In Silico Analysis and In Vitro Characterization of the Bioactive Profile of Three Novel Peptides Identified from 19 kDa α-Zein Sequences of Maize

In this study, we characterized three novel peptides derived from the 19 kDa α-zein, and determined their bioactive profile in vitro and developed a structural model in silico. The peptides, 19ZP1, 19ZP2 and 19ZP3, formed α-helical structures and had positive and negative electrostatic potential surfaces (range of −1 to +1). According to the in silico algorithms, the peptides displayed low probabilities for cytotoxicity (≤0.05%), cell penetration (10–33%) and antioxidant activities (9–12.5%). Instead, they displayed a 40% probability for angiotensin-converting enzyme (ACE) inhibitory activity. For in vitro characterization, peptides were synthesized by solid phase synthesis and tested accordingly. We assumed α-helical structures for 19ZP1 and 19ZP2 under hydrophobic conditions. The peptides displayed antioxidant activity and ACE-inhibitory activity, with 19ZP1 being the most active. Our results highlight that the 19 kDa α-zein sequences could be explored as a source of bioactive peptides, and indicate that in silico approaches are useful to predict peptide bioactivities, but more structural analysis is necessary to obtain more accurate data.     

Evaluation of quercetin as a potential β-lactamase CTX-M-15 inhibitor via the molecular docking,dynamics simulations,and MMGBSA

Antimicrobial resistance (AMR) threatens millions of people around the world and has been declared a global risk by the World Economic Forum. One of the important AMR mechanisms in Enterobacteriaceae is the production of extended-spectrum β-lactamases. The most common ESBL, CTX-M β-lactamases, is spread to the world by CTX-M-15 and CTX-M-14. Sulbactam, clavulanic acid, and tazobactam are first-generation β-lactamase inhibitors and avibactam is a new non-β-lactam β-lactamase inhibitor. We studied that avibactam, sulbactam, clavulanic acid, tazobactam, and quercetin natural flavonoids were docked to target protein CTXM-15. Subsequently, the complexes were simulated using the molecular dynamics simulations method during 100 ns for determining the final binding positions of ligands. Clavulanic acid left CTX-M-15 and other ligands remained in the binding site after the simulation. The estimated binding energies were calculated during 100 ns simulation by the MMGBSA-MMPBSA method. The estimated free binding energies of avibactam, sulbactam, quercetin, tazobactam, and clavulanic acid were sorted as –33.61 kcal/mol, –16.04 kcal/mol, –14 kcal/mol, –12.68 kcal/mol, and –2.95 kcal/mol. As a result of both final binding positions and free binding energy calculations, Quercetin may be evaluated an alternative candidate and a more potent β-lactamases inhibitor for new antimicrobial combinations to CTX-M-15. The results obtained in silico studies are predicted to be a preliminary study for in vitro studies for quercetin and similar bioactive natural compounds. These studies are notable for the discovery of natural compounds that can be used in the treatment of infections caused by β-lactamase-producing pathogens.     

Characterizing affinity epitopes between prion protein and β-amyloid using an epitope mapping immunoassay

Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that β-amyloid_1–42 oligomer causes neurotoxicity associated with Alzheimer''s disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer''s disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in β-amyloid. Residues 23–39 and 93–119 in the prion protein were involved in binding to β-amyloid_1–40 and _1–42, and monomers of this protein interacted with prion protein residues 93–113 and 123–166. Furthermore, β-amyloid antibodies against the C-terminus detected bound β-amyloid_1–42 at residues 23–40, 104–122 and 159–175. β-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to β-amyloid_1–40 and _1–42. The 3D structure appears to be necessary for β-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer''s disease.     

