Impact of Conjugation of the Reactive Oxygen Species (ROS)-Generating Catalytic Moiety with Membrane-Active Antimicrobial Peptoids: Promoting Multitarget Mechanism and Enhancing Selectivity.

Antimicrobial peptides (AMPs) represent promising therapeutic modalities against multidrug-resistant bacterial infections. As a mimic of natural AMPs, peptidomimetic oligomers like peptoids (i.e., oligo-N-substituted glycines) have been utilized for antimicrobials with resistance against proteolytic degradation. Here, we explore the conjugation of catalytic metal-binding motifs─the amino terminal Cu(II) and Ni(II) binding (ATCUN) motif─with cationic amphipathic antimicrobial peptoids to enhance their efficacy. Upon complexation with Cu(II) or Ni(II), the conjugates catalyzed hydroxyl radical generation, and 22 and 22-Cu exhibited over 10-fold improved selectivity compared to the parent peptoid, likely due to reduced hydrophobicity. Cu-ATCUN-peptoids caused bacterial membrane disruption, aggregation of intracellular biomolecules, DNA oxidation, and lipid peroxidation, promoting multiple killing mechanisms. In a mouse sepsis model, 22 demonstrated antimicrobial and anti-inflammatory efficacy with low toxicity. This study suggests a strategy to improve the potency of membrane-acting antimicrobial peptoids by incorporating ROS-generating motifs, thereby adding oxidative damage as a killing mechanism.

Real-Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label-Free Imaging with Optical Diffraction Tomography.

Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug-resistant bacteria. As a mimic of AMPs, peptoids with N-substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure-activity relationship (SAR) of a library of indole side chain-containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label-free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real-time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance-breaking novel antibiotic drug.

Structure-activity relationship analysis of activity-based probes targeting HTRA family of serine proteases.

High temperature requirement A serine proteases (HTRA) are ubiquitously expressed and participate in protein quality control and cellular stress responses. They are linked to several clinical illnesses, including bacterial infection, cancer, age-related macular degeneration, and neurodegenerative diseases. In addition, several recent studies have revealed HTRAs as important biomarkers and potential therapeutic targets, necessitating the development of an effective detection method to evaluate their functional states in various disease models. We developed a new series of HTRA-targeting activity-based probes with enhanced subtype selectivity and reactivity. In conjunction with our previously developed tetrapeptide probes, we established the structure-activity relationship of the new probes for different HTRA subtypes. Our probes are cell-permeable and have potent inhibitory effects against HTRA1 and HTRA2, making them valuable for identifying and validating HTRAs as an important biomarker.

Helicity Modulation Improves the Selectivity of Antimicrobial Peptoids.

The modulation of conformational flexibility in antimicrobial peptides (AMPs) has been investigated as a strategy to improve their efficacy against bacterial pathogens while reducing their toxicity. Here, we synthesized a library of helicity-modulated antimicrobial peptoids by the position-specific incorporation of helix-inducing monomers. The peptoids displayed minimal variations in hydrophobicity, which permitted the specific assessment of the effect of conformational differences on antimicrobial activity and selectivity. Among the moderately helical peptoids, the most dramatic increase in selectivity was observed in peptoid 17, providing more than a 20-fold increase compared to fully helical peptoid 1. Peptoid 17 had potent broad-spectrum antimicrobial activity that included clinically isolated multi-drug-resistant pathogens. Compared to pexiganan AMP, 17 showed superior metabolic stability, which could potentially reduce the dosage needed, alleviating toxicity. Dye-uptake assays and high-resolution imaging revealed that the antimicrobial activity of 17 was, as with many AMPs, mainly due to membrane disruption. However, the high selectivity of 17 reflected its unique conformational characteristics, with differential interactions between bacterial and erythrocyte membranes. Our results suggest a way to distinguish different membrane compositions solely by helicity modulation, thereby improving the selectivity toward bacterial cells with the maintenance of potent and broad-spectrum activity.

Activity-Based Probes for the High Temperature Requirement A Serine Proteases.

The high temperature requirement A (HTRA) family of serine proteases mediates protein quality control. These proteins process misfolded proteins in several diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). While their structures and activation mechanisms have been studied, the precise details of the regulation of their activity under physiological conditions have not been completely elucidated, partly due to the lack of suitable chemical probes. In the present study, we developed novel activity-based probes (ABPs) targeting the HTRAs and demonstrated their utility in the monitoring and quantification of changes in enzyme activity in live cells. Using our probes, we found the activity of HTRA1 to be highly elevated in an AD-like cell-based model. We also observed the active HTRA2 in live cells by using a mitochondrion-targeted probe. We believe that our probes can serve as a useful tool to study the role of human HTRAs in neurodegenerative diseases.

