A New Gramicidin S Analogue with Potent Antibacterial Activity and Negligible Hemolytic Toxicity.

Antibiotic resistance is an urgent threat to global health, with the decreasing efficacy of conventional drugs underscoring the urgency for innovative therapeutic strategies. Antimicrobial peptides present as promising alternatives to conventional antibiotics. Gramicidin S is one such naturally occurring antimicrobial peptide that is effective against Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of 4 µg/mL (3.6 µM). Despite this potent activity, its significant hemolytic toxicity restricts its clinical use to topical applications. Herein, we present rational modifications to the key ß-strand and ß-turn regions of gramicidin S to concurrently mitigate hemolytic effects, while maintaining potency. Critically, peptide 9 displayed negligible hemolytic toxicity, while possessing significant antibacterial potency against a panel of methicillin-sensitive and methicillin-resistant S. aureus clinical isolates (MIC of 8 µg/mL, 7.2 µM). Given the substantial antibacterial activity and near absence of cytotoxicity, 9 presents as a potential candidate for systemic administration in the treatment of S. aureus bacteremia/sepsis.

Smart Wearable Patches Using Light-Controlled Activation and Delivery of Photoswitchable Antimicrobial Peptides.

A novel strategy to treat Staphylococcus aureus (S. aureus) skin infections is presented, where UV light is used to facilitate concomitant light-controlled activation and delivery of an antimicrobial therapeutic agent. Specifically, a new photoswitchable gramicidin S analogue was immobilized onto a polymeric wearable patch via a photocleavable linker that undergoes photolysis at the same wavelength of light required for activation of the peptide. Unlike toxic gramicidin S, the liberated active photoswitchable peptide exhibits antimicrobial activity against S. aureus while being ostensibly non-haemolytic to red blood cells. Moreover, irradiation with visible light switches off the antimicrobial properties of the peptide within seconds, presenting an ideal strategy to regulate antibiotic activity for localized bacterial infections with the potential to mitigate resistance.

Designer D-peptides targeting the N-terminal region of α-synuclein to prevent parkinsonian-associated fibrilization and cytotoxicity.

The deposition of α-synuclein (αS) aggregates in the gut and the brain is ever present in cases of Parkinson's disease. While the central non-amyloidogenic-component (NAC) region of αS plays a critical role in fibrilization, recent studies have identified a specific sequence from within the N-terminal region (NTR, residues 36-42) as a key modulator of αS fibrilization. Due to the lack of effective therapeutics which specifically target αS aggregates, we have developed a strategy to prevent the aggregation and subsequent toxicity attributed to αS fibrilization utilizing NTR targeting peptides. In this study, L- and D-isoforms of a hexa- (VAQKTV-Aib, 77-82 NAC) and heptapeptide (GVLYVGS-Aib, 36-42 NTR) containing a self-recognition component unique to αS, as well as a C-terminal disruption element, were synthesized to target primary sequence regions of αS that modulate fibrilization. The D-peptide that targets the NTR (NTR-TP-D) was shown by ThT fluorescence assays and TEM to be the most effective at preventing fibril formation and elongation, as well as increasing the abundance of soluble monomeric αS. In addition, NTR-TP-D alters the conformation of destabilised monomers into a less aggregation-prone state and reduces the hydrophobicity of αS fibrils via fibril remodelling. Furthermore, both NTR-TP isoforms alleviate the cytotoxic effects of αS aggregates in both Neuro-2a and Caco-2 cells. Together, this study highlights how targeting the NTR of αS using D-isoform peptide inhibitors may effectively combat the deleterious effects of αS fibrilization and paves the way for future drug design to utilise such an approach to treat Parkinson's disease.

Mechanically Induced Switching between Two Discrete Conductance States: A Potential Single-Molecule Variable Resistor.

