Design, synthesis, and biological evaluation of 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides as a potential EGR-1 inhibitor for targeted therapy of atopic dermatitis.

Atopic dermatitis is a chronic inflammatory skin disease characterized by intense itching and frequent skin barrier dysfunctions. EGR-1 is a transcription factor that aggravates the pathogenesis of atopic dermatitis by promoting the production of various inflammatory cytokines. Three 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides (IT21, IT23, and IT25) were identified as novel inhibitors of EGR-1 DNA-binding activity. In silico docking experiments were performed to elucidate the binding conditions of the EGR-1 zinc-finger (ZnF) DNA-binding domain. Electrophoretic mobility shift assays confirmed the targeted binding effect on the EGR-1 ZnF DNA-binding domain, leading to dose-dependent dissociation of the EGR-1-DNA complex. At the functional cellular level, IT21, IT23, and IT25 effectively reduced mRNA expression of TNFα-induced EGR-1-regulated inflammatory genes, particularly in HaCaT keratinocytes inflamed by TNFα. In the in vivo efficacy study, IT21, IT23, and IT25 demonstrated the potential to alleviate atopic dermatitis-like skin lesions in the ear skin of BALB/c mice. These findings suggest that targeting the EGR-1 ZnF DNA-binding domain with 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide derivatives (IT21, IT23, and IT25) could serve as lead compounds for the development of potential therapeutic agents against inflammatory skin disorders, including atopic dermatitis.

The EGR1-Artemin Axis in Keratinocytes Enhances the Innervation of Epidermal Sensory Neurons during Skin Inflammation Induced by House Dust Mite Extract from Dermatophagoidesfarinae.

Epidermal hyperinnervation is a critical feature of pruritus during skin inflammation. However, the mechanisms underlying epidermal hyperinnervation are unclear. This study investigates the role of the transcription factor EGR1 in epidermal innervation by utilizing wild-type (Egr1+/+) and Egr1-null (Egr1‒/‒) mice topically applied Dermatophagoides farinae extract from dust mite. Our findings revealed that Egr1‒/‒ mice exhibited reduced scratching behaviors and decreased density of epidermal innervation compared with Egr1+/+ mice. Furthermore, we identified artemin, a neurotrophic factor, as an EGR1 target responsible for Dermatophagoides farinae extract-induced hyperinnervation. It has been demonstrated that Dermatophagoides farinae extract stimulates toll-like receptors in keratinocytes. To elucidate the cellular mechanism, we stimulated keratinocytes with Pam3CSK4, a toll-like receptor 1/2 ligand. Pam3CSK4 triggered a toll-like receptor 1/2-mediated signaling cascade involving IRAK4, IκB kinase, MAPKs, ELK1, EGR1, and artemin, leading to increased neurite outgrowth and neuronal migration. In addition, increased expression of EGR1 and artemin was observed in the skin tissues of patients with atopic dermatitis. These findings highlight the significance of the EGR1-artemin axis in keratinocytes, promoting the process of epidermal innervation and suggesting it as a potential therapeutic target for alleviating itch and pain associated with house dust mite-induced skin inflammation.

Effect of Hybrid Compounds of Stilbene and Pentadienone on Inhibition of Tubulin Polymerization.

INTRODUCTION: Tubulin polymerization inhibitors induce cancer cell death; therefore, they can be developed as chemotherapeutic agents. We hypothesized that hybrid compounds, including the trans-stilbene moiety contained in resveratrol and penta-1,4-dien-3-one contained in curcumin, could inhibit tubulin polymerization. METHODS: Twenty-six hybrid stilbene and pentadienone compounds were designed and synthesized. The cytotoxicity of the hybrid compounds against MDA-MB-231 human breast cancer cells was determined using a clonogenic long-term survival assay. The relationship between cytotoxicity and structural properties was evaluated. Biological activities, including inhibition of tubulin polymerization and cell cycle progression, were investigated to select compounds with excellent anticancer properties. The molecular binding mode between the selected compound and the α, ß-tubulin dimers was investigated. RESULTS: Twenty-six hybrid stilbene and pentadienone compounds were designed and synthesized. Among them, compound 13 exhibited the highest inhibitory effect on the clonogenicity of MDA-MB-231 cells. Compound 13 induced the destabilization of tubulins and inhibited cell cycle progression at the G2/M phase. Through in silico molecular docking analysis, compound 13 was predicted to bind to the colchicine binding site of α, ß-tubulin. CONCLUSION: The stilbene and pentadienone hybrid compound 13 has a binding mode similar to that of colchicine. Compound 13 inhibited the clonogenicity of MDA-MB-231 cells through a mechanism that destabilizes tubulin polymerization, leading to cell cycle arrest at the G2/M phase.

