Application of enzyme-linked immunosorbent assay to detect antimicrobial peptides in human intestinal lumen.

Intestinal transplantation is the definitive treatment for intestinal failure. However, tissue rejection and graft-versus-host disease are relatively common complications, necessitating aggressive immunosuppression that can itself pose further complications. Tracking intraluminal markers in ileal effluent from standard ileostomies may present a noninvasive and sensitive way to detect developing pathology within the intestinal graft. This would be an improvement compared to current assessments, which are limited by poor sensitivity and specificity, contributing to under or over-immunosuppression, respectively, and by the need for invasive biopsies. Herein, we report an approach to reproducibly analyze ileal fluid obtained through stoma sampling for antimicrobial peptide/protein concentrations, reasoning that these molecules may provide an assessment of intestinal homeostasis and levels of intestinal inflammation over time. Concentrations of lysozyme (LYZ), myeloperoxidase (MPO), calprotectin (S100A8/A9) and ß-defensin 2 (DEFB2) were assessed using adaptations of commercially available enzyme-linked immunosorbent assays (ELISAs). The concentration of α-defensin 5 (DEFA5) was assessed using a newly developed sandwich ELISA. Our data support that with proper preparation of ileal effluent specimens, precise and replicable determination of antimicrobial peptide/protein concentrations can be achieved for each of these target molecules via ELISA. This approach may prove to be reliable as a clinically useful assessment of intestinal homeostasis over time for patients with ileostomies.

A heterodimer of α and ß hemoglobin chains functions as an innate anticancer agent.

Metastatic (as well as tumor) microenvironments contain both cancer-promoting and cancer-restraining factors. The balance between these opposing forces determines the fate of cancer cells that disseminate to secondary organ sites. In search for microenvironmental drivers or inhibitors of metastasis, we identified, in a previous study, the beta subunit of hemoglobin (HBB) as a lung-derived antimetastatic factor. In the present study, exploring mechanisms regulating melanoma brain metastasis, we discovered that brain-derived factors restrain proliferation and induce apoptosis and necrosis of brain-metastasizing melanoma cells. Employing various purification procedures, we identified a heterodimer composed of hemoglobin alpha and beta chains that perform these antimetastatic functions. Neither the alpha nor the beta subunit alone was inhibitory. An alpha/beta chain dimer chemically purified from human hemoglobin inhibited the cell viability of primary melanomas, melanoma brain metastasis (MBM), and breast cancer cell lines. The dimer-induced DNA damage, cell cycle arrest at the SubG1 phase, apoptosis, and significant necrosis in four MBM cell lines. Proteomic analysis of dimer-treated MBM cells revealed that the dimer downregulates the expression of BRD4, GAB2, and IRS2 proteins, playing crucial roles in cancer cell sustainability and progression. Thus, we hypothesize that the hemoglobin dimer functions as a resistance factor against brain-metastasizing cancer cells.

Interferon ɛ restricts Zika virus infection in the female reproductive tract.

Interferon ɛ (IFNɛ) is a unique type I IFN that has been implicated in host defense against sexually transmitted infections. Zika virus (ZIKV), an emerging pathogen, can infect the female reproductive tract (FRT) and cause devastating diseases, particularly in pregnant women. How IFNɛ contributes to protection against ZIKV infection in vivo is unknown. In this study, we show that IFNɛ plays a critical role in host protection against vaginal ZIKV infection in mice. We found that IFNɛ was expressed not only by epithelial cells in the FRT but also by immune and stromal cells at baseline or after exposure to viruses or specific Toll-like receptor (TLR) agonists. IFNɛ-deficient mice exhibited abnormalities in the epithelial border and underlying tissue in the cervicovaginal tract, and these defects were associated with increased susceptibility to vaginal but not subcutaneous ZIKV infection. IFNɛ deficiency resulted in an increase in magnitude, duration, and depth of ZIKV infection in the FRT. Critically, intravaginal administration of recombinant IFNɛ protected Ifnɛ-/- mice and highly susceptible Ifnar1-/- mice against vaginal ZIKV infection, indicating that IFNɛ was sufficient to provide protection even in the absence of signals from other type I IFNs and in an IFNAR1-independent manner. Our findings reveal a potentially critical role for IFNɛ in mediating protection against the transmission of ZIKV in the context of sexual contact.

