A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms.

BACKGROUND: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. OBJECTIVES: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. METHODS: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. RESULTS: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. CONCLUSIONS: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.

Penetrating the Blood-Brain Barrier with New Peptide-Porphyrin Conjugates Having anti-HIV Activity.

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide-drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide-porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.

Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.

The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms.

OBJECTIVES: To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms. METHODS: Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths. RESULTS: Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified. CONCLUSIONS: Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.

Characterization of Tachyplesin Peptides and Their Cyclized Analogues to Improve Antimicrobial and Anticancer Properties.

Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic ß-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.

Potential of two delivery systems for nisin topical application to dental plaque biofilms in dogs.

BACKGROUND: Periodontal disease (PD) is caused by the development of a microbial biofilm (dental plaque) in the periodontium, affecting approximately 80% of dogs. Several bacterial species present in the canine oral cavity can be implicated in the development of this disease, including Enterococcus spp. To decrease antibiotic administration, a possible control strategy for dog's enterococcal PD may involve the use of the antimicrobial peptide (AMP) nisin. Nisin's inhibitory activity was evaluated against a collection of previously characterized enterococci obtained from the oral cavity of dogs with PD (n = 20), as well as the potential of a guar-gum gel and a veterinary toothpaste as topical delivery systems for this AMP. The Minimum Inhibitory (MIC) and Bactericidal Concentrations (MBC) and the Minimum Biofilm Eradication (MBEC) and Inhibitory Concentrations (MBIC) were determined for nisin and for the supplemented guar-gum gel. For the supplemented veterinary toothpaste an agar-well diffusion assay was used to evaluate its inhibitory potential. RESULTS: Nisin was effective against all isolates. Independently of being or not incorporated in the guar-gum gel, its inhibitory activity on biofilms was higher, with MBIC (12.46 ± 5.16 and 13.60 ± 4.31 µg/mL, respectively) and MBEC values (21.87 ± 11.33 and 42.34 ± 16.61 µg/mL) being lower than MIC (24.61 ± 4.64 and 14.90 ± 4.10 µg/mL) and MBC (63.09 ± 13.22 and 66.63 ± 19.55 µg/mL) values. The supplemented toothpaste was also effective, showing inhibitory activity against 95% of the isolates. CONCLUSIONS: The inhibitory ability of nisin when incorporated in the two delivery systems was maintained or increased, demonstrating the potential of these supplemented vehicles to be applied to PD control in dogs.

Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.

Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria.

Mining viral proteins for antimicrobial and cell-penetrating drug delivery peptides.

MOTIVATION: The need for more effective and safer pharmaceuticals is a persistent quest. Microbial adaptations create the need to permanently develop new antimicrobials (AMPs), for instance. Similarly, intracellular delivery of drugs is still a challenge and translocation of membranes for drug delivery is an area of intense research. Peptides can be used both as AMP drug leads and drug carrier systems for intracellular delivery. Multifunctional proteins are abundant in viruses but, surprisingly, have never been thoroughly screened for bioactive peptide sequences. RESULTS: Using the AMPA and CellPPD online tools, we have evaluated the propensity of viral proteins to comprise AMP or cell-penetrating peptides (CPPs). Capsid proteins from both enveloped and non-enveloped viruses, and membrane and envelope proteins from enveloped viruses, in a total of 272 proteins from 133 viruses, were screened to detect the presence of potential AMP and CPP sequences. A pool of 2444 and 426 CPP and AMP sequences, respectively, were discovered. The capsids of flaviviruses are the best sources of these peptides reaching more than 80% of CPP sequence coverage per protein. Selected sequences were tested experimentally and validated the results. Overall, this study reveals that viruses form a natural multivalent biotechnological platform still underexplored in drug discovery and the heterogeneous abundance of CPP/AMP sequences among viral families opens new avenues in viral biology research.

Shifting gear in antimicrobial and anticancer peptides biophysical studies: from vesicles to cells.