In Silico Approach of Glycinin and Conglycinin Chains of Soybean By-Product (Okara) Using Papain and Bromelain

This study explores utilization of a sustainable soybean by-product (okara) based on in silico approach. In silico approaches, as well as the BIOPEP database, PeptideRanker database, Peptide Calculator database (Pepcalc), ToxinPred database, and AllerTop database, were employed to evaluate the potential of glycinin and conglycinin derived peptides as a potential source of bioactive peptides. These major protein precursors have been found as protein in okara as a soybean by-product. Furthermore, primary structure, biological potential, and physicochemical, sensory, and allergenic characteristics of the theoretically released antioxidant peptides were predicted in this research. Glycinin and α subunits of β-conglycinin were selected as potential precursors of bioactive peptides based on in silico analysis. The most notable among these are antioxidant peptides. First, the potential of protein precursors for releasing bioactive peptides was evaluated by determining the frequency of occurrence of fragments with a given activity. Through the BIOPEP database analysis, there are several antioxidant bioactive peptides in glycinin and β and α subunits of β-conglycinin sequences. Then, an in silico proteolysis using selected enzymes (papain, bromelain) to obtain antioxidant peptides was investigated and then analyzed using PeptideRanker and Pepcalc. Allergenic analysis using the AllerTop revealed that all in silico proteolysis-derived antioxidant peptides are probably nonallergenic peptides. We also performed molecular docking against MPO (myeloperoxidases) for this peptide. Overall, the present study highlights that glycinin and β and α subunits of β-conglycinin could be promising precursors of bioactive peptides that have an antioxidant peptide for developing several applications.     

Isoorientin Inhibits Amyloid β25–35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway

Alzheimer’s disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aβ) peptides. Aβ induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aβ-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3′,4′,5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aβ-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aβ-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aβ_25–35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aβ_25–35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aβ_25–35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.     

Facile One-Pot Multicomponent Synthesis of Pyrazolo-Thiazole Substituted Pyridines with Potential Anti-Proliferative Activity: Synthesis,In Vitro and In Silico Studies

Pyrazolothiazole-substituted pyridine conjugates are an important class of heterocyclic compounds with an extensive variety of potential applications in the medicinal and pharmacological arenas. Therefore, herein, we describe an efficient and facile approach for the synthesis of novel pyrazolo-thiazolo-pyridine conjugate 4, via multicomponent condensation. The latter compound was utilized as a base for the synthesis of two series of 15 novel pyrazolothiazole-based pyridine conjugates (5–16). The newly synthesized compounds were fully characterized using several spectroscopic methods (IR, NMR and MS) and elemental analyses. The anti-proliferative impact of the new synthesized compounds 5–13 and 16 was in vitro appraised towards three human cancer cell lines: human cervix (HeLa), human lung (NCI-H460) and human prostate (PC-3). Our outcomes regarding the anti-proliferative activities disclosed that all the tested compounds exhibited cytotoxic potential towards all the tested cell lines with IC₅₀ = 17.50–61.05 µM, especially the naphthyridine derivative 7, which exhibited the most cytotoxic potential towards the tested cell lines (IC₅₀ = 14.62–17.50 µM) compared with the etoposide (IC50 = 13.34–17.15 µM). Moreover, an in silico docking simulation study was performed on the newly prepared compounds within topoisomerase II (3QX3), to suggest the binding mode of these compounds as anticancer candidates. The in silico docking results indicate that compound 7 was a promising lead anticancer compound which possesses high binding affinity toward topoisomerase II (3QX3) protein.     

Bioactive Polyketide and Diketopiperazine Derivatives from the Mangrove-Sediment-Derived Fungus Aspergillus sp. SCSIO41407

Ten polyketide derivatives (1–10), including a new natural product named (E)-2,4-dihydroxy-3-methyl-6-(2-oxopent-3-en-1-yl) benzaldehyde (1), and five known diketopiperazines (11–15), were isolated from the mangrove-sediment-derived fungus Aspergillus sp. SCSIO41407. The structures of 1–15 were determined via NMR and MS spectroscopic analysis. In a variety of bioactivity screening, 3 showed weak cytotoxicity against the A549 cell line, and 2 exhibited weak antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 3, 5, and 6 showed inhibition against acetylcholinesterase (AChE) with IC₅₀ values of 23.9, 39.9, and 18.6 μM. Compounds 11, 12, and 14 exhibited obvious inhibitory activities of lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) with IC₅₀ values of 19.2, 20.9, and 8.7 μM, and they also suppressed RANKL-induced osteoclast differentiation in bone marrow macrophages cells (BMMCs), with the concentration of 5 μM. In silico molecular docking with AChE and NF-κB p65 protein were also performed to understand the inhibitory activities, and 1, 11–14 showed obvious protein/ligand-binding effects to the NF-κB p65 protein.     

