The SpACE-CCM: A facile and versatile cell culture medium-based biosensor for detection of SARS-CoV-2 spike-ACE2 interaction.

The COVID-19 pandemic is an ongoing global public health threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and binding of the SARS-CoV-2 spike to its receptor, angiotensin-converting enzyme 2 (ACE2), on host cells is critical for viral infection. Here, we developed a luminescent biosensor that readily detects interactions of the spike receptor-binding domain (RBD) and ACE2 in cell culture medium ('SpACE-CCM'), which was based on bimolecular complementation of the split nanoluciferase-fused spike RBD and ectodomain of ACE2 and further engineered to be efficiently secreted from cells by adding a heterologous secretory signal peptide (SSP). Screening of various SSPs identified 'interferon-α+alanine-aspartate' as the SSP that induced the highest activity. The SpACE-CCM biosensor was validated by observing a marked reduction of the activity caused by interaction-defective mutations or in the presence of neutralizing antibodies, recombinant decoy proteins, or peptides. Importantly, the SpACE-CCM biosensor responded well in assay-validating conditions compared with conventional cell lysate-based NanoLuc Binary Technology, indicating its advantage. We further demonstrated the biosensor's versatility by quantitatively detecting neutralizing activity in blood samples from COVID-19 patients and vaccinated individuals, discovering a small molecule interfering with the spike RBD-ACE2 interaction through high-throughput screening, and assessing the cross-reactivity of neutralizing antibodies against SARS-CoV-2 variants. Because the SpACE-CCM is a facile and rapid one-step reaction biosensor that aptly recapitulates the native spike-ACE2 interaction, it would be advantageous in many experimental and clinical applications associated with this interaction.

Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) represent a major component of the tumor microenvironment and interplay with cancer cells by secreting cytokines, growth factors and extracellular matrix proteins. When estrogen receptor-negative breast cancer MDA-MB-231 cells were treated with the CAF-conditioned medium (CAF-CM), Akt and STAT3 involved in cell proliferation and survival were activated through phosphorylation. CAFs secrete fibroblast growth factor 2 (FGF2), thereby stimulating breast cancer cell progression. Akt activation induced by CAF-CM in MDA-MB-231 cells was abolished when FGF2-neutralizing antibody was added. Treatment of MDA-MB-231 cells directly with FGF2 enhanced the phosphorylation of Akt and the FGF receptor (FGFR) substrate, FRS2α. These events were abrogated by siRNA-mediated silencing of FGFR1. In a xenograft mouse model, co-injection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced activation of Akt. Stable knockdown of FGFR1 blunted Akt phosphorylation in xenograft tumors. MDA-MB-231 cells co-cultured with CAFs or directly stimulated with FGF2 exhibited enhanced nuclear localization of FGFR1. Notably, FGF2 stimulation produced reactive oxygen species (ROS) accumulation in MDA-MB-231 cells, and FGF2-induced nuclear accumulation of FGFR1 was abrogated by the ROS scavenging agent, N-acetylcysteine.

STAT3 as a Potential Target for Tumor Suppressive Effects of 15-Deoxy-Δ12,14-prostaglandin J2 in Triple Negative Breast Cancer.

STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

Aberrant activation of the CD45-Wnt signaling axis promotes stemness and therapy resistance in colorectal cancer cells.

Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes. Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models. Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the ß-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype. Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.

15-Deoxy-Δ12,14 -prostaglandin J2 binds and inactivates STAT3 via covalent modification of cysteine 259 in H-Ras-transformed human breast epithelial cells.

Signal transducer and activator of transcription 3 (STAT3) has been considered as a potential target for development of anticancer therapeutics. Here, we report a novel mechanism by which the cyclopentenone prostaglandin, 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ) functions as an allosteric inhibitor of STAT3. 15d-PGJ2 inhibits phosphorylation, dimerization, nuclear translocation, and transcriptional activity of STAT3 in H-Ras-transformed human mammary epithelial cells (MCF10A-Ras) through the Michael addition reaction at cysteine 259 of STAT3. Comparative studies with 15d-PGJ2 analogues reveal that both C12-C13 and C9-C10 double bonds conjugated to the carbonyl group in the cyclopentenone ring of 15d-PGJ2 are essential for STAT3 binding. Antiproliferative and pro-apoptotic activities of 15d-PGJ2 in MCF10A-Ras cells are attributable to covalent modification of STAT3 on Cys259, and mimic the effects induced by mutation of this amino acid.