Interferon ε restricts Zika virus infection in the female reproductive tract.

Interferon ε (IFNε) is a unique type I IFN that has been implicated in host defense against sexually transmitted infections (STIs). Zika virus (ZIKV), an emerging pathogen, can infect the female reproductive tract (FRT) and cause devastating diseases, particularly in pregnant women. How IFNε contributes to protection against ZIKV infection in vivo is unknown. Here, we show that IFNε plays a critical role in host protection against vaginal ZIKV infection in mice. We found that IFNε was expressed not only by epithelial cells in the FRT, but also by certain immune and other cells at baseline or after exposure to viruses or specific TLR agonists. IFNε-deficient mice exhibited abnormalities in the epithelial border and underlying tissue in the cervicovaginal tract, and these defects were associated with increased susceptibility to vaginal, but not subcutaneous ZIKV infection. IFNε-deficiency resulted in an increase in magnitude, duration, and depth of ZIKV infection in the FRT. Critically, intravaginal administration of recombinant IFNε protected Ifnε-/- mice and highly susceptible Ifnar1-/- mice against vaginal ZIKV infection, indicating that IFNε was sufficient to provide protection even in the absence of signals from other type I IFNs and in an IFNAR1-independent manner. Our findings reveal a potentially critical role for IFNε in mediating protection against transmission of ZIKV in the context of sexual contact.

Flagella-driven motility is a target of human Paneth cell defensin activity.

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Configuration-Specific Antibody for Bacterial Heptosylation: An Antiadhesion Therapeutic Strategy.

Alternative antibacterial therapies refractory to existing mechanisms of antibiotic resistance are urgently needed. One such attractive therapy is to inhibit bacterial adhesion and colonization. Ser O-heptosylation (Ser O-Hep) on autotransporters of Gram-negative bacteria is a novel glycosylation and has been proven to be essential for bacterial colonization. Herein, we chemically synthesized glycopeptides containing this atypical glycan structure and an absolute C6 configuration through the assembly of Ser O-Hep building blocks. Using glycopeptides as haptens, we generated first-in-class poly- and monoclonal antibodies, termed Anti-SerHep1a and Anti-SerHep1b, that stereoselectively recognize Ser O-heptosylation (d/l-glycero) with high specificity in vitro and in vivo. Importantly, these antibodies effectively blocked diffusely adhering Escherichia coli 2787 adhesion to HeLa cells and in mice in a dose- and Ser O-Hep-dependent manner. Together, these antibodies represent not only useful tools for the discovery of unknown serine O-heptosylated proteins bearing various C6 chiral centers but also a novel class of antiadhesion therapeutic agents for the treatment of bacterial infection.

Mouse α-Defensins: Structural and Functional Analysis of the 17 Cryptdin Isoforms Identified from a Single Jejunal Crypt.

Mouse α-defensins, better known as cryptdins, are host protective antimicrobial peptides produced in the intestinal crypt by Paneth cells. To date, more than 20 cryptdin mRNAs have been identified from mouse small intestine, of which the first six cryptdins (Crp1 to Crp6) have been isolated and characterized at the peptide level. We quantified bactericidal activities against Escherichia coli and Staphylococcus aureus of the 17 cryptdin isoforms identified by Ouellette and colleagues from a single jejunal crypt (A. J. Ouellette et al., Infect Immun 62:5040-5047, 1994), along with linearized analogs of Crp1, Crp4, and Crp14. In addition, we analyzed the most potent and weakest cryptdins in the panel with respect to their ability to self-associate in solution. Finally, we solved, for the first time, the high-resolution crystal structure of a cryptdin, Crp14, and performed molecular dynamics simulation on Crp14 and a hypothetical mutant, T14K-Crp14. Our results indicate that mutational effects are highly dependent on cryptdin sequence, residue position, and bacterial strain. Crp14 adopts a disulfide-stabilized, three-stranded ß-sheet core structure and forms a noncanonical dimer stabilized by asymmetrical interactions between the two ß1 strands in parallel. The killing of E. coli by cryptdins is generally independent of their tertiary and quaternary structures that are important for the killing of S. aureus, which is indicative of two distinct mechanisms of action. Importantly, sequence variations impact the bactericidal activity of cryptdins by influencing their ability to self-associate in solution. This study expands our current understanding of how cryptdins function at the molecular level.