Apoptotic and Cell Cycle Effects of Triterpenes Isolated from Phoradendron wattii on Leukemia Cell Lines

Current antineoplastic agents present multiple disadvantages, driving an ongoing search for new and better compounds. Four lupane-type triterpenes, 3α,24-dihydroxylup-20(29)-en-28-oic acid (1), 3α,23-dihydroxy-30-oxo-lup-20(29)-en-28-oic acid (2), 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (3), and 3α,23-dihydroxylup-20(29)-en-28-oic acid (4), previously isolated from Phoradendron wattii, were evaluated on two cell lines of chronic (K562) and acute (HL60) myeloid leukemia. Compounds 1, 2, and 4 decreased cell viability and inhibit proliferation, mainly in K562, and exhibited an apoptotic effect from 24 h of treatment. Of particular interest is compound 2, which caused arrest in active phases (G2/M) of the cell cycle, as shown by in silico study of the CDK1/Cyclin B/Csk2 complex by molecular docking. This compound [3α,23-dihydroxy-30-oxo-lup-20(29)-en-28-oic acid] s a promising candidate for incorporation into cancer treatments and deserves further study.     

Insight into Gentisic Acid Antidiabetic Potential Using In Vitro and In Silico Approaches

Numerous scientific studies have confirmed the beneficial therapeutic effects of phenolic acids. Among them gentisic acid (GA), a phenolic acid extensively found in many fruit and vegetables has been associated with an enormous confirmed health benefit. The present study aims to evaluate the antidiabetic potential of gentisic acid and highlight its mechanisms of action following in silico and in vitro approaches. The in silico study was intended to predict the interaction of GA with eight different receptors highly involved in the management and complications of diabetes (dipeptidyl-peptidase 4 (DPP4), protein tyrosine phosphatase 1B (PTP1B), free fatty acid receptor 1 (FFAR1), aldose reductase (AldR), glycogen phosphorylase (GP), α-amylase, peroxisome proliferator-activated receptor gamma (PPAR-γ) and α-glucosidase), while the in vitro study studied the potential inhibitory effect of GA against α-amylase and α-glucosidase. The results indicate that GA interacted moderately with most of the receptors and had a moderate inhibitory activity during the in vitro tests. The study therefore encourages further in vivo studies to confirm the given results.     

Anti-Malignant Ascites Effect of Total Diterpenoids from Euphorbiae ebracteolatae Radix Is Attributable to Alterations of Aquaporins via Inhibiting PKC Activity in the Kidney

This study evaluated the anti-ascites effect of total diterpenoids extracted from Euphorbiae ebracteolatae Radix (TDEE) on malignant ascitic mice and elucidated its underlying mechanism. TDEE was extracted by dichloromethane and subjected to column chromatography. The purity of six diterpenoids isolated from TDEE was determined to be 77.18% by HPLC. TDEE (3 and 0.6 g raw herbs/kg, p.o.) reduced ascites and increased urine output. Meanwhile, analysis of tumor cell viability, cycle and apoptosis indicated that TDEE had no antitumor activity. In addition, the expression levels of aquaporins (AQPs) and the membrane translocation levels of protein kinase C (PKC) α and PKCβ in kidney and cells were measured. TDEE reduced the levels of AQP1–4, and inhibited PKCβ expression in membrane fraction. Four main diterpenoids, except compound 2, reduced AQP1 level in human kidney-2 cells. Compounds 4 and 5 inhibited AQP2–4 expression in murine inner medullary collecting duct cells. The diterpenoid-induced inhibition of AQP1–4 expression was blocked by phorbol-12-myristate-13-acetate (PMA; agonist of PKC). The diterpenoids from TDEE are the main anti-ascites components. The anti-ascites effect of diterpenoids may be associated with alterations in AQPs in the kidneys to promote diuresis. The inhibition of AQP1–4 expression by TDEE is related to the inhibition of PKCβ activation.     