Interaction of Nrf2 with dimeric STAT3 induces IL-23 expression: Implications for breast cancer progression.

Persistent activation of STAT3 and Nrf2 is considered to stimulate the aggressive behavior of basal-like breast cancer (BLBC). However, the precise mechanism underlying sustained overactivation of these transcription factors and their roles in breast cancer progression remain elusive. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and Nrf2 mRNA were correlated with elevated expression of P-STAT3Y705 and Nrf2 target proteins in breast cancer patients. Our present study demonstrates a unique interaction between Nrf2 and STAT3 in the maintenance and progression of BLBC. RNA sequencing analysis identified the gene encoding IL-23A upregulated by concurrent binding of STAT3 and Nrf2 to its promoter. IL-23A depletion also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, the STAT3-Nrf2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer.

An Electrophilic Deguelin Analogue Inhibits STAT3 Signaling in H-Ras-Transformed Human Mammary Epithelial Cells: The Cysteine 259 Residue as a Potential Target.

Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signals that are often constitutively activated in many cancerous or transformed cells and some stromal cells in the tumor microenvironment. Persistent STAT3 activation in malignant cells stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. STAT3 undergoes activation through phosphorylation on tyrosine 705, which facilitates its dimerization. Dimeric STAT3 translocates to the nucleus, where it regulates the transcription of genes involved in cell proliferation, survival, etc. In the present study, a synthetic deguelin analogue SH48, discovered by virtual screening, inhibited the phosphorylation, nuclear translocation, and transcriptional activity of STAT3 in H-ras transformed human mammary epithelial MCF-10A cells (MCF10A-ras). We speculated that SH48 bearing an α,ß-unsaturated carbonyl group could interact with a thiol residue of STAT3, thereby inactivating this transcription factor. Non-electrophilic analogues of SH48 failed to inhibit STAT3 activation, lending support to the above supposition. By utilizing a biotinylated SH48, we were able to demonstrate the complex formation between SH48 and STAT3. SH48 treatment to MCF10A-ras cells induced autophagy, which was verified by staining with a fluorescent acidotropic probe, LysoTracker Red, as well as upregulating the expression of LC3II and p62. In conclusion, the electrophilic analogue of deguelin interacts with STAT3 and inhibits its activation in MCF10A-ras cells, which may account for its induction of autophagic death.

Curcumin induces stabilization of Nrf2 protein through Keap1 cysteine modification.

The present study was aimed to investigate the effects of curcumin, a representative chemopreventive phytochemical with pronounced antioxidant and anti-inflammatory properties, on activation of Nrf2 and expression of its target protein heme oxygenase-1 (HO-1) in mouse skin in vivo and in cultured murine epidermal cells. Treatment of mouse epidermal JB6 cells with curcumin resulted in the induction of HO-1 expression, and this was abrogated in cells transiently transfected with Nrf2 siRNA. While curcumin treatment increased protein expression of Nrf2, it did not alter the steady-state level of the Nrf2 mRNA transcript. Treatment of cells with curcumin stabilized Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of this transcription factor. Tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,ß-unsaturated carbonyl group, failed to induce HO-1 expression as well as nuclear translocation of Nrf2 and its binding to the antioxidant/electrophile response elements. Cells transfected with a mutant Keap1 protein in which cysteine 151 (Cys151) is replaced by serine exhibited marked reduction in curcumin-induced Nrf2 transactivation. Mass spectrometric analysis revealed that curcumin binds to Keap1 Cys151, supporting that this amino acid is a critical target for curcumin modification of Keap1, which facilitates the liberation of Nrf2. Thus, it is likely that the α,ß-unsaturated carbonyl moiety of curcumin is essential for its binding to Keap1 and stabilization of Nrf2 by hampering ubiquitination and proteasomal degradation.

17-Oxo-docosahexaenoic acid induces Nrf2-mediated expression of heme oxygenase-1 in mouse skin in vivo and in cultured murine epidermal cells.