Design of stapled peptide-based PROTACs for MDM2/MDMX atypical degradation and tumor suppression.

Rationale: Although stapled peptides offer a powerful solution to overcome the susceptibility of linear peptides to proteolytic degradation and improve their ability to cross membranes, an efficient and durable disease treatment strategy has not yet been developed due to the inevitable elimination of peptide inhibitors and rapid accumulation of target proteins. Methods: Herein we developed stapled peptide-based proteolysis-targeting chimeras (SP-PROTACs), that simultaneously exhibited improved cellular uptake and proteolytic stability attributed to the stapled peptides, and efficient target protein degradation promoted by the PROTACs. Based on the PMI peptide with dual specificity for both MDM2 and MDMX, a series of SP-PROTACs were designed. Results: Among them, the optimized SPMI-HIF2-1 exhibited similar binding affinity with MDM2 and MDMX but obviously higher helical contents, improved proteolytic stability, better cellular permeability, and a better pharmacokinetic profile compared with its linear counterpart. Importantly, SPMI-HIF2-1 could effectively kill cancer cells and inhibit tumor progression in subcutaneous and orthotopic colorectal cancer xenograft models through simultaneously promoting the atypical degradation of both MDM2 and MDMX and durable p53 activation. An FP-based binding assay and structural modeling analysis of the ternary complex suggested that SPMI-HIF2-1 simultaneously bound with the target protein and E3 ligase. Conclusion: Our findings not only provide a new class of anticancer drug candidates, but also bridge the gap and reduce the physical distance between peptides and PROTACs.

Virulence Factors of Pseudomonas Aeruginosa and Antivirulence Strategies to Combat Its Drug Resistance.

Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections in severely ill and immunocompromised patients. Ubiquitously disseminated in the environment, especially in hospitals, it has become a major threat to human health due to the constant emergence of drug-resistant strains. Multiple resistance mechanisms are exploited by P. aeruginosa, which usually result in chronic infections difficult to eradicate. Diverse virulence factors responsible for bacterial adhesion and colonization, host immune suppression, and immune escape, play important roles in the pathogenic process of P. aeruginosa. As such, antivirulence treatment that aims at reducing virulence while sparing the bacterium for its eventual elimination by the immune system, or combination therapies, has significant advantages over traditional antibiotic therapy, as the former imposes minimal selective pressure on P. aeruginosa, thus less likely to induce drug resistance. In this review, we will discuss the virulence factors of P. aeruginosa, their pathogenic roles, and recent advances in antivirulence drug discovery for the treatment of P. aeruginosa infections.

Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection.

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1ß and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Human defensin-inspired discovery of peptidomimetic antibiotics.

SignificanceWe report the development of peptidomimetic antibiotics derived from a natural antimicrobial peptide, human α-defensin 5. By engaging multiple bacterial targets, the lead compound is efficacious in vitro and in vivo against bacteria with highly inducible antibiotic resistance, promising a useful therapeutic agent for the treatment of infections caused by antibiotic-resistant bacteria.

Inhibition of SARS-CoV-2 Infection by Human Defensin HNP1 and Retrocyclin RC-101.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 pandemic. The emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that can inhibit the virus. One component of the innate immune system with broad antipathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The ability of defensins to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of proteins with high conformational plasticity. We found that human neutrophil α-defensin HNP1 binds to SARS-CoV-2 Spike protein with submicromolar affinity that is more than 20 fold stronger than its binding to serum albumin. As such, HNP1, as well as a θ-defensin retrocyclin RC-101, both interfere with Spike-mediated membrane fusion, Spike-pseudotyped lentivirus infection, and authentic SARS-CoV-2 infection in cell culture. These effects correlate with the abilities of the defensins to destabilize and precipitate Spike protein and inhibit the interaction of Spike with the ACE2 receptor. Serum reduces the anti-SARS-CoV-2 activity of HNP1, though at high concentrations, HNP1 was able to inactivate the virus even in the presence of serum. Overall, our results suggest that defensins can negatively affect the native conformation of SARS-CoV-2 Spike, and that α- and θ-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.

