Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD ...comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL‐6 confers a protective effect to lesional skin during ICD. How IL‐6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid‐specific knockout of the IL‐6Rα (IL‐6RαΔmyeloid) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant‐8 (JP8) fuel, two well‐characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL‐6RαΔmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro‐inflammatory cytokines IL‐1α and TNF‐α, but reduced expression of chemokine proteins including CCL2‐5, CCL7, CCL11, CXCL1 and CXCL10 in IL‐6RαΔmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL‐6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD.
It is imperative to explore the gigantic available chemical space to identify new scaffolds for drug lead discovery. Identifying potent hits from virtual screening of large chemical databases is ...challenging and computationally demanding. Rather than the traditional two-dimensional (2D)/three-dimensional (3D) approaches on smaller chemical libraries of a few hundred thousand compounds, we screened a ZINC library of 15 million compounds using multiple computational methods. Here, we present the successful application of a virtual screening methodology that identifies several chemotypes as starting hits against lactate dehydrogenase-A (LDHA). From 29 compounds identified from virtual screening, 17 (58%) showed IC
values < 63 μM, two showed single-digit micromolar inhibition, and the most potent hit compound had IC
down to 117 nM. We enriched the database and employed an ensemble approach by combining 2D fingerprint similarity searches, pharmacophore modeling, molecular docking, and molecular dynamics. WaterMap calculations were carried out to explore the thermodynamics of surface water molecules and gain insights into the LDHA binding pocket. The present work has led to the discovery of two new chemical classes, including compounds with a succinic acid monoamide moiety or a hydroxy pyrimidinone ring system. Selected hits block lactate production in cells and inhibit pancreatic cancer cell lines with cytotoxicity IC
down to 12.26 μM against MIAPaCa-2 cells and 14.64 μM against PANC-1, which, under normoxic conditions, is already comparable or more potent than most currently available known LDHA inhibitors.
Abstract
Irritant contact dermatitis (ICD), the most common occupational cutaneous illness, is an acute inflammatory response caused by topical irritant exposure. Multiple factors are associated with ...the manifestation and severity of ICD and contribute to the lack of effective prophylactic and treatment strategies. To determine the pathomechanism of ICD caused by the irritants, benzalkonium chloride (BKC) and JP-8 jet fuel, 2 mouse strains, C57BL/6 and Balb/c, were assessed due to their differential immune predispositions. Dermatitis lesions were obtained for histological examination, cytokine protein expression analysis, and determination of immune cell infiltration via flow cytometric analysis. Following acute (3-day) BKC exposure C57BL/6 skin displayed increased neutrophils and expression of 19 distinct cytokines, but fewer dendritic cells and lower expression of IL-1α and IL-9 as compared with Balb/c skin. Following prolonged (7-day) exposure to BKC, inflammatory cell populations trended similar to 3-day exposure; however, only 6 distinct cytokines were higher in C57BL/6, whereas Balb/c displayed higher expression of IL-27, 28, and 31. Following acute JP-8 exposure, C57BL/6 skin displayed higher levels of γδ T cell infiltration, G and M-CSF expression, but lower populations of neutrophils, monocytes, and dendritic cells compared with Balb/c skin. As with BKC, skin inflammatory cell populations following 7-day JP-8 exposure trended similar to 3-day exposure. However, C57BL/6 skin displayed higher levels of IL-6 and LIF, whereas Balb/c showed increased IL-1β, IL-27, G-CSF, TNFα, and 7 additional chemokines. These findings further define the pathology of ICD, partially explain individual variation of ICD, and offer insight into biomarkers for risk assessment.
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The design, synthesis, and biological evaluation of a series novel N1‑methyl pyrazolo4,3-dpyrimidines as inhibitors of tubulin polymerization and colchicine binding were described ...here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited 3Hcolchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11–13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42 μM, respectively. Compounds 14–16 inhibited the polymerization of tubulin with IC50s near ∼1 μM. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented βIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9–17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P < 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (βIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo4,3-dpyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel.
Diabetes currently affects over twenty-five million Americans. Annual health care cost of diabetes exceeds $254 billion and is associated with a distinct set of diabetic complications that include ...delayed wound healing and diabetic ulcers. Interleukin 6 (IL-6) plays an important role in wound healing and is known to be elevated in the serum of both type I and type II diabetes patients. This study assesses the expression and function of IL-6 in the hyperglycemic epidermis and keratinocyte culture. Streptozotocin-treated mice were wounded six weeks after induction of hyperglycemia. Wound closure, protein, and mRNA expression were assessed up to 13 days of postwounding. Wound closure was delayed 4-5 days in hyperglycemic animals. Hyperglycemic wounds displayed greater IL-6 and IL-6Rα protein expression at 1, 7, and 10 days of postwounding compared to euglycemic control. However, IL-6Rα mRNA expression was reduced at all time points beyond day 1, while IL-6 mRNA expression did not significantly differ at any time point. SOCS3 mRNA expression was higher in the hyperglycemic skin at every time point. Imaging of fluorescent immunohistology also revealed significantly lower expression of SOCS3, but higher nuclear pSTAT3 in the epidermis of the hyperglycemic skin. Primary mouse keratinocytes cultured in high glucose for 7 days displayed 2-fold higher IL-6Rα mRNA and higher rmIL-6-induced nuclear pSTAT3, but lower SOCS3 basal levels compared to normal glucose-cultured cells. Thus, it appears that delayed diabetic skin wound healing is associated with increased induction and expression of IL-6 and its receptor, but its function in epidermal keratinocytes may be impaired.