Recently, growing attention has been given to new classes of bioactive lipid mediators derived from ω-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), especially in the context of their role as endogenous signal modulators. One such molecule is 17-oxo-DHA, generated from DHA by the action of COX2 and a dehydrogenase. The redox-sensitive transcription factor, Nrf2 plays a key role in cellular stress responses. In the present study, the effects of 17-oxo-DHA on Nrf2-mediated expression of cytoprotective enzymes were examined in mouse skin in vivo and cultured murine epidermal JB6 cells. Topical application of 17-oxo-DHA markedly elevated the nuclear localization of Nrf2 and expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 in hairless mouse skin. In contrast to 17-oxo-DHA, the non-electrophilic metabolic precursor 17-hydroxy-DHA was a much weaker inducer of Nrf2 activation and its target protein expression. Likewise, 17-oxo-DHA significantly enhanced nuclear translocation and transcriptional activity of Nrf2 with concomitant upregulation of HO-1 expression in cultured JB6 cells. 17-Oxo-DHA was a much stronger inducer of Nrf2-mediated antioxidant response than its parent molecule, DHA. HO-1 expression was abolished in Nrf2 knockdown JB6 cells or embryo fibroblasts from Nrf2 knock out mice. 17-Oxo-DHA also markedly reduced the level of Keap1 protein by inducing ubiquitination. Mutation of Cys151 and Cys273 in Keap1 abrogated 17-oxo-DHA-induced ubiquitination and proteasome-mediated degradation of Keap1 as well as HO-1 expression, suggesting that these cysteine residues are putative sites for 17-oxo-DHA binding. Further, Keap1 degradation stimulated by 17-oxo-DHA coincided with accumulation of the autophagy substrate, p62/SQSTM1.

Acetazolamide-based [18F]-PET tracer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized novel [18F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models other than SKRC-52. [18F]-acetazolamide (1) was found to be insufficient for the specific accumulation in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Direct interaction with 14-3-3γ promotes surface expression of Best1 channel in astrocyte.

BACKGROUND: Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer's disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells. METHODS: The protein-protein interaction between Best1 and 14-3-3γ was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3γ on Best1-mediated current was measured by whole-cell patch clamp technique. RESULTS: We identified 14-3-3γ as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3γ was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3γ. We also revealed that interaction between Best1 and 14-3-3γ was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3γ without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3γ-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3γ-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3γ. CONCLUSIONS: 14-3-3γ promotes surface expression of Best1 channel in astrocytes through direct interaction.

Design and synthesis of new 2-anilinoquinolines bearing N-methylpicolinamide moiety as potential antiproliferative agents.

A series of new 2-anilinoquinolines 6a-o possessing the substantial N-methylpicolinamide motif at C5 has been designed and synthesized as sorafenib analogs. The antiproliferative activities of the target compounds were preliminarily appraised against a panel of three human cancer cell lines (MCF-7, SK-BR3, and HCT116), and a selected array was further tested over a panel of approximately 60 cancer cell lines at NCI at 10 µM concentration. Interestingly, compounds 6c, 6d, 6j, 6k, and 6l showed promising selective anticancer activities (growth inhibition >80%) toward certain cancer cells at 10 µM testing dose. Compounds 6d and 6j were advanced to five-dose testing mode to determine their GI50 values and compared with our previously reported ureidoquinoline B and sorafenib as reference compounds. The 4-chloro-3-trifluoromethylaniline derivative 6j manifested superior potency than both compound B and sorafenib over eleven and eight cell lines, respectively. It showed GI50 values of 0.36, 0.66, 0.68, and 0.60 µM against the breast MDA-MB-468, renal A498, and melanoma SK-MEL-5 and UACC-62 cell lines, respectively. Moreover, both 6d and 6j exerted low cytotoxic effects against HFF-1 normal cell line. Furthermore, compounds 6d and 6j were tested against both B-RafV600E and C-Raf kinases and displayed modest inhibitory activities, which were justified by molecular docking study. Compound 6j could serve as a promising candidate for further development of potent anticancer chemotherapeutics.

Pharmacophore-based virtual screening, biological evaluation and binding mode analysis of a novel protease-activated receptor 2 antagonist.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR2 antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR2 antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22 µM against PAR2. In next step, a PAR2 homology model was constructed using the crystal structure of the PAR1 as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR2 agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe243(5.39) is crucial for PAR2 ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR2 antagonist blocked a conformational rearrangement of Na(+) allosteric site in contrast to PAR2 agonist that showed Na(+) relocation upon GPCR activation. In conclusion, we identified new a PAR2 antagonist together with its binding mode, which provides useful insights for the design and development of PAR2 ligands.