Despite the intensive study on the mechanism of action of membrane-active molecules such as antimicrobial and anticancer peptides, most of the biophysical work has been performed using artificial model systems, mainly lipid vesicles. The use of these systems allows full control of the experimental parameters, and to obtain molecular-level detail on the action of peptides, the correlation with biological action is intangible. Recently, several biophysical methodologies have been translated to studies using bacterial and cancer cells. Here, we review biophysical studies on the mechanism of action of antimicrobial and anticancer peptides performed directly on cells. The data in these studies allow to correlate vesicle-based and cell-based studies and fill the vesicle-cell interdisciplinary gap.

Cell-penetrating peptides: A tool for effective delivery in gene-targeted therapies.

The current landscapes of novel therapeutic approaches rely mostly on gene-targeted technologies, enabling to fight rare genomic diseases, from infections to cancer and hereditary diseases. Although, reaching the action-site for this novel treatments requires to deliver nucleic acids, or other macromolecules into cells, which may pose difficult tasks to pharmaceutical companies. To overcome this technological limitation, a wide variety of vectors have been developed in the past decades and have proven to be successful in delivering various therapeutics. Cell-penetrating peptides (CPP) have been one of the technologies widely studied and have been increasingly used to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into cells. Despite the already proved huge potential that these peptide-based approaches may suggest, few advances have been put to pharmacological or clinical use. This review will describe the origin, development, and usage of CPP to deliver therapeutic agents into cells, with special emphasis on their current application to gene-therapies. Specifically, we will describe the current trials being conducted to treat cancer, gene disorders, and autoimmune diseases using CPP-based therapies. © 2014 IUBMB Life, 66(3):182-194, 2014.

Antimicrobial hydrogels for the treatment of infection.

The increasing prevalence of microbial infections, especially those associated with impaired wound healing and biomedical implant failure has spurred the development of new materials having antimicrobial activity. Hydrogels are a class of highly hydrated material finding use in diverse medical applications such as drug delivery, tissue engineering, as wound fillers, and as implant coatings, to name a few. The biocompatible nature of many gels make them a convenient starting platform to develop selectively active antimicrobial materials. Hydrogels with antimicrobial properties can be obtained through the encapsulation or covalent immobilization of known antimicrobial agents, or the material itself can be designed to possess inherent antimicrobial activity. In this review we present an overview of antimicrobial hydrogels that have recently been developed and when possible provide a discussion relevant to their mechanism of action.

Peptides as models for the structure and function of viral capsid proteins: Insights on dengue virus capsid.

The structural organization of viral particles is among the most astonishing examples of molecular self-assembly in nature, involving proteins, nucleic acids, and, sometimes, lipids. Proper assembly is essential to produce well structured infectious virions. A great variety of structural arrangements can be found in viral particles. Nucleocapsids, for instance, may display highly ordered geometric shapes or consist in macroscopically amorphous packs of the viral genome. Alphavirus and flavivirus are viral genera that exemplify these extreme cases, the former comprising viral particles structured with a T = 4 icosahedral symmetry, whereas flavivirus capsids have no regular geometry. Dengue virus is a member of flavivirus genus and is used in this article to illustrate how viral protein-derived peptides can be used advantageously over full-length proteins to unravel the foundations of viral supramolecular assemblies. Membrane- and viral RNA-binding data of capsid protein-derived dengue virus peptides are used to explain the amorphous organization of the viral capsid. Our results combine bioinformatic and spectroscopic approaches using two- or three-component peptide and/or nucleic acid and/or lipid systems.

Quantifying molecular partition of cell-penetrating peptide-cargo supramolecular complexes into lipid membranes: optimizing peptide-based drug delivery systems.

One of the major challenges in the drug development process is biodistribution across epithelia and intracellular drug targeting. Cellular membrane heterogeneity is one of the major drawbacks in developing efficient and sustainable drug delivery systems, which brings the need to study their interaction with lipids in order to unravel their mechanisms of action and improve their delivery capacities. Cell penetrating peptides (CPPs) are able to translocate almost any cell membrane carrying cargo molecules. However, different CPP use different entry mechanisms, which are often concentration-dependent and cargo-dependent. Being able to quantify the lipid affinity of CPP is of obvious importance and can be achieved by studying the partition extent of CPP into lipid bilayers. The partition constant (Kp) reflects the lipid-water partition extent. However, all currently available methodologies are only suitable to determine the partition of single molecules into lipid membranes or entities, being unsuitable to determine the partition of bimolecular or higher order supramolecular complexes. We derived and tested a mathematical model to determine the Kp of supramolecular CPP-cargo complexes from fluorescence spectroscopy data, using DNA oligomers as a model cargo. As a proof-of-concept example, the partition extent of two new membrane active peptides derived from dengue virus capsid protein (DENV C protein) with potential CPP properties, in both scenarios (free peptide and complexed with a molecular cargo), were tested. We were able to identify the lipid affinity of these CPP:DNA complexes, thus gaining valuable insights into better CPP formulations.

