Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease.

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Lipopolysaccharide induces a stromal-epithelial signalling axis in a rat model of chronic periodontitis.

AIM: Lipopolysaccharide is a bacterial virulence factor implicated in chronic periodontitis, which may penetrate the junctional epithelial barrier and basement membrane to insult underlying stroma. We sought to identify lipopolysaccharide-induced global gene expression changes responsible for signalling between stroma and epithelium during disease onset. MATERIALS AND METHODS: Using a rat lipopolysaccharide periodontitis model, junctional epithelium and underlying stromal tissue were separately collected from healthy and diseased animals by laser-capture microdissection and subject to gene expression microarray analysis. Key gene products identified were validated in gingival epithelial and fibroblast cell cultures. RESULTS: Global gene expression patterns distinguishing health versus disease were found in and between both tissue types. In stroma, the most significantly altered gene ontology function group (Z ≥ 4.00) was cytokines, containing most significantly (±2-fold; p < 0.05) upregulated genes amphiregulin, IL1-ß and Fas ligand, all positive, diffusible modulators of the epithelial growth factor receptor pathway. In epithelium, the most significant changes were in downregulated FOS-related antigen-1 gene, somatostatin receptor-2 gene and mucin-4 gene, all negative modulators of the epithelial growth factor receptor pathway. CONCLUSION: These results establish a periodontitis model for studying gene product interactions and suggests that the onset of junctional epithelial disease hyperproliferation involves a concerted stromal-epithelial signalling axis.

Lipopolysaccharide-induced epithelial monoamine oxidase mediates alveolar bone loss in a rat chronic wound model.

Reactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated >/=4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H(2)O(2), as well as MAO/B and tumor necrosis factor (TNF)-alpha levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H(2)O(2) production and TNF-alpha expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H(2)O(2) levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H(2)O(2) and TNF-alpha activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss.

Mechanical induction of an epithelial cell chymase associated with wound edge migration.

Chymase is a chymotrypsin-like serine protease predominantly produced by mast cells. In this study, human cutaneous and gingival keratinocytes, ovary surface epithelia, and a porcine epithelial cell line were assayed by homology-based cloning, and the amplified DNA fragment was identified as a chymase. In vitro, chymase could not be induced by serum or cytokine treatment alone. Chymase was activated 3-fold within 60 min in basal media by scratch wounding cultured monolayers and further potentiated over 10-fold at 18 h by additional serum and cytokine treatment. Chymase activity was cell-associated and found to peak within 24 h of wounding and then steadily decreased as cultures healed, reaching baseline levels before confluence was reestablished. Affinity column purified enzyme effectively degraded fibronectin and was found by Western blot analysis using a human chymase antibody to be of about 30 kDa. Immunostaining revealed chymase activation at the wound edge colocalizing with reactive oxygen species generation. Specifically, chymase activation was attenuated by inhibition of nitric oxide, superoxide, and peroxynitrite. Exogenous peroxynitrite but not hydrogen peroxide also resulted in chymase activation in unwounded monolayers. Disruption of cytoskeletal stress fibers by cytochalasin D attenuated both wound-activated chymase and reactive oxygen species generation. Chymase inhibitor chymostatin reduced the loss of cell-cell contacts and the onset of porcine and human skin epithelial cell migration at the wound edge. This shows that an epithelial chymase is rapidly activated by a ligand-independent mechanism following mechanical stress via cytoskeletal and reactive oxygen species signaling and is associated with the onset of epithelial cell migration.

RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation.