Defensins: The natural peptide antibiotic.

Defensins are a family of cationic antimicrobial peptides active against a broad range of infectious microbes including bacteria, viruses and fungi, playing important roles as innate effectors and immune modulators in immunological control of microbial infection. Their antibacterial properties and unique mechanisms of action have garnered considerable interest in developing defensins into a novel class of natural antibiotic peptides to fend off pathogenic infection by bacteria, particularly those resistant to conventional antibiotics. However, serious pharmacological and technical obstacles, some of which are unique to defensins and others are common to peptide drugs in general, have hindered the development and clinical translation of defensins as anti-infective therapeutics. To overcome them, several technologies have been developed, aiming for improved functionality, prolonged circulation time, enhanced proteolytic stability and bioavailability, and efficient and controlled delivery and release of defensins to the site of infection. Additional challenges include the alleviation of potential toxicity of defensins and their cost-effective manufacturing. In this review, we briefly introduce defensin biology, focus on various transforming strategies and practical techniques developed for defensins and their derivatives as antibacterial therapeutics, and conclude with a summation of future challenges and possible solutions.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression.

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.

Human Defensins Inhibit SARS-CoV-2 Infection by Blocking Viral Entry.

Innate immunity during acute infection plays a critical role in the disease severity of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and is likely to contribute to COVID-19 disease outcomes. Defensins are highly abundant innate immune factors in neutrophils and epithelial cells, including intestinal Paneth cells, and exhibit antimicrobial and immune-modulatory activities. In this study, we investigated the effects of human α- and ß-defensins and RC101, a θ-defensin analog, on SARS-CoV-2 infection. We found that human neutrophil peptides (HNPs) 1-3, human defensin (HD) 5 and RC101 exhibited potent antiviral activity against pseudotyped viruses expressing SARS-CoV-2 spike proteins. HNP4 and HD6 had weak anti-SARS-CoV-2 activity, whereas human ß-defensins (HBD2, HBD5 and HBD6) had no effect. HNP1, HD5 and RC101 also inhibited infection by replication-competent SARS-CoV-2 viruses and SARS-CoV-2 variants. Pretreatment of cells with HNP1, HD5 or RC101 provided some protection against viral infection. These defensins did not have an effect when provided post-infection, indicating their effect was directed towards viral entry. Indeed, HNP1 inhibited viral fusion but not the binding of the spike receptor-binding domain to hACE2. The anti-SARS-CoV-2 effect of defensins was influenced by the structure of the peptides, as linear unstructured forms of HNP1 and HD5 lost their antiviral function. Pro-HD5, the precursor of HD5, did not block infection by SARS-CoV-2. High virus titers overcame the effect of low levels of HNP1, indicating that defensins act on the virion. HNP1, HD5 and RC101 also blocked viral infection of intestinal and lung epithelial cells. The protective effects of defensins reported here suggest that they may be useful additives to the antivirus arsenal and should be thoroughly studied.

Human Beta-Defensin 2 and 3 Inhibit HIV-1 Replication in Macrophages.

Human beta-defensins (hBDs) are broad-spectrum antimicrobial peptides, secreted by epithelial cells of the skin and mucosae, and astrocytes, which we and others have shown to inhibit HIV-1 in primary CD4+ T cells. Although loss of CD4+ T cells contributes to mucosal immune dysfunction, macrophages are a major source of persistence and spread of HIV and also contribute to the development of various HIV-associated complications. We hypothesized that, besides T cells, hBDs could protect macrophages from HIV. Our data in primary human monocyte-derived macrophages (MDM) in vitro show that hBD2 and hBD3 inhibit HIV replication in a dose-dependent manner. We determined that hBD2 neither alters surface expression of HIV receptors nor induces expression of anti-HIV cytokines or beta-chemokines in MDM. Studies using a G-protein signaling antagonist in a single-cycle reporter virus system showed that hBD2 suppresses HIV at an early post-entry stage via G-protein coupled receptor (GPCR)-mediated signaling. We find that MDM express the shared chemokine-hBD receptors CCR2 and CCR6, albeit at variable levels among donors. However, cell surface expression analyses show that neither of these receptors is necessary for hBD2-mediated HIV inhibition, suggesting that hBD2 can signal via additional receptor(s). Our data also illustrate that hBD2 treatment was associated with increased expression of APOBEC3A and 3G antiretroviral restriction factors in MDM. These findings suggest that hBD2 inhibits HIV in MDM via more than one CCR thus adding to the potential of using ß-defensins in preventive and therapeutic approaches.