The fabrication of solid-state single-molecule switches with high on-off conductance ratios has been proposed to advance conventional technology in areas such as molecular electronics. Herein, we employed the scanning tunneling microscope break junction (STM-BJ) technique to modulate conductance in single-molecule junctions using mechanically induced stretching. Compound 1a possesses two dihydrobenzothiophene (DHBT) anchoring groups at the opposite ends linked with rigid alkyne side arms to form a gold-molecule-gold junction, while 1b contains 4-pyridine-anchoring groups. The incorporation of ferrocene into the backbone of each compound allows rotational freedom to the cyclopentadienyl (Cp) rings to give two distinct conductance states (high and low) for each. Various control experiments and suspended junction compression/retraction measurements indicate that these high- and low-conductance plateaus are the results of conformational changes within the junctions (extended and folded states) brought about by mechanically induced stretching. A high-low switching factor of 42 was achieved for 1a, whereas an exceptional conductance ratio in excess of 2 orders of magnitude (205) was observed for 1b. To the best of our knowledge, this is the highest experimental on-off conductance switching ratio for a single-molecule junction exploiting the mechanically induced STM-BJ method. Computational studies indicated that the two disparate conductance states observed for 1a and 1b result from mechanically induced conformational changes due to an interplay between conductance and the dihedral angles associated with the electrode-molecule interfaces. Our study reveals the structure-function relationship that determines conductance in such flexible and dynamic systems and promotes the development of a single-molecule variable resistor with high on-off switching factors.

Unravelling Structural Dynamics within a Photoswitchable Single Peptide: A Step Towards Multimodal Bioinspired Nanodevices.

The majority of the protein structures have been elucidated under equilibrium conditions. The aim herein is to provide a better understanding of the dynamic behavior inherent to proteins by fabricating a label-free nanodevice comprising a single-peptide junction to measure real-time conductance, from which their structural dynamic behavior can be inferred. This device contains an azobenzene photoswitch for interconversion between a well-defined cis, and disordered trans isomer. Real-time conductance measurements revealed three distinct states for each isomer, with molecular dynamics simulations showing each state corresponds to a specific range of hydrogen bond lengths within the cis isomer, and specific dihedral angles in the trans isomer. These insights into the structural dynamic behavior of peptides may rationally extend to proteins. Also demonstrated is the capacity to modulate conductance which advances the design and development of bioinspired electronic nanodevices.

Short Photoswitchable Antibacterial Peptides.

Three photoswitchable tetrapeptides, based on a known synthetic antibacterial, were designed and synthesized to determine activity against Staphylococcus aureus. Each peptide contains an azobenzene photoswitch incorporated into either the N-terminal side chain (1), C-terminal side chain (2), or the C-terminus (3) to allow reversible switching between cis- and trans-enriched photostationary states. Biological assays revealed that the C-terminus azobenzene (3) possessed the most potent antibacterial activity, with an MIC of 1 µg/mL. In this study, net positive charge, hydrophobicity, position of the azobenzene, secondary structure, and amphiphilicity were all found to contribute to antibacterial activity, with each of these factors likely facilitating the peptide to disrupt the negatively charged bacterial lipid membrane. Hence, these short photoswitchable antibacterial tetrapeptides provide insights for the future design and synthesis of antibiotics targeting S. aureus.

Constructing Dual-Molecule Junctions to Probe Intermolecular Crosstalk.

Understanding and controlling charge transport across multiple parallel molecules are fundamental to the creation of innovative functional electronic components, as future molecular devices will likely be multimolecular. The smallest possible molecular ensemble to address this challenge is a dual-molecule junction device, which has potential to unravel the effects of intermolecular crosstalk on electronic transport at the molecular level that cannot be elucidated using either conventional single-molecule or self-assembled monolayer (SAM) techniques. Herein, we demonstrate the fabrication of a scanning tunneling microscopy (STM) dual-molecule junction device, which utilizes noncovalent interactions and allows for direct comparison to the conventional STM single-molecule device. STM-break junction (BJ) measurements reveal a decrease in conductance of 10% per molecule from the dual-molecule to the single-molecule junction device. Quantum transport simulations indicate that this decrease is attributable to intermolecular crosstalk (i.e., intermolecular π-π interactions), with possible contributions from substrate-mediated coupling (i.e., molecule-electrode). This study provides the first experimental evidence to interpret intermolecular crosstalk in electronic transport at the STM-BJ level and translates the experimental observations into meaningful molecular information to enhance our fundamental knowledge of this subject matter. This approach is pertinent to the design and development of future multimolecular electronic components and also to other dual-molecular systems where such crosstalk is mediated by various noncovalent intermolecular interactions (e.g., electrostatic and hydrogen bonding).