The skin functions as a protective barrier to inhibit the entry of foreign pathogens, all the while hosting a diverse milieu of microorganisms. Over time, skin cells, immune cells, cytokines, and ...microbes interact to integrate the processes of maintaining the skin's physical and immune barrier. In the present study, the basal expression of two immunologically divergent mouse strains C57BL/6 and BALB/c, as well as a strain on the C57 background lacking IL-6, was characterized. Additionally, cutaneous antimicrobial gene expression profiles and skin bacterial microbiome were assessed between strains. Total RNA sequencing was performed on untreated C57BL/6 (control), BALB/c, and IL-6-deficient skin samples and found over 3,400 genes differentially modulated between strains. It was found that each strain modulated its own transcriptional "profile" associated with skin homeostasis and also influenced the overall bacterial colonization as indicated by the differential phyla present on each strain. Together, these data not only provide a comprehensive view of the transcriptional changes in homeostatic skin of different mouse strains but also highlight the possible influence of the strain differences (e.g., Th1/Th2 balance) as well as a role for IL-6 in overall skin immunity and resident microbial populations.
Irritant Contact Dermatitis (ICD) is characterized by epidermal hyperplasia and inflammatory cytokine release. IL-6 has been shown to be involved in the pathogenesis of ICD; however, the involvement ...of the IL-22/IL-22Rα axis and its relation to IL-6 in the inflammatory response following irritant exposure are unknown. Using a chemical model of ICD, it was observed that mice with a keratinocyte-specific knockout of IL-6Rα (IL-6RαΔker) presented with increased inflammation and IL-22Rα and IL-22 protein expression relative to WT following irritant exposure, indicating that IL-6Rα deficiency in epidermal keratinocytes leads to the upregulation of IL-22Rα and its ligand during ICD. Furthermore, it was shown that IL-6 negatively regulates the expression of IL-22Rα on epidermal keratinocytes. This effect is functional as the effects of IL-22 on keratinocyte proliferation and differentiation were markedly reduced when keratinocytes were pretreated with IL-6 prior to IL-22 treatment. These results show that IL-6 modulates the IL-22/IL-22Rα axis in the skin and suggest that this occurrence may be associated with the increased epidermal hyperplasia and exacerbated inflammatory response observed in IL-6RαΔker mice during ICD.
Irritant contact dermatitis (ICD) is a cutaneous inflammatory response to a variety of triggers that requires no sensitization and accounts for up to 80% of occupational dermatitis cases. IL-6 has ...been alternately associated with both allergic and irritant dermatitis and is closely linked to skin wound healing, therefore making it an ideal candidate to investigate in the mechanism of ICD.
Despite being a well-known pro-inflammatory cytokine, IL-6 deficient (IL-6KO) mice show much more severe ICD than controls. Transcriptome analysis was employed to examine irritant-exposed and control skin samples from C57BL/6 and IL-6KO mice. Over 1900 transcripts were found differentially modulated between C57 (1184 total) and IL-6KO (802 total) mice with the magnitude of expression significantly disparate. Overall gene ontology revealed metabolic and cellular enriched functional processes but numerous pro-inflammatory and immune associated genes (Cxcl2, Cxcl3, Cxcl5, Acod, Hamp, c-Lectins, for example), keratin associated genes (Krt6b and various Krtaps), and members of the Sprr and Lce family, which promote skin barrier integrity and keratinocyte functions, were also differentially modulated.
The altered expression of these genes may provide a potential mechanism to explain the increased ICD severity in IL-6-deficient mice. Overall, this study offers new insight into the pathogenesis of ICD, indicates new mediators/biomarkers that may influence the variability of responses to irritants and provides potential targets for therapeutic development.
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A series of methoxy naphthyl substituted cyclopentadpyrimidine compounds, 4–10, were designed and synthesized to study the influence of the 3-D conformation on microtubule ...depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6ʹ-methoxynaphthyl-1ʹ-amino)-cyclopentadpyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopentadpyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5ʹ-methoxynaphthyl-2ʹ-amino)-cyclopentadpyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopentadpyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC50 values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and have identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopentadpyrimidine class of microtubule targeting agents.
It been shown that IL‐6 modulates TGF‐β1 expression in fibroblasts, however, what role IL‐6 plays concerning TGF‐βR expression and function in skin is unknown. Therefore, the aim of this study was to ...investigate the mechanism by which IL‐6 might modulates TGF‐β receptors in skin. Skin from WT, IL‐6 over‐expressing mice and IL‐6 treated keratinocyte cultures was analysed for TGF‐βRI and TGF‐βRII expression via histology, PCR and flow cytometry. Receptor function was assessed by cell migration, bromodeoxyuridine (BrdU) proliferation assays, and Smad7 expression and Smad2/3 phosphorylation. Receptor localization within the membrane was determined by co‐immunoprecipitation. IL‐6 overexpression and treatment increased TGF‐βRII expression in the epidermis. IL‐6 treatment of keratinocytes induced TGF‐βRI and II expression and augmented TGF‐β1‐induced function as demonstrated through increased migration and decreased proliferation. Additionally, IL‐6 treatment of keratinocytes altered receptor activity as indicated by altered Smad2/3 phosphorylation and increased Smad7 and membrane localization. These results suggest that IL‐6 regulates keratinocyte function by modulating TGF‐βRI and II expression and signal transduction via trafficking of the receptor to lipid raft pools.
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