Simulation-Guided Rational de Novo Design of a Small Pore-Forming Antimicrobial Peptide.

In the age of failing small-molecule antibiotics, tapping the near-infinite structural and chemical repertoire of antimicrobial peptides (AMPs) offers one of the most promising routes toward developing next-generation antibacterial compounds. One of the key impediments en route is the lack of methodologies for systematic rational design and optimization of new AMPs. Here we present a new simulation-guided rational design approach and apply it to develop a potent new AMP. We show that unbiased atomic detail molecular dynamics (MD) simulations are able to predict structures formed by evolving peptide designs enabling structure-based rational fine-tuning of functional properties. Starting from a 14-residue poly leucine template we demonstrate the design of a minimalistic potent new AMP. Consisting of only four types of amino acids (LDKA), this peptide forms large pores in microbial membranes at very low peptide-to-lipid ratios (1:1000) and exhibits low micromolar activity against common Gram-positive and Gram-negative pathogenic bacteria. Remarkably, the four amino acids were sufficient to encode preferential poration of bacterial membranes with negligible damage to red blood cells at bactericidal concentrations. As the sequence is too short to span cellular membranes, pores are formed by stacking of channels in each bilayer leaflet.

Ebola Virus Delta Peptide Is a Viroporin.

The Ebola virus (EBOV) genome encodes a partly conserved 40-residue nonstructural polypeptide, called the delta peptide, that is produced in abundance during Ebola virus disease (EVD). The function of the delta peptide is unknown, but sequence analysis has suggested that delta peptide could be a viroporin, belonging to a diverse family of membrane-permeabilizing small polypeptides involved in replication and pathogenesis of numerous viruses. Full-length and conserved C-terminal delta peptide fragments permeabilize the plasma membranes of nucleated cells of rodent, dog, monkey, and human origin; increase ion permeability across confluent cell monolayers; and permeabilize synthetic lipid bilayers. Permeabilization activity is completely dependent on the disulfide bond between the two conserved cysteines. The conserved C-terminal portion of the peptide is biochemically stable in human serum, and most serum-stable fragments have full activity. Taken together, the evidence strongly suggests that Ebola virus delta peptide is a viroporin and that it may be a novel, targetable aspect of Ebola virus disease pathology.IMPORTANCE During the unparalleled West African outbreak of Ebola virus disease (EVD) that began in late 2013, the lack of effective countermeasures resulted in chains of serial infection and a high mortality rate among infected patients. A better understanding of disease pathology is desperately needed to develop better countermeasures. We show here that the Ebola virus delta peptide, a conserved nonstructural protein produced in large quantities by infected cells, has the characteristics of a viroporin. This information suggests a critical role for the delta peptide in Ebola virus disease pathology and as a possible target for novel countermeasures.

A novel, tomographic imaging probe for rapid diagnosis of fungal keratitis.

Fungal keratitis is a leading cause of ocular morbidity and blindness in developing countries. Diagnosing fungal keratitis currently relies on a comparative evaluation of corneal biopsy or scraping using a direct microscopy and culture results. These methods not only carry the risk of developing complications due to the invasive tissue sampling but also are largely limited by diagnostic speed and accuracy, making it difficult to initiate timely appropriate antifungal therapy. Therefore, rapid and noninvasive diagnostic tools are a pressing need for improved outcomes for fungal keratitis. Taking advantage of the highly specific fungal cell targeting properties of caspofungin, we have developed a fluorescent chemical probe with high selectivity against fungal pathogens. Utilizing fluorescence imaging technology, we have demonstrated a highly specific and sensitive detection of Aspergillus in a fungal keratitis model in mice as early as 5 min post-topical application of the probe. Our results indicate that a fluorescence-mediated platform can be used as a rapid (<10 min) alternative to conventional methods for detecting Aspergillus, and potentially other fungi, in fungal infections of the cornea.

First principles design of a core bioenergetic transmembrane electron-transfer protein.

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

pH-Triggered, Macromolecule-Sized Poration of Lipid Bilayers by Synthetically Evolved Peptides.

pH-triggered membrane-permeabilizing peptides could be exploited in a variety of applications, such as to enable cargo release from endosomes for cellular delivery, or as cancer therapeutics that selectively permeabilize the plasma membranes of malignant cells. Such peptides would be especially useful if they could enable the movement of macromolecules across membranes, a rare property in membrane-permeabilizing peptides. Here we approach this goal by using an orthogonal high-throughput screen of an iterative peptide library to identify peptide sequences that have the following two properties: (i) little synthetic lipid membrane permeabilization at physiological pH 7 at high peptide concentration and (ii) efficient formation of macromolecule-sized defects in synthetic lipid membranes at acidic pH 5 and low peptide concentration. The peptides we selected are remarkably potent macromolecular sized pore-formers at pH 5, while having little or no activity at pH 7, as intended. The action of these peptides likely relies on tight coupling between membrane partitioning, α-helix formation, and electrostatic repulsions between acidic side chains, which collectively drive a sharp pH-triggered transition between inactive and active configurations with apparent pKa values of 5.5-5.8. This work opens new doors to developing applications that utilize peptides with membrane-permeabilizing activities that are triggered by physiologically relevant decreases in pH.

Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides.

In this work, we sought to rationally design membrane-active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane-active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6, by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macromolecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH sensitivity into its sequence. We conclude that the properties of pHlip, GALA, and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane-active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field.

Absorption and folding of melittin onto lipid bilayer membranes via unbiased atomic detail microsecond molecular dynamics simulation.

Unbiased molecular simulation is a powerful tool to study the atomic details driving functional structural changes or folding pathways of highly fluid systems, which present great challenges experimentally. Here we apply unbiased long-timescale molecular dynamics simulation to study the ab initio folding and partitioning of melittin, a template amphiphilic membrane active peptide. The simulations reveal that the peptide binds strongly to the lipid bilayer in an unstructured configuration. Interfacial folding results in a localized bilayer deformation. Akin to purely hydrophobic transmembrane segments the surface bound native helical conformer is highly resistant against thermal denaturation. Circular dichroism spectroscopy experiments confirm the strong binding and thermostability of the peptide. The study highlights the utility of molecular dynamics simulations for studying transient mechanisms in fluid lipid bilayer systems. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

A High-Capacitance Salt-Free Dielectric for Self-Healable, Printable, and Flexible Organic Field Effect Transistors and Chemical Sensor.

Printable and flexible electronics attract sustained attention for their low cost, easy scale up, and potential application in wearable and implantable sensors. However, they are susceptible to scratching, rupture, or other damage from bending or stretching due to their "soft" nature compared to their rigid counterparts (Si-based electronics), leading to loss of functionality. Self-healing capability is highly desirable for these "soft" electronic devices. Here, a versatile self-healing polymer blend dielectric is developed with no added salts and it is integrated into organic field transistors (OFETs) as a gate insulator material. This polymer blend exhibits an unusually high thin film capacitance (1400 nF cm -2 at 120 nm thickness and 20-100 Hz). Furthermore, it shows pronounced electrical and mechanical self-healing behavior, can serve as the gate dielectric for organic semiconductors, and can even induce healing of the conductivity of a layer coated above it together with the process of healing itself. Based on these attractive properties, we developed a self-healable, low-voltage operable, printed, and flexible OFET for the first time, showing promise for vapor sensing as well as conventional OFET applications.

The electrical response of bilayers to the bee venom toxin melittin: evidence for transient bilayer permeabilization.

Melittin is a 26-residue bee venom peptide that folds into amphipathic α-helix and causes membrane permeabilization via a mechanism that is still disputed. While an equilibrium transmembrane pore model has been a central part of the mechanistic dialogue for decades, there is growing evidence that a transmembrane pore is not required for melittin's activity. In part, the controversy is due to limited experimental tools to probe the bilayer's response to melittin. Electrochemical impedance spectroscopy (EIS) is a technique that can reveal details of molecular mechanism of peptide activity, as it yields direct, real-time measurements of membrane resistance and capacitance of supported bilayers. In this work, EIS was used in conjunction with vesicle leakage studies to characterize the response of bilayers of different lipid compositions to melittin. Experiments were carried out at low peptide to lipid ratios between 1:5000 and 1:100. The results directly demonstrate that the response of the bilayer to melittin at these concentrations cannot be explained by an equilibrium transmembrane pore model.

Highly efficient macromolecule-sized poration of lipid bilayers by a synthetically evolved peptide.

Peptides that self-assemble, at low concentration, into bilayer-spanning pores which allow the passage of macromolecules would be beneficial in multiple areas of biotechnology. However, there are few, if any, natural or designed peptides that have this property. Here we show that the 26-residue peptide "MelP5", a synthetically evolved gain-of-function variant of the bee venom lytic peptide melittin identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers under conditions where melittin and other pore-forming peptides do not. In surface-supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at peptide:lipid ratios as low as 1:25000. The increase in bilayer conductance due to MelP5 is dramatically higher, per peptide, than the increase due to the parent sequence of melittin or other peptide pore formers. Here we also develop two novel assays for macromolecule leakage from vesicles, and we use them to characterize MelP5 pores in bilayers. We show that MelP5 allows the passage of macromolecules across vesicle membranes at peptide:lipid ratios as low as 1:500, and under conditions where neither osmotic lysis nor gross vesicle destabilization occur. The macromolecule-sized, equilibrium pores formed by MelP5 are unique as neither melittin nor other pore-forming peptides release macromolecules significantly under the same conditions. MelP5 thus appears to belong to a novel functional class of peptide that could form the foundation of multiple potential biotechnological applications.

