Structure-Based Site of Metabolism (SOM) Prediction of Ligand for CYP3A4 Enzyme: Comparison of Glide XP and Induced Fit Docking (IFD)

Metabolism is one of the prime reasons where most of drugs fail to accomplish their clinical trials. The enzyme CYP3A4, which belongs to the superfamily of cytochrome P450 enzymes (CYP), helps in the metabolism of a large number of drugs in the body. The enzyme CYP3A4 catalyzes oxidative chemical processes and shows a very broad range of ligand specificity. The understanding of the compound’s structure where oxidation would take place is crucial for the successful modification of molecules to avoid unwanted metabolism and to increase its bioavailability. For this reason, it is required to know the site of metabolism (SOM) of the compounds, where compounds undergo enzymatic oxidation. It can be identified by predicting the accessibility of the substrate’s atom toward oxygenated Fe atom of heme in a CYP protein. The CYP3A4 enzyme is highly flexible and can take significantly different conformations depending on the ligand with which it is being bound. To predict the accessibility of substrate atoms to the heme iron, conventional protein-rigid docking methods failed due to the high flexibility of the CYP3A4 protein. Herein, we demonstrated and compared the ability of the Glide extra precision (XP) and Induced Fit docking (IFD) tool of Schrodinger software suite to reproduce the binding mode of co-crystallized ligands into six X-ray crystallographic structures. We extend our studies toward the prediction of SOM for compounds whose experimental SOM is reported but the ligand-enzyme complex crystal structure is not available in the Protein Data Bank (PDB). The quality and accuracy of Glide XP and IFD was determined by calculating RMSD of docked ligands over the corresponding co-crystallized bound ligand and by measuring the distance between the SOM of the ligand and Fe atom of heme. It was observed that IFD reproduces the exact binding mode of available co-crystallized structures and correctly predicted the SOM of experimentally reported compounds. Our approach using IFD with multiple conformer structures of CYP3A4 will be one of the effective methods for SOM predictionAuthor A.P.S. is grateful to the University Grants Commission, New Delhi for the financial assistance under the major research project (42-677/2013 (SR)) and Dr. Babasaheb Ambedkar Marathwada University, Aurangabad for the research grant (STAT/VI/RG/Dept/2019-20/309-10)S

Facile synthesis of novel coumarin derivatives, antimicrobial analysis, enzyme assay, docking study, ADMET prediction and toxicity study

The work reports the synthesis under solvent-free condition using the ionic liquid [Et3NH][HSO4] as a catalyst of fifteen novel 3-((dicyclohexylamino)(substituted phenyl/heteryl)-methyl)-4-hydroxy-2H-chromen-2-onederivatives 4a–o as potential antimicrobial agents. The structures of the synthesized compounds were confirmed by IR, 1H-NMR, 13C-NMR, mass spectral studies and elemental analyses. All the synthesized compounds were evaluated for their in vitro antifungal and antibacterial activity. The compound 4k bearing 4-hydroxy-3-ethoxy group on the phenyl ring was found to be the most active antifungal agent. The compound 4e bearing a 2,4-difluoro group on the phenyl ring was found to be the most active antibacterial agent. The mode of action of the most promising antifungal compound 4k was established by an ergosterol extraction and quantitation assay. From the assay it was found that 4k acts by inhibition of ergosterol biosynthesis in C. albicans. Molecular docking studies revealed a highly spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase, which suggests that the tested compounds inhibit the synthesis of this enzyme. The synthesized compounds were analyzed for in silico ADMET properties to establish oral drug like behavior and showed satisfactory results. To establish the antimicrobial selectivity and safety, the most active compounds 4e and 4k were further tested for cytotoxicity against human cancer cell line HeLa and were found to be non-cytotoxic in nature. An in vivo acute oral toxicity study was also performed for the most active compounds 4e and 4k and results indicated that the compounds are non-toxic.S

Sterol Intermediates of Cholesterol Biosynthesis Inhibit Hair Growth and Trigger an Innate Immune Response in Cicatricial Alopecia