OBJECTIVE: The structural organization of oral soft tissue and its relationship with highly calcified teeth are difficult to preserve unless tissues are decalcified, paraffin embedded and subsequently sectioned. However, enamel decalcification time and its negative impact on RNA integrity makes it difficult to effectively analyse in situ gene expression. This study examined the impact of microwave-enhanced decalcification on processing time, RNA integrity and detection of in situ mRNA expression in hard and soft tissue for cell type specific markers of Keratinocyte growth factor receptor, Scleraxis and Osteonectin. DESIGN: Maxillas and mandibles were obtained from three male Wistar strain rats. Right side tissues were decalcified using a microwave plus 10% EDTA solution (M+) while left side tissues were decalcified in 10% EDTA solution alone (M-). RESULTS: Microwave use reduced decalcification time by up to 50% and had no significant impact on morphology, RNA quality and in situ detection of gene expression relative to the M-group. CONCLUSIONS: In situ RT-PCR gene expression of microwave decalcified paraffin-embedded oral tissues is an effective technique to localize in situ gene expression while maintaining excellent soft and hard tissue architecture.

Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptosis in vitro.

The ability of keratinocyte growth factor 1 to modulate apoptosis in the absence of proliferation was studied in vitro. A HaCaT scrape wound model was developed in which dense monolayers prior to wounding were cultured to quiescence in defined media with hydroxyurea at concentrations that blocked proliferation without loss of cell viability. Scrape wounding was then found to induce apoptosis, originating at the wound edge, but subsequently radiating away over a 24 h period to encompass areas not originally damaged. Keratinocyte growth factor 1 inhibited this radial progression of apoptosis in a concentration-dependent manner up to 20 ng per mL with induced migration present at the wound edge. The extent of this rescue was modulated by the concentration of Ca2+ prior to wounding. In control wound cultures apoptotic bodies were found in cells adjacent to the wound interface but were greatly reduced in keratinocyte-growth-factor-1-treated groups. Keratinocyte growth factor 1 receptor expression was significantly induced within two to three cell widths of the scraped wound edge, at levels far exceeding those found at the leading edge of a nonwounded epithelial sheet. Tumor necrosis factor alpha (1-5 ng per mL) or Escherichia coli lipopolysaccharide (10-50 ng per mL) exacerbated scrape-induced early apoptosis (1-4 h), but was largely ameliorated by coculture with keratinocyte growth factor 1. Keratinocyte growth factor 1 protection was associated with a reduction in both caspase-3 activation and cytokeratin-19 loss. Protected wound edges were also associated with the maintenance of e-cadherin expression and induction of beta1 integrin and actin stress fiber organization. These results suggest that keratinocyte growth factor 1 may play a role in limiting mechanically induced apoptotic processes at the epithelial wound edge in a manner that is distinct from its proliferative function.

Keratinocyte growth factor (KGF)-1 and -2 protein and gene expression in human gingival fibroblasts.

The onset and progression of periodontal disease is associated with significant changes in the epithelial component of the attachment complex. From the early to the advanced stages of periodontal disease increased epithelial cell proliferation, migration and invasion into the surrounding connective tissue takes place. Concomitantly there is a significant increase in proinflammatory cytokine expression in periodontal tissue and quantitative and qualitative changes in the subgingival microflora, including an increase in gram-negative microorganisms. One of the most significant virulence factors of these bacteria is lipopolysaccharide (LPS) connected to the outer membrane. Two important growth factors controlling epithelial behavior are Keratinocyte Growth Factor-1 (KGF-1) and -2 (KGF-2). Connective tissue cells express these growth factors, but only epithelial cells respond to them. We studied the effect of proinflammatory cytokines and LPS on gingival fibroblast expression of KGF-1 and KGF-2 in vitro. Gingival fibroblasts were found to express KGF-1 and -2 in culture but only KGF-1 protein and gene expression was stimulated by serum, in a concentration-dependent manner by proinflammatory cytokines IL-1alpha, IL-1beta, TNF-alpha and IL-6 and LPS isolated from Porphyromonas gingivalis and Escherichia coli. The local increase in proinflammatory cytokine expression and the accumulation of LPS in disease sites may therefore stimulate gingival fibroblast expression of KGF-1. We hypothesize that this local increase in KGF-1 expression may, via a paracrine mechanism, stimulate local epithelial cell proliferation, migration and invasion during the onset and progression of periodontitis.