(Bhunia et al. , 2010;;Domadia et al. 2010).PMAP-23 adopts amphipathic helix, hinge, and amphipathic helix.The initial binding of PMAP-23 to the anionic membrane surface is mainly due to the N-terminal amphipathic helix.On the other hand, the efficient and rapid insertion of the peptide into the hydrophobic core of the membrane largely depends on the C-terminal hydrophobic helix.The central hinge region, which is an important structural component, enables a separation between the N-terminal electrostatic interaction and the C-terminal hydrophobic interaction with the target cell membranes.Based on the interaction of PMAP-23 with membranes, therefore, we designed PMAP-NC to have a more amphipathic at N-terminus and more hydrophobic at C-terminus.
In bacterial growth inhibition tests, PMAP-23 and PMAP-NC acted similarly against Gram-positive S. aureus and Gram-negative E. coli.In bacterial killing assays, however, PMAP-NC killed both S. aureus and E. coli rapidly, whereas the same concentration of PMAP-23 was negligible even after 120 min of contact with the bacterial strains (Fig. 2).Compared with PMAP-23, PMAP-NC dissipated membrane potential to a greater extent in S. aureus (Fig. 3a).Likewise, PMAP-NC induced greater leakage of calcein from LUVs than PMAP-23 (Fig. 3b).Considering that PMAP-C lead to efficient membrane depolarization and permeabilization and exerted more improved the bactericidal activity than PMAP-N, the insertion of hydrophobic C-terminal helix into the membrane is more critical step than the binding of amphipathic N-terminal helix to the membrane in the process of bactericidal activity.Therefore, the rationally designed PMAP-NC showed the strongest and fastest bactericidal activity due to efficient membrane disruption.
In recent years, AMPs have garnered extensive attention for their potential as a new class of anticancer drugs to overcome tumor resistance to conventional chemotherapy (Gaspar et al. 2013;Jafari et al. 2022;Deslouches and Di 2017).While AMPs are believed to interact with the membrane surface of cancer cells, disrupting their membranes and causing cancer cell death, the structural and physicochemical parameters that determine their anticancer activity are still poorly understood.In order to design anticancer peptides without cytotoxic activity against normal cells, great efforts are being made to understand the difference between normal cells and cancer cells.We showed that PMAP-NC significantly reduced cell viability of cancer cells with low cytotoxicity against human erythrocytes.The membrane permeability of PMAP-NC was dependent on the PS content, which explains the selective anticancer activity.The unique structure of PMAP-23, which forms an amphiphilic N-helix and a hydrophobic C-helix connected by a central hinge, plays a key role in performing selective membrane disruption of PS-containing liposomes.These results may provide a good strategy for developing selective anticancer peptides.
In conclusion, PMAP-23 showed only antibacterial activity, whereas PMAP-NC was toxic to bacterial and cancer cells.PMAP-23 required a delay time for its bactericidal effect, but PMAP-NC was able to kill bacteria rapidly.The fast kinetics of bactericidal activity caused by PMAP-NC are linked to efficient membrane depolarization and permeability, indicating that membrane disruption is the key event in bacterial killing for PMAP-NC.Comparison of PMAP-N and PMAP-C demonstrated that the hydrophobic C-terminal α-helix was more important for the membrane disruption ability of PMAP-NC than the amphipathic N-terminal α-helix.Importantly, we found that PMAP-NC disrupted PS-containing liposomes as a function of PS concentration, which explains its relatively strong anticancer activity.Taken together, our results suggest that a unique structure with an amphipathic N-helix, a central hinge, and a hydrophobic C-helix is responsible for bactericidal and anticancer activity of PMAP-NC.As the lipopolysaccharide (LPS) outer membrane plays a crucial role in the mode of action of AMPs against Gram-negative bacteria (Bhattacharjya et al. 2022;Bhattacharjya 2016), we intend to investigate this aspect in our future studies.We propose that detailed knowledge of the interaction of peptides with various helix-hinge-helix structures with lipid bilayers may facilitate the design of highly selective and potent AMPs as effective antimicrobial and/or anticancer drugs in the future.
Handling editor: R. Dave.Hyunhee Lee and Sung-Heui Shin have contributed equally to this work.

Fig. 1
Fig. 1 Membrane interaction mode and structural characteristics of PMAP-23.(a) Schematic representation of the interaction of PMAP-23 with lipid bilayers: i) random in buffer; ii) association of the N-terminal α-helix to the outer leaflet; iii) insertion of the C-terminal α-helix into the core of lipid bilayers.(b) Amino acid sequences of

Fig. 3
Fig. 3 Membrane depolarization, disruption, and translocation of PMAP-23 and derivatives.(a) Dose-dependent peptide-induced membrane depolarization of S. aureus.Full membrane depolarization was induced by adding Gramicidin D. (b) Membrane disruption induced by peptides.Peptide-induced membrane disruption is defined

Fig. 4
Fig. 4 Cytotoxicity of peptides against cancer cells and human erythrocytes.Dose-response curves for the anticancer activity against (a) MDA361 (human breast cancer cell line) and (b) A549 (human lung cancer cell line).Cell viability was measured by the MTS assay.(c) Dose-dependent hemolysis of human erythrocytes.(d)The PS-dependent membrane disruption induced by peptides.Peptide-induced membrane disruption is defined as the percent calcein leakage from zwitterionic PC with increasing negatively charged PS concentrations (0, 5, 10, 15, and 20 mol%).Data are shown as mean ± SD ( n = 3)

Table 2
Tryptophan fluorescence emission maxima (λ max ) and Stern-Volmer constants (K SV ) for PMAP-23 and its derivatives in buffer and in