Quantitative Imaging of the Action of vCPP2319, an Antimicrobial Peptide from a Viral Scaffold, against Staphylococcus aureus Biofilms of a Clinical Isolate.

The formation of biofilms is a common virulence factor that makes bacterial infections difficult to treat and a major human health problem. Biofilms are bacterial communities embedded in a self-produced matrix of extracellular polymeric substances (EPS). In this work, we show that vCPP2319, a polycationic peptide derived from the capsid protein of Torque teno douroucouli virus, is active against preformed Staphylococcus aureus biofilms produced by both a reference strain and a clinical strain isolated from a diabetic foot infection, mainly by the killing of biofilm-embedded bacteria. The direct effect of vCPP2319 on bacterial cells was imaged using atomic force and confocal laser scanning microscopy, showing that the peptide induces morphological changes in bacterial cells and membrane disruption. Importantly, vCPP2319 exhibits low toxicity toward human cells and high stability in human serum. Since vCPP2319 has a limited effect on the biofilm EPS matrix itself, we explored a combined effect with α-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact, α-amylase decreases the density of S. aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimaging data shows that vCPP2319 partially restores biofilm compactness after digestion of the polysaccharides, probably due to electrostatic cross-bridging of the matrix nucleic acids, which explains why α-amylase fails to improve the antibacterial action of the peptide.

Anticancer peptide SVS-1: efficacy precedes membrane neutralization.

Anticancer peptides are polycationic amphiphiles capable of preferentially killing a wide spectrum of cancer cells relative to noncancerous cells. Their primary mode of action is an interaction with the cell membrane and subsequent activation of lytic effects; however, the exact mechanism responsible for this mode of action remains controversial. Using zeta potential analyses we demonstrate the interaction of a small anticancer peptide with membrane model systems and cancer cells. Electrostatic interactions have a pivotal role in the cell killing process, and in contrast to the antimicrobial peptides action cell death occurs without achieving full neutralization of the membrane charge.

Anticancer ß-hairpin peptides: membrane-induced folding triggers activity.

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells electronegative relative to the surface of noncancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but to preferentially fold at the surface of cancer cells, adopting an amphiphilic ß-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charged membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma), and MDA-MB-436 (breast carcinoma). However, the cytotoxicity toward noncancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays, and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells.

In Vivo Evaluation of the Efficacy of a Nisin-Biogel as a New Approach for Canine Periodontal Disease Control.

Periodontal disease (PD) is a common oral disease in dogs. Recent in vitro research revealed that nisin−biogel is a promising compound for canine PD control. In this work, a clinical trial was developed to assess the in vivo efficacy of nisin−biogel in dogs by determining the dental plaque index (DPI), gingivitis index (GI), and periodontal pocket depth (PPD) after dental administration. The biogel's influence on aerobic bacteria counts was also evaluated, as well as its acceptance/adverse effects in dogs. Twenty animals were allocated to one of two groups: a treatment group (TG) subjected to a dental topical application of nisin−biogel for 90 days and a control group (CG) with no treatment. Besides daily monitoring, on day 1 (T0) and at the end of the assay (T90), animals were subjected to blood analysis, periodontal evaluation, dental plaque sampling, scaling, and polishing. Statistical analysis with mixed models showed a significant reduction in mean PPD (estimate = −0.371, p-value < 0.001) and DPI (estimate = −0.146, p-value < 0.05) in the TG animals at T90. A reduction in the GI (estimate = −0.056, p-value > 0.05) was also observed but with no statistical significance. No influence on total bacterial counts was observed, and no adverse effects were detected. The nisin−biogel was revealed to be a promising compound for canine PD control.

Parainfluenza Fusion Peptide Promotes Membrane Fusion by Assembling into Oligomeric Porelike Structures.