(E)-3-(1,3-Diphenyl-1H-pyrazol-4-yl)-1-(thia-zol-2-yl)prop-2-en-1-one.

In the title mol-ecule, C21H15N3OS, the C5=C6 double bond in the central enone group adopts a trans configuration. The dihedral angle between planes of the thia-zole and pyrazole rings is 6.6 (2)°. In the crystal, pairs of C-H⋯O hydrogen bonds generate inversion dimers and another pair of C-H⋯N hydrogen bonds link the dimers into chains propagating along a-axis direction.

(E)-1-(2-Hy-droxy-4,6-di-meth-oxy-phen-yl)-3-(naph-thalen-1-yl)prop-2-en-1-one.

In the title compound, C21H18O4, the relative conformation of the C=C and C=O double bonds in the central enone group is s-cisoid; there is a trans configuration about the C=C bond. The dihedral angle formed by the naphthalene ring system and the benzene ring is 16.80 (2)°. The meth-oxy groups at the ortho and para positions of the benzene ring are tilted to the ring by 169.8 (1) and 174.5 (1)°, respectively. The hy-droxy group in the benzene ring participates in an intra-molecular O-H⋯O hydrogen bond. In the crystal, C-H⋯O inter-actions link mol-ecules into linear chains along the a-axis direction.

Design, synthesis, and biological activities of 3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-ones.

Novel (Z)-3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-one derivatives were designed and synthesized to find chemotherapeutic agents. Derivative 9 was selected based on its clonogenicity against cancer cells and synthetic yield for further biological experiments. It showed decreases in aurora kinase A, B, and C phosphorylation from western blot analysis. Derivative 9 upregulated the expression of G1 cell cycle inhibitory proteins including p21 and p27, and G1 progressive cyclin D1, and downregulated G1-to-S progressive cyclins, resulting in cell cycle arrest at the G1/S boundary. It stimulated the cleavage of caspase-9, -3, -7, and poly (ADP-ribose) polymerase, resulting in triggering apoptosis through a caspase-dependent pathway. In addition, derivative 9 inhibited in vivo tumor growth in a syngeneic tumor implantation mouse model. The findings of this study suggest that derivative 9 can be considered as a lead compound for chemotherapeutic agents.

Inhibition of EGR-1-dependent MMP1 transcription by ethanol extract of Ageratum houstonianum in HaCaT keratinocytes.

Matrix metalloproteinase 1 (MMP-1) initiates the breakdown of matrix networks by cleaving fibrillar collagen during the pathophysiological progression of skin aging. Ageratum houstonianum ethanol extract (AHE) has been used as a traditional herbal medicine to treat external wounds and skin diseases. However, the mechanism of action underlying A. houstonianum-mediated modulation of skin aging has not been investigated. In this study, we evaluated the effect of AHE on MMP-1 expression in HaCaT keratinocytes. Gene expression was analyzed by Reverse transcription-PCR (RT-PCR), Quantitative real-time PCR (Q-PCR), gene promoter-reporter assay, and immunoblotting. We found that AHE abrogated TNFα-induced MMP1 expression at the transcriptional level via the suppression of ERK1/2 mitogen-activated protein kinase (MAPK)-mediated Early Growth Response 1 (EGR1) expression. We also demonstrated that ß-caryophyllene, a cannabinoid receptor 2 (CB2) agonist, is a functional component of the AHE that inhibits TNFα-induced EGR-1 and MMP1 expression. AHE exerts inhibitory activity on TNFα-induced MMP1 expression at the transcription level through EGR-1 downregulation in keratinocytes. ß-Caryophyllene is a bioactive ingredient of AHE that is responsible for the inhibition of TNFα-induced EGR1 expression. ß-Caryophyllene can be used as a potential agent to prevent inflammation-induced skin aging.

