SARS-CoV-2 Mpro oligomerization as a potential target for therapy.

The main protease (Mpro) of SARS-CoV-2 is critical in the virus's replication cycle, facilitating the maturation of polyproteins into functional units. Due to its conservation across taxa, Mpro is a promising target for broad-spectrum antiviral drugs. Targeting Mpro with small molecule inhibitors, such as nirmatrelvir combined with ritonavir (Paxlovid™), which the FDA has approved for post-exposure treatment and prophylaxis, can effectively interrupt the replication process of the virus. A key aspect of Mpro's function is its ability to form a functional dimer. However, the mechanics of dimerization and its influence on proteolytic activity remain less understood. In this study, we utilized biochemical, structural, and molecular modelling approaches to explore Mpro dimerization. We evaluated critical residues, specifically Arg4 and Arg298, that are essential for dimerization. Our results show that changes in the oligomerization state of Mpro directly affect its enzymatic activity and dimerization propensity. We discovered a synergistic relationship influencing dimer formation, involving both intra- and intermolecular interactions. These findings highlight the potential for developing allosteric inhibitors targeting Mpro, offering promising new directions for therapeutic strategies.

Identification of a new genetic variant (G231N, E232T, N235D) of peptidylarginine deiminase from P. gingivalis in advanced periodontitis.

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis.

Ursolic Acid Formulations Effectively Induce Apoptosis and Limit Inflammation in the Psoriasis Models In Vitro.

Psoriasis, a prevalent inflammatory skin disorder affecting a significant percentage of the global population, poses challenges in its management, necessitating the exploration of novel cost-effective and widely accessible therapeutic options. This study investigates the potential of ursolic acid (UA), a triterpenoid known for its anti-inflammatory and pro-apoptotic properties, in addressing psoriasis-related inflammation and keratinocyte hyperproliferation. The research involved in vitro models employing skin and immune cells to assess the effects of UA on psoriasis-associated inflammation. The presented research demonstrates the limiting effects of UA on IL-6 and IL-8 production in response to the inflammatory stimuli and limiting effects on the expression of psoriatic biomarkers S100A7, S100A8, and S100A9. Further, the study reveals promising outcomes, demonstrating UA's ability to mitigate inflammatory responses and hyperproliferation of keratinocytes by the induction of non-inflammatory apoptosis, as well as a lack of the negative influence on other cell types, including immune cells. Considering the limitations of UA's poor solubility, hybrid systems were designed to enhance its bioavailability and developed as hybrid nano-emulsion and bi-gel topical systems to enhance bioavailability and effectiveness of UA. One of them in particular-bi-gel-demonstrated high effectiveness in limiting the pathological response of keratinocytes to pro-psoriatic stimulation; this was even more prominent than with ursolic acid alone. Our results indicate that topical formulations of ursolic acid exhibit desirable anti-inflammatory activity in vitro and may be further employed for topical psoriasis treatment.

Topical Formulations Based on Ursolic Acid-Loaded Nanoemulgel with Potential Application in Psoriasis Treatment.

Psoriasis is a chronic disorder that causes a rash with itchy, scaly patches. It affects nearly 2-5% of the worldwide population and has a negative effect on patient quality of life. A variety of therapeutic approaches, e.g., glucocorticoid topical therapy, have shown limited efficacy with systemic adverse reactions. Therefore, novel therapeutic agents and physicochemical formulations are in constant need and should be obtained and tested in terms of effectiveness and minimization of side effects. For that reason, the aim of our study was to design and obtain various hybrid systems, nanoemulgel-macroemulsion and nanoemulgel-oleogel (bigel), as vehicles for ursolic acid (UA) and to verify their potential as topical formulations used in psoriasis treatment. Obtained topical formulations were characterized by conducting morphological, rheological, texture, and stability analysis. To determine the safety and effectiveness of the prepared ursolic acid carriers, in vitro studies on human keratinocyte cell-like HaCaT cells were performed with cytotoxicity analysis for individual components and each formulation. Moreover, a kinetic study of ursolic acid release from the obtained systems was conducted. All of the studied UA-loaded systems were well tolerated by keratinocyte cells and had suitable pH values and stability over time. The obtained formulations exhibit an apparent viscosity, ensuring the appropriate time of contact with the skin, ease of spreading, soft consistency, and adherence to the skin, which was confirmed by texture tests. The release of ursolic acid from each of the formulations is followed by a slow, controlled release according to the Korsmeyer-Peppas and Higuchi models. The elaborated systems could be considered suitable vehicles to deliver triterpene to psoriatic skin.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Novel inhibitors of HSV-1 protease effective in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen known to cause infections of diverse severity, ranging from mild ulceration of mucosal and dermal tissues to life-threatening viral encephalitis. In most cases, standard treatment with acyclovir is sufficient to manage the disease progression. However, the emergence of ACV-resistant strains drives the need for new therapeutics and molecular targets. HSV-1 VP24 is a protease indispensable for the assembly of mature virions and, as such, constitutes an interesting target for the therapy. In this study, we present novel compounds, KI207M and EWDI/39/55BF, that block the activity of VP24 protease and consequently inhibit HSV-1 infection in vitro and in vivo. The inhibitors were shown to prevent the egress of viral capsids from the cell nucleus and suppress the cell-to-cell spread of the infection. They were also proven effective against ACV-resistant HSV-1 strains. Considering their low toxicity and high antiviral potency, the novel VP24 inhibitors could provide an alternative for treating ACV-resistant infections or a drug to be used in combined, highly effective therapy.