α-Pinene, a Major Constituent of Pine Tree Oils, Enhances Non-Rapid Eye Movement Sleep in Mice through GABAA-benzodiazepine Receptors.

α-Pinene is a major monoterpene of the pine tree essential oils. It has been reported that α-pinene shows anxiolytic and hypnotic effects upon inhaled administration. However, hypnotic effect by oral supplementation and the molecular mechanism of α-pinene have not been determined yet. By combining in vivo sleep behavior, ex vivo electrophysiological recording from brain slices, and in silico molecular modeling, we demonstrate that (-)-α-pinene shows sleep enhancing property through a direct binding to GABAA-benzodiazepine (BZD) receptors by acting as a partial modulator at the BZD binding site. The effect of (-)-α-pinene on sleep-wake profiles was evaluated by recording electroencephalogram and electromyogram. The molecular mechanism of (-)-α-pinene was investigated by electrophysiology and molecular docking study. (-)-α-pinene significantly increased the duration of non-rapid eye movement sleep (NREMS) and reduced the sleep latency by oral administration without affecting duration of rapid eye movement sleep and delta activity. (-)-α-pinene potentiated the GABAA receptor-mediated synaptic response by increasing the decay time constant of sIPSCs in hippocampal CA1 pyramidal neurons. These effects of (-)-α-pinene on sleep and inhibitory synaptic response were mimicked by zolpidem, acting as a modulator for GABAA-BZD receptors, and fully antagonized by flumazenil, an antagonist for GABAA-BZD receptor. (-)-α-pinene was found to bind to aromatic residues of α1- and -γ2 subunits of GABAA-BZD receptors in the molecular model. We conclude that (-)-α-pinene enhances the quantity of NREMS without affecting the intensity of NREMS by prolonging GABAergic synaptic transmission, acting as a partial modulator of GABAA-BZD receptors and directly binding to the BZD binding site of GABAA receptor.

Discovery of a Nanomolar Multikinase Inhibitor (KST016366): A New Benzothiazole Derivative with Remarkable Broad-Spectrum Antiproliferative Activity.

Herein we report the discovery of compound 6 [KST016366; 4-((2-(3-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-6-yl)oxy)picolinamide] as a new potent multikinase inhibitor through minor structural modification of our previously reported RAF kinase inhibitor A. In vitro anticancer evaluation of 6 showed substantial broad-spectrum antiproliferative activity against 60 human cancer cell lines. In particular, it showed GI50 values of 51.4 and 19 nm against leukemia K-562 and colon carcinoma KM12 cell lines, respectively. Kinase screening of compound 6 revealed its nanomolar-level inhibitory activity of certain oncogenic kinases implicated in both tumorigenesis and angiogenesis. Interestingly, 6 displays IC50 values of 0.82, 3.81, and 53 nm toward Tie2, TrkA, and ABL-1 (wild-type and T315I mutant) kinases, respectively. Moreover, 6 is orally bioavailable with a favorable in vivo pharmacokinetic profile. Compound 6 may serve as a promising candidate for further development of potent anticancer chemotherapeutics.

Novel 5-substituted-2-anilinoquinolines with 3-(morpholino or 4-methylpiperazin-1-yl)propoxy moiety as broad spectrum antiproliferative agents: Synthesis, cell based assays and kinase screening.

A series of new 2-anilinoquinolines possessing 3-(morpholino or 4-methylpiperazin-1-yl)propoxy moiety at C5 of quinoline has been designed and synthesized as potential anticancer agents. Their antiproliferative activities were evaluated against a panel of 60 cancer cell lines at NCI and compared with gefitinib as a reference compound. Most of the tested compounds displayed potent and broad spectrum antiproliferative activities. Compounds 7d, 7f and 7g showed strong inhibitory and lethal effects at 10µM concentration. Moreover, they manifested superior potencies and efficacies than gefitinib across the most tested cell lines. Compound 7d, with 4-chloro-3-trifluoromethylphenyl group, proved to be the most potent and efficacious derivative in this series, with mean GI50 and TGI values of 1.62µM and 3.47µM, respectively. Kinase screening of 7d against a panel of 47 oncogenic kinases revealed its selective inhibitory effect (96% inhibition) towards TrkA kinase. Furthermore, the most potent compounds showed low cytotoxic effects against HFF-1 normal cell line.