3-(4-Methyl-phen-yl)-4H-chromen-4-one.

In the title compound, C16H12O2, the dihedral angle formed between the plane of the chromenone ring system (r.m.s. deviation = 0.031 Å) and the pendant benzene ring is 31.09 (5)°. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into C(6) chains propagating along the a-axis direction.

1-[5-(3,5-Di-meth-oxy-phen-yl)-3-(2-meth-oxy-phen-yl)-4,5-di-hydro-1H-pyrazol-1-yl]ethanone.

In the title compound, C20H22N2O4, two benzene rings bearing meth-oxy substituents are connected by a central acetyl-pyrazoline ring: the dihedral angle between the benzene rings is 83.7 (1)°. In the crystal, pairwise C-H⋯O hydrogen bonds generate inversion dimers and additional weak C-H⋯O inter-actions link the dimers into chains propagating along the c-axis direction.

Chrysin Inhibits NF-κB-Dependent CCL5 Transcription by Targeting IκB Kinase in the Atopic Dermatitis-Like Inflammatory Microenvironment.

Chrysin (5,7-dihydroxyflavone) is a natural polyphenolic compound that induces an anti-inflammatory response. In this study, we investigated the molecular mechanism underlying the chrysin-induced suppression of C-C motif chemokine ligand 5 (CCL5) gene expression in atopic dermatitis (AD)-like inflammatory microenvironment. We showed that chrysin inhibited CCL5 expression at the transcriptional level through the suppression of nuclear factor kappa B (NF-κB) in the inflammatory environment. Chrysin could bind to the ATP-binding pocket of the inhibitor of κB (IκB) kinase (IKK) and, subsequently, prevent IκB degradation and NF-κB activation. The clinical efficacy of chrysin in targeting IKK was evaluated in 2,4-dinitrochlorobenzene-induced skin lesions in BALB/c mice. Our results suggested that chrysin prevented CCL5 expression by targeting IKK to reduce the infiltration of mast cells to the inflammatory sites and at least partially attenuate the inflammatory responses. These findings suggested that chrysin might be useful as a platform for the design and synthesis of small-molecule IKK-targeting drugs for the treatment of chronic inflammatory diseases, such as AD.

A Novel Synthetic Compound (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile Inhibits TNFα-Induced MMP9 Expression via EGR-1 Downregulation in MDA-MB-231 Human Breast Cancer Cells.

Breast cancer is a common malignancy among women worldwide. Gelatinases such as matrix metallopeptidase 2 (MMP2) and MMP9 play crucial roles in cancer cell migration, invasion, and metastasis. To develop a novel platform compound, we synthesized a flavonoid derivative, (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile (named DK4023) and characterized its inhibitory effects on the motility and MMP2 and MMP9 expression of highly metastatic MDA-MB-231 breast cancer cells. We found that DK4023 inhibited tumor necrosis factor alpha (TNFα)-induced motility and F-actin formation of MDA-MB-231 cells. DK4023 also suppressed the TNFα-induced mRNA expression of MMP9 through the downregulation of the TNFα-extracellular signal-regulated kinase (ERK)/early growth response 1 (EGR-1) signaling axis. These results suggest that DK4023 could serve as a potential platform compound for the development of novel chemopreventive/chemotherapeutic agents against invasive breast cancer.

(E)-1-(4-Fluoro-2-hy-droxy-phen-yl)-3-(2,3,4-tri-meth-oxy-phen-yl)prop-2-en-1-one.

In the title mol-ecule, C18H17FO5, the conformation about the C=C bond of the central enone group is trans. The dihedral angle between the benzene rings is 13.08 (3)°. The hy-droxy group attached to the benzene ring is involved in an intra-molecular O-H⋯O hydrogen bond. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into chains along [001].

Diethyl 4-(1H-imidazol-2-yl)-2,6-dimethyl-1,4-di-hydro-pyridine-3,5-di-carboxyl-ate.

In the title compound, C16H21N3O4, the 1,4-di-hydro-pyridine ring adopts a flattened boat conformation, with the imidazole substituent in an axial orientation [dihedral angle between ring planes = 82.9 (6)°]. In the crystal structure, pairs of N-H⋯O and N-H⋯N hydrogen bonds with graph-set notation R 2 2(14) connect the mol-ecules into chains running along the c-axis direction.