WITHDRAWN: Photobuforin II, a fluorescent photoswitchable peptide.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.bbadva.2023.100106. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal.

Photobuforin II, a fluorescent photoswitchable peptide.

Antimicrobial peptide buforin II translocates across the cell membrane and binds to DNA. Its sequence is identical to a portion of core histone protein H2A making it a highly charged peptide. Buforin II has a proline residue in the middle of its sequence that creates a helix-hinge-helix motif which has been found to play a key role in its ability to translocate across the cell membrane. To explore the structure-function relationship of this proline residue this study has replaced P11 with a meta-substituted azobenzene amino acid (Z). The resultant peptide, photobuforin II, retained the secondary structure and membrane activity of the naturally occurring peptide while gaining new spectroscopic properties. Photobuforin II can be isomerized from its trans to cis isomer upon irradiation with ultra-violet (UV) light and from its cis to trans isomer upon irradiation with visible (VL). Photobuforin II is also fluorescent with an emission peak at 390 nm. The intrinsic fluorescence of the peptide was used to determine binding to the membrane and to DNA. VL-treated photobuforin II has a 2-fold lower binding constant compared to UV-treated photobuforin and causes 11-fold more membrane leakage in 3:1 POPC:POPG vesicles. Photobuforin II provides insights into the importance of structure function relationships in membrane active peptides while also demonstrating that azobenzene can be used in certain peptide sequences to produce intrinsic fluorescence.

Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development.

Cryptococcus neoformans is a major fungal pathogen that often causes life-threatening meningitis in immunocompromised populations. This yeast pathogen is highly resistant to the echinocandin drug caspofungin. Previous studies showed that Cryptococcus lipid translocase (flippase) is required for the caspofungin resistance of that fungus. Mutants with a deleted subunit of lipid flippase, Cdc50, showed increased sensitivity to caspofungin. Here we designed an antifungal peptide targeting the P4-ATPase function. We synthesized stable peptides based on the Cdc50 loop region to identify peptides that can sensitize caspofungin by blocking flippase function and found that myristylated peptides based on the "AS15 sequence" was effective at high concentrations. A modified peptide, "AW9-Ma" showed a MIC of 64 µg/mL against H99 wild type and a fractional inhibitory concentration (FIC) index value of 0.5 when used in combination with caspofungin. Most notably, in the presence of the AW9-Ma peptide, C. neoformans wild type was highly sensitive to caspofungin with a MIC of 4 µg/mL, the same as the cdc50Δ mutant. Further assays with flow cytometry showed inhibition of the lipid flippase enzyme activity and significant accumulation of phosphatidylserine on the cell membrane surface. Using a fluorescently labeled peptide, we confirmed that the peptide co-localized with mCherry-tagged P4-ATPase protein Apt1 in C. neoformans. Structure-activity relationship studies of the AW9 sequence showed that two lysine residues on the peptide are likely responsible for the interaction with the P4-ATPase, hence critical for its antifungal activity. IMPORTANCE The authors have developed a lead compound peptide antifungal drug targeting a protein from the organism Cryptococcus neoformans. Binding of the drug to the target fungal protein causes charged lipid molecules to be retained on the surface. This peptide works in synergy with the existing antifungal drug caspofungin. Echinocandin drugs like caspofungin are one of the few classes of existing antifungals. Due to the high concentrations needed, caspofungin is rarely used to treat C. neoformans infections. The authors believe that their new compound provides a way to lower the concentration of caspofungin needed to treat such infections, thus opening the possibility for greater utility of these antifungal.

Alternative Antibiotics in Dentistry: Antimicrobial Peptides.

The rise of antibiotic resistant bacteria due to overuse and misuse of antibiotics in medicine and dentistry is a growing concern. New approaches are needed to combat antibiotic resistant (AR) bacterial infections. There are a number of methods available and in development to address AR infections. Dentists conventionally use chemicals such as chlorohexidine and calcium hydroxide to kill oral bacteria, with many groups recently developing more biocompatible antimicrobial peptides (AMPs) for use in the oral cavity. AMPs are promising candidates in the treatment of (oral) infections. Also known as host defense peptides, AMPs have been isolated from animals across all kingdoms of life and play an integral role in the innate immunity of both prokaryotic and eukaryotic organisms by responding to pathogens. Despite progress over the last four decades, there are only a few AMPs approved for clinical use. This review summarizes an Introduction to Oral Microbiome and Oral Infections, Traditional Antibiotics and Alternatives & Antimicrobial Peptides. There is a focus on cationic AMP characteristics and mechanisms of actions, and an overview of animal-derived natural and synthetic AMPs, as well as observed microbial resistance.