Synthesis and Cytotoxic Activity of 1,2,4-Triazolo-Linked Bis-Indolyl Conjugates as Dual Inhibitors of Tankyrase and PI3K

A series of new 1,2,4-triazolo-linked bis-indolyl conjugates (15a–r) were prepared by multistep synthesis and evaluated for their cytotoxic activity against various human cancer cell lines. It was observed that they were more susceptible to colon and breast cancer cells. Conjugates 15o (IC₅₀ = 2.04 μM) and 15r (IC₅₀ = 0.85 μM) illustrated promising cytotoxicity compared to 5-fluorouracil (5-FU, IC₅₀ = 5.31 μM) against the HT-29 cell line. Interestingly, 15o and 15r induced cell cycle arrest at the G₀/G₁ phase and disrupted the mitochondrial membrane potential. Moreover, these conjugates led to apoptosis in HT-29 at 2 μM and 1 μM, respectively, and also enhanced the total ROS production as well as the mitochondrial-generated ROS. Immunofluorescence and Western blot assays revealed that these conjugates reduced the expression levels of the PI3K-P85, β-catenin, TAB-182, β-actin, AXIN-2, and NF-κB markers that are involved in the β-catenin pathway of colorectal cancer. The results of the in silico docking studies of 15r and 15o further support their dual inhibitory behaviour against PI3K and tankyrase. Interestingly, the conjugates have adequate ADME-toxicity parameters based on the calculated results of the molecular dynamic simulations, as we found that these inhibitors (15r) influenced the conformational flexibility of the 4OA7 and 3L54 proteins.     

The Antiviral Effects of Jasminin via Endogenous TNF-α and the Underlying TNF-α-Inducing Action

Previous studies have reported that recombinant tumor necrosis factor (TNF)-α has powerful antiviral activity but severe systematic side effects. Jasminin is a common bioactive component found in Chinese herbal medicine beverage “Jasmine Tea”. Here, we report that jasminin-induced endogenous TNF-α showed antiviral activity in vitro. The underlying TNF-α-inducing action of jasminin was also investigated in RAW264.7 cells. The level of endogenous TNF-α stimulated by jasminin was first analyzed by an enzyme-linked immunosorbent assay (ELISA) from the cell culture supernatant of RAW264.7 cells. The supernatants were then collected to investigate the potential antiviral effect against herpes simplex virus 1 (HSV-1). The antiviral effects of jasminin alone or its supernatants were evaluated by a plaque reduction assay. The potential activation of the PI3K–Akt pathway, three main mitogen-activated protein kinases (MAPKs), and nuclear factor (NF)–κB signaling pathways that induce TNF-α production were also investigated. Jasminin induces TNF-α protein expression in RAW264.7 cells without additional stimuli 10-fold more than the control. No significant up-expression of type I, II, and III interferons; interleukins 2 and 10; nor TNF-β were observed by the jasminin stimuli. The supernatants, containing jasminin-induced-TNF-α, showed antiviral activity against HSV-1. The jasminin-stimulated cells caused the simultaneous activation of the Akt, MAPKs, and NF–κB signal pathways. Furthermore, the pretreatment of the cells with the Akt, MAPKs, and NF–κB inhibitors effectively suppressed jasminin-induced TNF-α production. Our research provides evidence that endogenous TNF-α can be used as a strategy to encounter viral infections. Additionally, the Akt, MAPKs, and NF–κB signaling pathways are involved in the TNF-α synthesis that induced by jasminin.     

Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone from Syzygium nervosum Seeds on Antiproliferative,DNA Damage,Cell Cycle Arrest,and Apoptosis in Human Cervical Cancer Cell Lines

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a natural product derived from Syzygium nervosum A. Cunn. ex DC., was investigated for its inhibitory activities against various cancer cell lines. In this work, we investigated the effects of DMC and available anticervical cancer drugs (5-fluorouracil, cisplatin, and doxorubicin) on three human cervical cancer cell lines (C-33A, HeLa, and SiHa). DMC displayed antiproliferative cervical cancer activity in C-33A, HeLa, and SiHa cells, with IC₅₀ values of 15.76 ± 1.49, 10.05 ± 0.22, and 18.31 ± 3.10 µM, respectively. DMC presented higher antiproliferative cancer activity in HeLa cells; therefore, we further investigated DMC-induced apoptosis in this cell line, including DNA damage, cell cycle arrest, and apoptosis assays. As a potential anticancer agent, DMC treatment increased DNA damage in cancer cells, observed through fluorescence inverted microscopy and a comet assay. The cell cycle assay showed an increased number of cells in the G₀/G₁ phase following DMC treatment. Furthermore, DMC treatment-induced apoptosis cell death was approximately three- to four-fold higher compared to the untreated group. Here, DMC represented a compound-induced apoptosis for cell death in the HeLa cervical cancer cell line. Our findings suggest that DMC, a phytochemical agent, is a potential candidate for antiproliferative cervical cancer drug development.     