Rationally designed peptide-based inhibitor of Aß42 fibril formation and toxicity: a potential therapeutic strategy for Alzheimer's disease.

Amyloid beta peptide (Aß42) aggregation in the brain is thought to be responsible for the onset of Alzheimer's disease, an insidious condition without an effective treatment or cure. Hence, a strategy to prevent aggregation and subsequent toxicity is crucial. Bio-inspired peptide-based molecules are ideal candidates for the inhibition of Aß42 aggregation, and are currently deemed to be a promising option for drug design. In this study, a hexapeptide containing a self-recognition component unique to Aß42 was designed to mimic the ß-strand hydrophobic core region of the Aß peptide. The peptide is comprised exclusively of D-amino acids to enhance specificity towards Aß42, in conjunction with a C-terminal disruption element to block the recruitment of Aß42 monomers on to fibrils. The peptide was rationally designed to exploit the synergy between the recognition and disruption components, and incorporates features such as hydrophobicity, ß-sheet propensity, and charge, that all play a critical role in the aggregation process. Fluorescence assays, native ion-mobility mass spectrometry (IM-MS) and cell viability assays were used to demonstrate that the peptide interacts with Aß42 monomers and oligomers with high specificity, leading to almost complete inhibition of fibril formation, with essentially no cytotoxic effects. These data define the peptide-based inhibitor as a potentially potent anti-amyloid drug candidate for this hitherto incurable disease.

Unravelling electron transfer in peptide-cation complexes: a model for mimicking redox centres in proteins.

Metalloproteins are crucial to many biological processes, such as photosynthesis, respiration, and efficient electron transport. Zinc is the most common transition metal found in proteins and is critical for structure, function and stability, however the effects from the electronic properties of a bound zinc ion on electron transfer are not clearly defined. Here, a series of ß-strand and 310-helical peptides, capable of binding Zn2+via suitably positioned His residues, was synthesized and their ability to undergo electron transfer in the presence and absence of Zn2+ studied by electrochemical and computational means. The ß-strand peptide was shown to be conformationally pre-organized, with this geometry maintained on complexation with zinc. Electrochemical studies show a significant increase in charge transport, following binding of the zinc ion to the ß-strand peptide. In contrast, complexation of zinc to the helical peptide disrupts the intramolecular hydrogen bonding network known to facilitate electron transfer and leads to a loss of secondary structure, resulting in a decrease in charge transfer. These experimental and computational studies reveal an interplay, which demonstrates that bound zinc enhances charge transfer by changing the electronic properties of the peptide, and not simply by influencing secondary structure.

A hypoxia-activated antibacterial prodrug.

A prodrug based on a known antibacterial compound is reported to target Staphylococcus aureus and Escherichia coli under reductive conditions. The prodrug was prepared by masking the N-terminus and side chain amines of a component lysine residue as 4-nitrobenzyl carbamates. Activation to liberate the antibacterial was demonstrated on treatment with a model reductant, tin(II) chloride. The bioactivity of 1 was confirmed in antibacterial susceptibility assays whereas prodrug 2 was inactive.

Backbone-Constrained Peptides: Temperature and Secondary Structure Affect Solid-State Electron Transport.

The primary sequence and secondary structure of a peptide are crucial to charge migration, not only in solution (electron transfer, ET), but also in the solid-state (electron transport, ETp). Hence, understanding the charge migration mechanisms is fundamental to the development of biomolecular devices and sensors. We report studies on four Aib-containing helical peptide analogues: two acyclic linear peptides with one and two electron-rich alkene-based side chains, respectively, and two peptides that are further rigidified into a macrocycle by a side bridge constraint, containing one or no alkene. ETp was investigated across Au/peptide/Au junctions, between 80 and 340 K in combination with the molecular dynamic (MD) simulations. The results reveal that the helical structure of the peptide and electron-rich side chain both facilitate the ETp. As temperature increases, the loss of helical structure, change of monolayer tilt angle, and increase of thermally activated fluctuations affect the conductance of peptides. Specifically, room temperature conductance across the peptide monolayers correlates well with previously observed ET rate constants, where an interplay between backbone rigidity and electron-rich side chains was revealed. Our findings provide new means to manipulate electronic transport across solid-state peptide junctions.