Substituting azobenzene for proline in melittin to create photomelittin: A light-controlled membrane active peptide.

In this article we present the synthesis and characterization of a new form of the membrane active peptide melittin: photomelittin. This peptide was created by substituting the proline residue in melittin for a synthetic azobenzene amino acid derivative. This azobenzene altered the membrane activity of the peptide while retaining much of the secondary structure. Furthermore, the peptide demonstrates added light-dependent activity in leakage assays. There is a 1.5-fold increase in activity when exposed to UV light as opposed to visible light. The peptides further exhibit light-dependent hemolytic activity against human red blood cells. This will enable future studies optimizing photomelittin and other azobenzene-containing membrane active peptides for uses in medicine, drug delivery, and other biotechnological applications.

Applications and evolution of melittin, the quintessential membrane active peptide.

Melittin, the main venom component of the European Honeybee, is a cationic linear peptide-amide of 26 amino acid residues with the sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-NH2. Melittin binds to lipid bilayer membranes, folds into amphipathic α-helical secondary structure and disrupts the permeability barrier. Since melittin was first described, a remarkable array of activities and potential applications in biology and medicine have been described. Melittin is also a favorite model system for biophysicists to study the structure, folding and function of peptides and proteins in membranes. Melittin has also been used as a template for the evolution of new activities in membranes. Here we overview the rich history of scientific research into the many activities of melittin and outline exciting future applications.

Synergies with and Resistance to Membrane-Active Peptides.

Membrane-active peptides (MAPs) have long been thought of as the key to defeating antimicrobial-resistant microorganisms. Such peptides, however, may not be sufficient alone. In this review, we seek to highlight some of the common pathways for resistance, as well as some avenues for potential synergy. This discussion takes place considering resistance, and/or synergy in the extracellular space, at the membrane, and during interaction, and/or removal. Overall, this review shows that researchers require improved definitions of resistance and a more thorough understanding of MAP-resistance mechanisms. The solution to combating resistance may ultimately come from an understanding of how to harness the power of synergistic drug combinations.

A Novel, Rapid, and Low-Volume Assay for Therapeutic Drug Monitoring of Posaconazole and Other Long-Chain Azole-Class Antifungal Drugs.

Clinicians need a better way to accurately monitor the concentration of antimicrobials in patient samples. In this report, we describe a novel, low-sample-volume method to monitor the azole-class antifungal drug posaconazole, as well as certain other long-chain azole-class antifungal drugs in human serum samples. Posaconazole represents an important target for therapeutic drug monitoring (TDM) due to its widespread use in treating invasive fungal infections and well-recognized variability of pharmacokinetics. The current "gold standard" requires trough and peak monitoring through high-pressure liquid chromatography (HPLC) or liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Other methods include bioassays that use highly susceptible strains of fungi in culture plates or 96-well formats to monitor concentrations. Currently, no method exists that is both highly accurate in detecting free drug concentrations and is also rapid. Herein, we describe a new method using reduced graphene oxide (rGO) and a fluorescently labeled aptamer, which can accurately assess clinically relevant concentrations of posaconazole and other long-chain azole-class drugs in little more than 1 h in a total volume of 100 µl.IMPORTANCE This work describes an effective assay for TDM of long-chain azole-class antifungal drugs that can be used in diluted human serum samples. This assay will provide a quick, cost-effective method for monitoring concentrations of drugs such as posaconazole that exhibit well-documented pharmacokinetic variability. Our rGO-aptamer assay has the potential to improve health care for those struggling to treat fungal infections in rural or resource-limited setting.

An Aptamer-Based Biosensor for the Azole Class of Antifungal Drugs.

This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring. Modified synthetic evolution of ligands through exponential enrichment (SELEX) was used to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding to its target molecule, as shown through fluorescence anisotropy-based binding measurements. Experiments using circular dichroism spectroscopy revealed a unique G-quadruplex structure that was essential and specific for binding to the azole antifungal target. Aptamer-functionalized graphene field effect transistor (GFET) devices were created and used to measure the strength of binding of azole antifungals to this surface. In total, this aptamer and the supporting sensing platform provide a valuable tool for therapeutic drug monitoring of patients with invasive fungal infections. IMPORTANCE We have developed the first aptamer directed toward the azole class of antifungal drugs and a functional biosensor for these drugs. This aptamer has a unique secondary structure that allows it to bind to highly hydrophobic drugs. The aptamer works as a capture component of a graphene field effect transistor device. These devices can provide a quick and easy assay for determining drug concentrations. These will be useful for therapeutic drug monitoring of azole antifungal drugs, which is necessary to deal with the complex drug dosage profiles.