(E)-3-{4,6-Dimeth-oxy-2-[(E)-4-meth-oxy-styr-yl]-3-methyl-phen-yl}-1-(2-hy-droxy-5-meth-oxy-phen-yl)prop-2-en-1-one.

In the title compound, C28H28O6, the benzene rings in the resveratrol moiety are connected by a trans C=C double bond, and the hydroxyl group containing a benzene ring and the central benzene ring are linked through a C=(O)-C=C (enone) moiety to form a chalcone unit. An intra-molecular O-H⋯O hydrogen bond generates an S(6) ring motif. In the crystal, pairs of C-H⋯O hydrogen bonds generate dimers and additional weak C-H⋯O inter-actions link the dimers into chains propagating along the b-axis direction.

Overcoming multidrug resistance by activating unfolded protein response of the endoplasmic reticulum in cisplatin-resistant A2780/CisR ovarian cancer cells.

Cisplatin is a widely used anti-cancer agent. However, the effectiveness of cisplatin has been limited by the commonly developed drug resistance. This study aimed to investigate the potential effects of endoplasmic reticulum (ER) stress to overcome drug resistance using the cisplatin-resistant A2780/CisR ovarian cancer cell model. The synthetic chalcone derivative (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (named DPP23) is an ER stress inducer. We found that DPP23 triggered apoptosis in both parental cisplatinsensitive A2780 and cisplatin-resistant A2780/CisR ovarian cancer cells due to activation of reactive oxygen species (ROS)-mediated unfolded protein response (UPR) pathway in the endoplasmic reticulum. This result suggests that ROSmediated UPR activation is potential in overcoming drug resistance. DPP23 can be used as a target pharmacophore for the development of novel chemotherapeutic agents capable of overcoming drug resistance in cancer cells, particularly ovarian cancer cells. [BMB Reports 2020; 53(2): 88-93].

Design, synthesis, and biological evaluation of polyphenols with 4,6-diphenylpyrimidin-2-amine derivatives for inhibition of Aurora kinase A.

BACKGROUND: Several 4,6-diarylpyrimidin-2-amine derivatives show anticancer properties. However, their mode of action is not fully characterized. To develop potent anticancer chemotherapeutic agents, we designed and synthesized 25 4,6-diphenylpyrimidin-2-amine derivatives containing a guanidine moiety. METHODS: Clonogenic long-term survival assays were performed to screen anticancer compounds. To derive the structural conditions showing good cytotoxicities against cancer cells, quantitative structure-activity relationships (QSAR) were calculated. Biological activities were determined by flow cytometry for cell cycle analysis and by immunoblot analysis for the detection of Aurora kinase A (AURKA) activity. Because 2-(2-Amino-6-(2,4-dimethoxyphenyl)pyrimidin-4-yl) phenol (derivative 12) selectively inhibited AURKA activity from the kinome assay, in silico docking experiments were performed to elucidate the molecular binding mode between derivative 12 and AURKA. RESULTS: The pharmacophores were derived based on the QSAR calculations. Derivative 12 inhibited AURKA activity and reduced phosphorylation of AURKA at Thr283 in HCT116 human colon cancer cells. Derivative 12 caused the accumulation of the G2/M phase of the cell cycle and triggered the cleavages of caspase-3, caspase -7, and poly(ADP-ribose) polymerase. The binding energies of 30 apo-AURKA - derivative 12 complexes obtained from in silico docking ranged from -16.72 to -11.63 kcal/mol. CONCLUSIONS: Derivative 12 is an AURKA inhibitor, which reduces clonogenicity, arrests the cell cycle at the G2/M phase, and induces caspase-mediated apoptotic cell death in HCT116 human colon cancer cells. In silico docking demonstrated that derivative 12 binds to AURKA well. The structure-activity relationship calculations showed hydrophobic substituents and 1-naphthalenyl group at the R2 position increased the activity. The existence of an H-bond acceptor at C-2 of the R1 position increased the activity, too. Graphical abstract Derivative 12 inhibits Aurora kinase A activity and causes the G2/M phase arrest of the cell cycle.