Photopharmacological Control of Cyclic Antimicrobial Peptides.

Gramicidin S is a naturally occurring antimicrobial cyclic peptide. Herein, we present a series of cyclic peptides based on gramicidin S that contain an azobenzene photoswitch to reversibly control secondary structure and, hence, antimicrobial activity. 1 H NMR spectroscopy and density functional theory calculations revealed a ß-sheet/ß-turn secondary structure for the cis configuration of each peptide, and an ill-defined conformation for all associated trans structures. The cis-enriched and trans-enriched photostationary states (PSSs) for peptides 1-3 were assayed against Staphylococcus aureus to reveal a clear relationship between well-defined secondary structure, amphiphilicity and optimal antimicrobial activity. Most notably, peptides 2 a and 2 b exhibited a fourfold difference in antimicrobial activity in the cis-enriched PSS over the trans-enriched equivalent. This photopharmacological approach allows antimicrobial activity to be regulated through photochemical control of the azobenzene photoswitch, thereby opening new avenues in the design and synthesis of future antibiotics.

Peptides as Bio-Inspired Electronic Materials: An Electrochemical and First-Principles Perspective.

Molecular electronics is at the forefront of interdisciplinary research, offering a significant extension of the capabilities of conventional silicon-based technology as well as providing a possible stand-alone alternative. Bio-inspired molecular electronics is a particularly intriguing paradigm, as charge transfer in proteins/peptides, for example, plays a critical role in the energy storage and conversion processes for all living organisms. However, the structure and conformation of even the simplest protein is extremely complex, and therefore, synthetic model peptides comprising well-defined geometry and predetermined functionality are ideal platforms to mimic nature for the elucidation of fundamental biological processes while also enhancing the design and development of single-peptide electronic components. In this Account, we first present intramolecular electron transfer within two synthetic peptides, one with a well-defined helical conformation and the other with a random geometry, using electrochemical techniques and computational simulations. This study reveals two definitive electron transfer pathways (mechanisms), the natures of which are dependent on secondary structure. Following on from this, electron transfer within a series of well-defined helical peptides, constrained by either Huisgen cycloaddition, ring-closing metathesis, or a lactam bridge, was determined. The electrochemical results indicate that each constrained peptide, in contrast to a linear counterpart, exhibits a remarkable shift of the formal potential to the positive (>460 mV) and a significant reduction of the electron transfer rate constant (up to 15-fold), which represent two distinct electronic "on/off" states. High-level calculations demonstrate that the additional backbone rigidity provided by the side-bridge constraints leads to an increased reorganization energy barrier, which impedes the vibrational fluctuations necessary for efficient intramolecular electron transfer through the peptide backbone. Further calculations reveal a clear mechanistic transition from hopping to superexchange (tunneling) stemming from side-bridge gating. We then extended our research to fine-tuning of the electronic properties of peptides through both structural and chemical manipulation, to reveal an interplay between electron-rich side chains and backbone rigidity on electron transfer. Further to this, we explored the possibility that the side-bridge constraints present in our synthetic peptides provide an additional electronic transport pathway, which led to the discovery of two distinct forms of quantum interferometer. The effects of destructive quantum interference appear essentially through both the backbone and an alternative tunneling pathway provided by the side bridge in the constrained ß-strand peptide, as evidenced by a correlation between electrochemical measurements and conductance simulations for both linear and constrained ß-strand peptides. In contrast, an interplay between quantum interference effects and vibrational fluctuations is revealed in the linear and constrained 310-helical peptides. Collectively, these exciting findings augment our fundamental knowledge of charge transfer dynamics and kinetics in peptides and also open up new avenues to design and develop functional bio-inspired electronic devices, such as on/off switches and quantum interferometers, for practical applications in molecular electronics.

Photoswitchable peptide-based 'on-off' biosensor for electrochemical detection and control of protein-protein interactions.