Design and Synthesis of New Benzo[d]oxazole-Based Derivatives and Their Neuroprotective Effects on β-Amyloid-Induced PC12 Cells

A series of novel synthetic substituted benzo[d]oxazole-based derivatives (5a–5v) exerted neuroprotective effects on β-amyloid (Aβ)-induced PC12 cells as a potential approach for the treatment of Alzheimer’s disease (AD). In vitro studies show that most of the synthesized compounds were potent in reducing the neurotoxicity of Aβ_25-35-induced PC12 cells at 5 μg/mL. We found that compound 5c was non-neurotoxic at 30 μg/mL and significantly increased the viability of Aβ_25-35-induced PC12 cells at 1.25, 2.5 and 5 μg/mL. Western blot analysis showed that compound 5c promoted the phosphorylation of Akt and glycogen synthase kinase (GSK-3β) and decreased the expression of nuclear factor-κB (NF-κB) in Aβ_25-35-induced PC12 cells. In addition, our findings demonstrated that compound 5c protected PC12 cells from Aβ_25-35-induced apoptosis and reduced the hyperphosphorylation of tau protein, and decreased the expression of receptor for AGE (RAGE), β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), inducible nitric oxide synthase (iNOS) and Bcl-2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2) via Akt/GSK-3β/NF-κB signaling pathway. In vivo studies suggest that compound 5c shows less toxicity than donepezil in the heart and nervous system of zebrafish.     

Anti-Inflammatory,Antidiabetic Properties and In Silico Modeling of Cucurbitane-Type Triterpene Glycosides from Fruits of an Indian Cultivar of Momordica charantia L.

Diabetes mellitus is a chronic disease and one of the fastest-growing health challenges of the last decades. Studies have shown that chronic low-grade inflammation and activation of the innate immune system are intimately involved in type 2 diabetes pathogenesis. Momordica charantia L. fruits are used in traditional medicine to manage diabetes. Herein, we report the purification of a new 23-O-β-d-allopyranosyl-5β,19-epoxycucurbitane-6,24-diene triterpene (charantoside XV, 6) along with 25ξ-isopropenylchole-5(6)-ene-3-O-β-d-glucopyranoside (1), karaviloside VI (2), karaviloside VIII (3), momordicoside L (4), momordicoside A (5) and kuguaglycoside C (7) from an Indian cultivar of Momordica charantia. At 50 µM compounds, 2–6 differentially affected the expression of pro-inflammatory markers IL-6, TNF-α, and iNOS, and mitochondrial marker COX-2. Compounds tested for the inhibition of α-amylase and α-glucosidase enzymes at 0.87 mM and 1.33 mM, respectively. Compounds showed similar α-amylase inhibitory activity than acarbose (0.13 mM) of control (68.0–76.6%). Karaviloside VIII (56.5%) was the most active compound in the α-glucosidase assay, followed by karaviloside VI (40.3%), while momordicoside L (23.7%), A (33.5%), and charantoside XV (23.9%) were the least active compounds. To better understand the mode of binding of cucurbitane-triterpenes to these enzymes, in silico docking of the isolated compounds was evaluated with α-amylase and α-glucosidase.     

The dopamine D1–D2DR complex in the rat spinal cord promotes neuropathic pain by increasing neuronal excitability after chronic constriction injury