Human intelectin-1 (ITLN1) genetic variation and intestinal expression.

Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan ß-D-galactofuranose or protein-protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.

A nano-predator of pathological MDMX construct by clearable supramolecular gold(I)-thiol-peptide complexes achieves safe and potent anti-tumor activity.

As alternatives to small-molecular proteolysis-targeting chimeras (PROTAC), peptide-based molecular glues (MG) are a broad range of dual-functional ligands that simultaneously bind with targetable proteins and E3 ligases by mimicking proteinprotein interaction (PPI) partners. Methods: Herein, we design a peptide-derived MG to target a tumor-driving protein, MDMX, for degradation, and nanoengineered it into a supramolecular gold(I)-thiol-peptide complex (Nano-MP) to implement the proteolysis recalcitrance, cellular internalization, and glutathione-triggered release. To optimize the tumor targeting, a pH-responsive macromolecule termed polyacryl sulfydryl imidazole (PSI) was synthesized to coat Nano-MP. Results: As expected, Nano-MP@PSI induced the MDMX degradation by ubiquitination and subsequently restored the anti-cancer function of p53 and p73. Nano-MP@PSI revealed potent anti-cancer activities in an orthotopic xenograft mouse model of retinoblastoma by intraocular injection and a patient-derived xenograft model of malignant pancreatic cancer by systemic injection, while maintaining a favorable safety profile and showing a highly favorable clearable profile of excretion from the living body. Conclusion: Collectively, this work not only provided a clinically viable paradigm for the treatment of a wide variety of tumors by multiple administration types, but, more importantly, it bridged the chasm between peptides and PROTACs, and likely reinvigorated the development of peptide-derived proteolysis-targeting chimeras for a great variety of diseases.

Mechanism through Which Retrocyclin Targets Flavivirus Multiplication.

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

Nanodefensin-encased hydrogel with dual bactericidal and pro-regenerative functions for advanced wound therapy.

Background: Host defense peptides (HDPs) have emerged as a novel therapeutic paradigm for wound management; however, their clinical applications remain a challenge owing to their poor pharmacological properties and lack of suitable pharmaceutical formulations. Nanodefensin (ND), a nanoengineered human α-defensin 5 (HD5), has shown improved pharmacological properties relative to the parent compound. In this study, we engineered a nanodefensin-encased hydrogel (NDEFgel), investigated the effects of NDEFgel on wound healing, and elucidated underlying mechanisms. Method: ND was chemically synthesized and tested functions by in vitro antimicrobial and scratch assays and western blotting. Different NDEFgels were evaluated by in vitro characterizations including degradation, drug release and antimicrobial activity. In full-thickness excisional murine models, the optimal NDEFgel was directly applied onto wound sites, and the efficacy was assessed. Moreover, the underlying mechanisms of pro-regenerative effect developed by NDEFgel were also explored. Results: Apart from bactericidal effects, ND modulated fibroblast behaviors by promoting migration and differentiation. Among the tested hydrogels, the Pluronic F127 (Plu) hydrogel represented the most desirable carrier for ND delivery owing to its favorable controlled release and compatibility with ND. Local treatment of NDEFgel on the wound bed resulted in accelerated wound regeneration and attenuated bacterial burden. We further demonstrated that NDEFgel therapy significantly upregulated genes related to collagen deposition and fibroblasts, and increased the expression of myofibroblasts and Rac1. We therefore found that Rac1 is a critical factor in the ND-induced modulation of fibroblast behaviors in vitro through a Rac1-dependent cytoskeletal rearrangement. Conclusion: Our results indicate that NDEFgel may be a promising dual-action therapeutic option for advanced wound management in the future.