Neuronal nitric oxide synthase (nNOS) is an enzyme responsible for catalyzing the production of the crucial cellular signalling molecule, nitric oxide (NO), through its interaction with the PDZ domain of α-syntrophin protein. In this study, a novel light-driven photoswitchable peptide-based biosensor, modelled on the nNOS ß-finger, is used to detect and control its interaction with α-syntrophin. An azobenzene photoswitch incorporated into the peptide backbone allows reversible switching between a trans photostationary state devoid of secondary structure, and a cis photostationary state possessing a well-defined antiparallel ß-strand geometry, as revealed by molecular modelling. Electrochemical impedance spectroscopy (EIS) is used to successfully detect the interaction between the gold electrode bound peptide in its cis photostationary state and a wide range of concentrations of α-syntrophin protein, highlighting both the qualitative and quantitative properties of the sensor. Furthermore, EIS demonstrates that the probe in its random trans photostationary state does not bind to the target protein. The effectiveness of the biosensor is further endorsed by the high thermal stability of the photostationary state of the cis-isomer, and the ability to actively control biomolecular interactions using light. This approach allows detection and control of binding to yield a regenerable on-off biosensor.

The correlation of electrochemical measurements and molecular junction conductance simulations in ß-strand peptides.

Understanding the electronic properties of single peptides is not only of fundamental importance, but it is also paramount to the realization of peptide-based molecular electronic components. Electrochemical and theoretical studies are reported on two ß-strand-based peptides, one with its backbone constrained with a triazole-containing tether introduced by Huisgen cycloaddition (peptide 1) and the other a direct linear analogue (peptide 2). Density functional theory (DFT) and non-equilibrium Green's function were used to investigate conductance in molecular junctions containing peptides 3 and 4 (analogues of 1 and 2). Although the peptides share a common ß-strand conformation, they display vastly different electronic transport properties due to the presence (or absence) of the side-bridge constraint and the associated effect on backbone rigidity. These studies reveal that the electron transfer rate constants of 1 and 2, and the conductance calculated for 3 and 4, differ by approximately one order of magnitude, thus providing two distinctly different conductance states and what is essentially a molecular switch. A definitive correlation of electrochemical measurements and molecular junction conductance simulations is demonstrated using two different charge transfer techniques. This study furthers our understanding of the electronic properties of peptides at the molecular level, which provides an opportunity to fine-tune their molecular orbital energies through suitable structural manipulation.

Unraveling the interplay of backbone rigidity and electron rich side-chains on electron transfer in peptides: the realization of tunable molecular wires.

Electrochemical studies are reported on a series of peptides constrained into either a 310-helix (1-6) or ß-strand (7-9) conformation, with variable numbers of electron rich alkene containing side chains. Peptides (1 and 2) and (7 and 8) are further constrained into these geometries with a suitable side chain tether introduced by ring closing metathesis (RCM). Peptides 1, 4 and 5, each containing a single alkene side chain reveal a direct link between backbone rigidity and electron transfer, in isolation from any effects due to the electronic properties of the electron rich side-chains. Further studies on the linear peptides 3-6 confirm the ability of the alkene to facilitate electron transfer through the peptide. A comparison of the electrochemical data for the unsaturated tethered peptides (1 and 7) and saturated tethered peptides (2 and 8) reveals an interplay between backbone rigidity and effects arising from the electron rich alkene side-chains on electron transfer. Theoretical calculations on ß-strand models analogous to 7, 8 and 9 provide further insights into the relative roles of backbone rigidity and electron rich side-chains on intramolecular electron transfer. Furthermore, electron population analysis confirms the role of the alkene as a "stepping stone" for electron transfer. These findings provide a new approach for fine-tuning the electronic properties of peptides by controlling backbone rigidity, and through the inclusion of electron rich side-chains. This allows for manipulation of energy barriers and hence conductance in peptides, a crucial step in the design and fabrication of molecular-based electronic devices.

The effect of a macrocyclic constraint on electron transfer in helical peptides: a step towards tunable molecular wires.

Two helical peptides, one constrained by a covalent side-chain staple, exhibit vastly different electronic properties despite adopting essentially the same backbone conformation. High level calculations confirm that these differences are due to the additional backbone rigidity imparted by the macrocyclic constraint.