Dopamine D1 receptor (D1DR) and D2 receptor (D2DR) are closely associated with pain modulation, but their exact effects on neuropathic pain and the underlying mechanisms remain to be identified. Our research revealed that intrathecal administration of D1DR and D2DR antagonists inhibited D1–D2DR complex formation and ameliorated mechanical and thermal hypersensitivity in chronic constriction injury (CCI) rats. The D1–D2DR complex was formed in the rat spinal cord, and the antinociceptive effects of D1DR and D2DR antagonists could be reversed by D1DR, D2DR, and D1–D2DR agonists. Gαq, PLC, and IP3 inhibitors also alleviated CCI-induced neuropathic pain. D1DR, D2DR, and D1–D2DR complex agonists all increased the intracellular calcium concentration in primary cultured spinal neurons, and this increase could be reversed by D1DR, D2DR antagonists and Gαq, IP3, PLC inhibitors. D1DR and D2DR antagonists significantly reduced the expression of p-PKC γ, p-CaMKII, p-CREB, and p-MAPKs. Levo-corydalmine (l-CDL), a monomeric compound in Corydalis yanhusuo W.T. Wang, was found to obviously suppress the formation of the spinal D1–D2DR complex to alleviate neuropathic pain in CCI rats and to decrease the intracellular calcium concentration in spinal neurons. l-CDL-induced inhibition of p-PKC γ, p-MAPKs, p-CREB, and p-CaMKII was also reversed by D1DR, D2DR, and D1–D2DR complex agonists. In conclusion, these results indicate that D1DR and D2DR form a complex and in turn couple with the Gαq protein to increase neuronal excitability via PKC γ, CaMKII, MAPK, and CREB signaling in the spinal cords of CCI rats; thus, they may serve as potential drug targets for neuropathic pain therapy.Subject terms: Molecular neuroscience, Cellular neuroscience     

GPR183 Regulates 7α,25-Dihydroxycholesterol-Induced Oxiapoptophagy in L929 Mouse Fibroblast Cell

7α,25-dihydroxycholesterol (7α,25-DHC) is an oxysterol synthesized from 25-hydroxycholesterol by cytochrome P450 family 7 subfamily B member 1 (CYP7B1) and is a monooxygenase (oxysterol-7α-hydroxylase) expressed under inflammatory conditions in various cell types. In this study, we verified that 7α,25-DHC-induced oxiapoptophagy is mediated by apoptosis, oxidative stress, and autophagy in L929 mouse fibroblasts. MTT assays and live/dead cell staining revealed that cytotoxicity was increased by 7α,25-DHC in L929 cells. Consequentially, cells with condensed chromatin and altered morphology were enhanced in L929 cells incubated with 7α,25-DHC for 48 h. Furthermore, apoptotic population was increased by 7α,25-DHC exposure through the cascade activation of caspase-9, caspase-3, and poly (ADP-ribose) polymerase in the intrinsic pathway of apoptosis in these cells. 7α,25-DHC upregulated reactive oxygen species (ROS) in L929 cells. Expression of autophagy biomarkers, including beclin-1 and LC3, was significantly increased by 7α,25-DHC treatment in L929 cells. 7α,25-DHC inhibits the phosphorylation of Akt associated with autophagy and increases p53 expression in L929 cells. In addition, inhibition of G-protein-coupled receptor 183 (GPR183), a receptor of 7α,25-DHC, using GPR183 specific antagonist NIBR189 suppressed 7α,25-DHC-induced apoptosis, ROS production, and autophagy in L929 cells. Collectively, GPR183 regulates 7α,25-DHC-induced oxiapoptophagy in L929 cells.     

Synthesis of 2-Aminopyrimidine Derivatives and Their Evaluation as β-Glucuronidase Inhibitors: In Vitro and In Silico Studies

Currently the discovery and development of potent β-glucuronidase inhibitors is an active area of research due to the observation that increased activity of this enzyme is associated with many pathological conditions, such as colon cancer, renal diseases, and infections of the urinary tract. In this study, twenty-seven 2-aminopyrimidine derivatives 1–27 were synthesized by fusion of 2-amino-4,6-dichloropyrimidine with a variety of amines in the presence of triethylamine without using any solvent and catalyst, in good to excellent yields. All synthesized compounds were characterized by EI-MS, HREI-MS and NMR spectroscopy. Compounds 1–27 were then evaluated for their β-glucuronidase inhibitory activity, and among them, compound 24 (IC₅₀ = 2.8 ± 0.10 µM) showed an activity much superior to standard D-saccharic acid 1,4-lactone (IC₅₀ = 45.75 ± 2.16 µM). To predict the binding mode of the substrate and β-glucuronidase, in silico study was performed. Conclusively, this study has identified a potent β-glucuronidase inhibitor that deserves to be further studied for the development of pharmaceutical products.