Paramyxoviruses are enveloped viruses harboring a negative-sense RNA genome that must enter the host's cells to replicate. In the case of the parainfluenza virus, the cell entry process starts with the recognition and attachment to target receptors, followed by proteolytic cleavage of the fusion glycoprotein (F) protein, exposing the fusion peptide (FP) region. The FP is responsible for binding to the target membrane, and it is believed to play a crucial role in the fusion process, but the mechanism by which the parainfluenza FP (PIFP) promotes membrane fusion is still unclear. To elucidate this matter, we performed biophysical experimentation of the PIFP in membranes, together with coarse grain (CG) and atomistic (AA) molecular dynamics (MD) simulations. The simulation results led to the pinpointing of the most important PIFP amino acid residues for membrane fusion and show that, at high concentrations, the peptide induces the formation of a water-permeable porelike structure. This structure promotes lipid head intrusion and lipid tail protrusion, which facilitates membrane fusion. Biophysical experimental results validate these findings, showing that, depending on the peptide/lipid ratio, the PIFP can promote fusion and/or membrane leakage. Our work furthers the understanding of the PIFP-induced membrane fusion process, which might help foster development in the field of viral entry inhibition.

Targeting Zika Virus with New Brain- and Placenta-Crossing Peptide-Porphyrin Conjugates.

Viral disease outbreaks affect hundreds of millions of people worldwide and remain a serious threat to global health. The current SARS-CoV-2 pandemic and other recent geographically- confined viral outbreaks (severe acute respiratory syndrome (SARS), Ebola, dengue, zika and ever-recurring seasonal influenza), also with devastating tolls at sanitary and socio-economic levels, are sobering reminders in this respect. Among the respective pathogenic agents, Zika virus (ZIKV), transmitted by Aedes mosquito vectors and causing the eponymous fever, is particularly insidious in that infection during pregnancy results in complications such as foetal loss, preterm birth or irreversible brain abnormalities, including microcephaly. So far, there is no effective remedy for ZIKV infection, mainly due to the limited ability of antiviral drugs to cross blood-placental and/or blood-brain barriers (BPB and BBB, respectively). Despite its restricted permeability, the BBB is penetrable by a variety of molecules, mainly peptide-based, and named BBB peptide shuttles (BBBpS), able to ferry various payloads (e.g., drugs, antibodies, etc.) into the brain. Recently, we have described peptide-porphyrin conjugates (PPCs) as successful BBBpS-associated drug leads for HIV, an enveloped virus in which group ZIKV also belongs. Herein, we report on several brain-directed, low-toxicity PPCs capable of targeting ZIKV. One of the conjugates, PP-P1, crossing both BPB and BBB, has shown to be effective against ZIKV (IC50 1.08 µM) and has high serum stability (t1/2 ca. 22 h) without altering cell viability at all tested concentrations. Peptide-porphyrin conjugation stands out as a promising strategy to fill the ZIKV treatment gap.

Using zeta-potential measurements to quantify peptide partition to lipid membranes.

Many cellular phenomena occur on the biomembranes. There are plenty of molecules (natural or xenobiotics) that interact directly or partially with the cell membrane. Biomolecules, such as several peptides (e.g., antimicrobial peptides) and proteins, exert their effects at the cell membrane level. This feature makes necessary investigating their interactions with lipids to clarify their mechanisms of action and side effects necessary. The determination of molecular lipid/water partition constants (K ( p )) is frequently used to quantify the extension of the interaction. The determination of this parameter has been achieved by using different methodologies, such as UV-Vis absorption spectrophotometry, fluorescence spectroscopy and ζ-potential measurements. In this work, we derived and tested a mathematical model to determine the K ( p ) from ζ-potential data. The values obtained with this method were compared with those obtained by fluorescence spectroscopy, which is a regular technique used to quantify the interaction of intrinsically fluorescent peptides with selected biomembrane model systems. Two antimicrobial peptides (BP100 and pepR) were evaluated by this new method. The results obtained by this new methodology show that ζ-potential is a powerful technique to quantify peptide/lipid interactions of a wide variety of charged molecules, overcoming some of the limitations inherent to other techniques, such as the need for fluorescent labeling.