Bestatin as a treatment modality in experimental periodontitis.

BACKGROUND: Chronic periodontitis (CP), the most prevalent dysbiotic bacteria-driven chronic inflammatory disease, is an underestimated global health problem in itself, and due to a causative relationship with other disorders such as cardiovascular diseases or Alzheimer disease. The CP pathogenesis is primarily driven by Porphyromonas gingivalis in humans, and Porphyromonas gulae in dogs. These microorganisms initiate a pathogenic shift in the composition of the tooth-surface microflora. Our objective was to evaluate antimicrobial effects of bestatin, a potential CP drug candidate. METHODS: We evaluated bestatin bacteriostatic efficiency against periodontopathogens in planktonic cultures via microplate assay, and mono- and multispecies oral biofilm models. Neutrophil bactericidal activities, such as phagocytosis, were investigated in vitro using granulocytes isolated from the peripheral blood. The therapeutic efficacy and the immunomodulatory function of bestatin was assessed in a murine model of CP. RESULTS: Bestatin exhibited bacteriostatic activity against both P. gingivalis and P. gulae, and controlled the formation and species composition of the biofilm. We demonstrated that bestatin promotes the phagocytosis of periodontopathogens by neutrophils. Finally, we found that providing bestatin in the animal feed prevented alveolar bone resorption. CONCLUSIONS: We show that in a murine model of CP bestatin not only shifted the biofilm species composition from pathogenic to a commensal one, but also promoted bacteria clearance by immune cells and alleviated inflammation. Taken together, these results suggest that bestatin is a promising drug choice for the treatment and/or prevention of periodontitis and clinical trials are required to fully evaluate its potency.

Bioactive Lavandula angustifolia essential oil-loaded nanoemulsion dressing for burn wound healing. In vitro and in vivo studies.

The aim of this study was to develop a dressing with bioactive lavender in a new form of nanoemulsion, and to verify its biosafety and effectiveness in burn wound healing. As part of this research, the composition of the bioactive carrier of lavender oil in the form of a nanoemulsion obtained using ultrasound was optimised. The mean particle size of the internal phase and polydispersity were determined using the dynamic light scattering method using a Zestasizer NanoZS by Malvern and using cryo-transmission electron microscopy (TEM). These studies confirmed that the selected formulation had a particle size of approximately 180 nm and remained stable over time. The preparation was also subjected to rheological analysis (viscosity approximately 480 mPa·s) and a pH test (approximately 6). A macroemulsion (ME) with the same qualitative composition was developed as a reference. Nanoformulations and MEs were tested for skin penetration using Raman spectroscopy in an in vitro model. Research has shown that both formulations deliver oil to living layers of the skin. Subsequently, studies were conducted to confirm the effect of lavender oil in emulsion systems on the mitigation of the inflammatory reaction and its pro-regenerative effect on the wound healing process in an in vitro cell culture model. The safe concentration of the oil in the emulsion preparation was also determined based on preliminary in vivo tests of skin sensitisation and irritation as well as an hemocompatibility test of the preparation.

Enhanced Biological Activity of a Novel Preparation of Lavandula angustifolia Essential Oil.

Lavandula angustifolia, one of the most popular medicinal plants, is the source of a bioactive essential oil characterized by a wide spectrum of biological activity, e.g., antiseptic, analgesic, and anticancer effects. In dermatology, the oil helps to relieve skin inflammation and exhibit wound healing potential. However, the mechanism of action of the lavender oil depends on its composition, which in turn is dependent on the origin and growing conditions. Our study aimed to compare the composition and proregenerative properties of the commercially-available narrow-leaved lavender oil produced in Provence, France, with the oil obtained from the narrow-leaved lavender cultivated locally in Poland. GC/MS analysis showed that self-manufactured essential oil had lower linalool content than commercial oil (23.2 vs. 40.2%), comparable linalyl acetate content (40.6 vs. 44%), while the proportion of lavandulyl acetate was significantly higher (23.2 vs. 5.5%). To determine the influence of lavender oil on the production of proinflammatory cytokines and proregenerative growth factors, gene expression of the selected signaling molecules by HaCaT cells was investigated using real-time PCR. Results showed a concentration-dependent effect of lavender oils on the production of IL-6, IL-8, and VEGF by the keratinocyte cell line. Finally, the potential of the lavender oil to increase the production of VEGF, the most important angiogenic factor, with the in-house preparation performing significantly better in the in vitro cell models was identified.