Mitochondrion-Targeting Peptides and Peptidomimetics: Recent Progress and Design Principles.

Mitochondria are multifunctional subcellular organelles whose operations encompass energy production, signal transduction, and metabolic regulation. Given their wide range of roles, they have been studied extensively as a potential therapeutic target for the treatment of various diseases, including cancer, diabetes, and neurodegenerative diseases. Mitochondrion-mediated pathways have been identified as promising targets in the context of these diseases. However, the delivery of specific probes and drugs to the mitochondria is one of the major problems that remains to be solved. Over the past decade, much effort has been devoted to developing mitochondrion-targeted delivery methods based on the membrane characteristics and the protein import machinery of mitochondria. While various methods utilizing small molecules to polymeric particles have been introduced, it is notable that many of these compounds share common structural elements and physicochemical properties for optimal selectivity and efficiency. In this Perspective, we will review the most recently developed mitochondrion-targeting peptides and peptidomimetics to outline the key aspects of structural requirements and design principles. We will also discuss successful and potential applications of mitochondrial delivery to assess opportunities and challenges in the targeting of mitochondria.

Development of DNA Aptamers against the Nucleocapsid Protein of Severe Fever with Thrombocytopenia Syndrome Virus for Diagnostic Application: Catalytic Signal Amplification using Replication Protein A-Conjugated Liposomes.

Most prevalent infectious diseases worldwide are caused by mediators such as insects and characterized by high mortality and morbidity, thereby creating a global public health concern. Therefore, a sensitive, selective detection platform for diagnosing diseases in the early stages of infection is needed to prevent disease spread and to protect public health. Here, we developed novel DNA aptamers specific to the nucleocapsid protein (NP) of the severe fever with thrombocytopenia syndrome (SFTS) virus and synthesized ssDNA-binding protein-conjugated liposomes encapsulated with horseradish peroxidase (HRP) for application in a simple and universal platform. This platform achieved highly sensitive detection of the NP by measuring the colorimetric signal following lysis of the HRP encapsulated liposomes, mediated by a mixture of 3,3',5,5'-tetramethylbenzidine and H2O2 solution. The limit of detection was 0.009 ng·mL-1, and NP was successfully detected in diluted human serum with a high recovery rate. Moreover, this method was specific and did not exhibit cross-reactivity among NPs of other virus types. These results demonstrated the efficacy of the proposed method as a highly sensitive, specific, and universal diagnostic tool for potential application in monitoring of the early stages of infectious diseases.

Mitochondria-Targeting Peptoids.

Mitochondria-specific delivery methods offer a valuable tool for studying mitochondria-related diseases and provide breakthroughs in therapeutic development. Although several small-molecule and peptide-based transporters have been developed, peptoids, proteolysis-resistant peptidomimetics, are a promising alternative to current approaches. We designed a series of amphipathic peptoids and evaluated their cellular uptake and mitochondrial localization. Two peptoids with cyclohexyl residues demonstrated highly efficient cell penetration and mitochondrial localization without significant adverse effects on the cells and mitochondria. These mitochondria-targeting peptoids could facilitate the selective and robust targeted delivery of bioactive compounds, such as drugs, antioxidants, and photosensitizers, with minimal off-target effects.

Development of a smart activity-based probe to detect subcellular activity of asparaginyl endopeptidase in living cells.

We developed a smart activity-based probe that detects the activity of asparaginyl endopeptidase (AEP) in live cells to monitor the dynamics of enzyme regulation. The newly designed probe generated a turn-on fluorescence signal in response to the activity of AEP in living cells without compromising the labelling efficiency or selectivity. Our probe closely reflected the enzyme activity in its native state, detecting subcellular AEP activity in colon cancer cells and neuronal cells.

Postsynthetic modification of peptoids via the Suzuki-Miyaura cross-coupling reaction.

We developed a new method for modifying the side chains of peptoids on a solid phase resin, employing the palladium-catalyzed Suzuki-Miyaura cross-coupling reaction. Optimized conditions using Pd(PPh3 )4 and K2 CO3 in the presence of Buchwald's SPhos ligand provided a high conversion in the coupling reaction. The usefulness of this method was demonstrated by synthesis of a two pyrene-conjugated peptoid helix, which exhibited an interesting excimer formation.