Design and synthesis of new potent anticancer benzothiazole amides and ureas featuring pyridylamide moiety and possessing dual B-Raf(V600E) and C-Raf kinase inhibitory activities.

A new series of benzothiazole amide and urea derivatives tethered with the privileged pyridylamide moiety by ether linkage at the 6-position of benzothiazole (22 final compounds) has been designed and synthesized as potent anticancer sorafenib analogs. A selected group of twelve derivatives was appraised for its antiproliferative activity over a panel of 60 human cancer cell lines at a single dose concentration of 10 µM at National Cancer Institute (NCI, USA). Compounds 4b, 5a, 5b and 5d exhibited promising growth inhibitions and thus were further tested in advanced 5-dose testing assay to determine their GI50 values. The cellular based assay results revealed that 3,5-bis-trifluoromethylphenyl (5b) urea member is the best derivative with superior potency and efficacy compared to sorafenib as well as notable extended spectrum activity covering 57 human cancer cell lines. Kinase screening of compound 5b showed its kinase inhibitory effect against both B-Raf(V600E) and C-Raf. Moreover, the most potent derivatives in cells were investigated for their RAF inhibitory activities, and the results were rationalized with the molecular docking study. Profiling of CYP450 and hERG channel inhibitory effects for the active compounds revealed their low possibilities to exhibit undesirable drug-drug interactions and cardiac side effects.

Immunomodulatory Activities of the Benzoxathiole Derivative BOT-4-One Ameliorate Pathogenic Skin Inflammation in Mice.

T-cell-mediated immune responses play an important role in body protection. However, aberrantly activated immune responses are responsible for inflammatory and autoimmune diseases. The regulation of pathologic immune responses may be a potential therapeutic strategy for the treatment of these diseases. Despite that multiple pharmacologic properties of benzoxathiole derivatives have been defined, the molecular mechanisms underlying these properties remain to be clarified. Here, we demonstrated the benzoxathiole derivative 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one (BOT-4-one) regulated immune responses and ameliorated experimentally induced inflammatory skin diseases both in vitro and in vivo. BOT-4-one inhibited the differentiation of CD4(+) T-cell subsets by regulating the expression and production of T-cell lineage-specific master transcription factors and cytokines and activating the signal transducer and activator of transcription proteins. In addition, BOT-4-one inhibited TCR-mediated Akt and NF-κB signaling. Topical application of BOT-4-one ameliorated experimentally induced inflammatory skin diseases in mice models such as 2,4,6-trinitrochlorobenzene-induced contact and atopic dermatitis and IL-23-induced psoriasis-like skin inflammation. Our study demonstrated that BOT-4-one ameliorates inflammatory skin diseases by suppressing the pathogenic CD4(+) T cell differentiation and overall immune responses.

Novel Scaffold Identification of mGlu1 Receptor Negative Allosteric Modulators Using a Hierarchical Virtual Screening Approach.

Metabotropic glutamate receptor 1 (mGluR1) is considered as an attractive drug target for neuropathic pain treatments. The hierarchical virtual screening approach for identifying novel scaffolds of mGluR1 allosteric modulators was performed using a homology model built with the dopamine D3 crystal structure as template. The mGluR1 mutagenesis data, conserved amino acid sequences across class A and class C GPCRs, and previously reported multiple sequence alignments of class C GPCRs to the rhodopsin template, were employed for the sequence alignment to overcome difficulties of model generation with low sequence identity of mGluR1 and dopamine D3. The structures refined by molecular dynamics simulations were employed for docking of Asinex commercial libraries after hierarchical virtual screening with pharmacophore and naïve Bayesian models. Five of 35 compounds experimentally evaluated using a calcium mobilization assay exhibited micromolar activities (IC50) with chemotype novelty that demonstrated the validity of our methods. A hierarchical structure and ligand-based virtual screening approach with homology model of class C GPCR based on dopamine D3 class A GPCR structure was successfully performed and applied to discover novel negative mGluR1 allosteric modulators.