Primary cicatricial alopecia (PCA) is a group of inflammatory hair disorders that cause scarring and permanent hair loss. Previous studies have implicated PPARγ, a transcription factor that integrates lipogenic and inflammatory signals, in the pathogenesis of PCA. However, it is unknown what triggers the inflammatory response in these disorders, whether the inflammation is a primary or secondary event in disease pathogenesis, and whether the inflammatory reaction reflects an autoimmune process. In this paper, we show that the cholesterol biosynthetic pathway is impaired in the skin and hair follicles of PCA patients. Treatment of hair follicle cells with BM15766, a cholesterol biosynthesis inhibitor, or 7-dehydrocholesterol (7-DHC), a sterol precursor, stimulates the expression of pro-inflammatory chemokine genes. Painting of mouse skin with 7-DHC or BM15766 inhibits hair growth, causes follicular plugging and induces the infiltration of inflammatory cells into the interfollicular dermis. Our results demonstrate that cholesterologenic changes within hair follicle cells trigger an innate immune response that is associated with the induction of toll-like receptor (TLR) and interferon (IFN) gene expression, and the recruitment of macrophages that surround the hair follicles and initiate their destruction. These findings reveal a previously unsuspected role for cholesterol precursors in PCA pathogenesis and identify a novel link between sterols and inflammation that may prove transformative in the diagnosis and treatment of these disorders

Correlation between phosphorylated H1 histone and condensed chromatin in Planococcus citri

Histones of the mealybug Planococcus citri were isolated and characterized. Although no major differences were obsd. between the core histones of male and female insects, the pattern of H1 histones was significantly different between the sexes. A specific hyperphosphorylated H1 was found only in male mealybugs, where the paternal chromosomes are condensed and transcriptionally inactive

Ionic Liquid-Promoted Synthesis of Novel Chromone-Pyrimidine Coupled Derivatives, Antimicrobial Analysis, Enzyme Assay, Docking Study and Toxicity Study

Herein, we report an environmentally friendly, rapid, and convenient ionic liquid ([Et3NH][HSO4])-promoted facile synthesis of ethyl 4-(6-substituted-4-oxo-4H-chromen-3-yl)-6-methyl-2-thioxo/oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 4(a–f) and 4-(6-substituted-4-oxo-4H-chromen-3-yl)-6-methyl-2-thioxo/oxo-1,2,3,4-tetrahydropyrimidine-5- carbohydrazide derivatives 6(a–f). All the synthesized derivatives 4(a–f) and 6(a–f) were evaluated for their in vitro antifungal and antibacterial activity, by method recommended by National Committee for Clinical Laboratory Standards (NCCLS). The compound 6c bearing a fluoro group on the chromone ring and oxygen and a hydrazino group (–NHNH2) on the pyrimidine ring, was found to be the most potent antibacterial compound amongst the synthesized derivatives. The compound 6f bearing a methoxy group (–OCH3) on the chromone ring and sulphur group on the pyrimidine ring, was found to exhibit equipotent antifungal activity when compared with the standard drug miconazole. A d-alanine-d-alanine ligase (DdlB) enzyme assay study and an ergosterol extraction and quantitation assay study were performed to predict the mode of action of the synthesized compounds. A molecular docking study was performed to predict the binding interactions with receptors and mode of action of the synthesized derivatives. Further, analysis of the ADMET parameters for the synthesized compounds has shown that these compounds have good oral drug-like properties and can be developed as oral drug candidates. To establish the antimicrobial selectivity and safety, the most active compounds 6c and 6f were further tested for cytotoxicity against the human cancer cell line HeLa and were found to be non-cytotoxic in nature. An in vivo acute oral toxicity study was also performed for the most active compounds 6c and 6f and the results indicated that the compounds are non-toxic in nature

Innate immune genes are upregulated in PCA.

<p>Real-time PCR validation of (A) <i>TLR4,</i> (B) <i>TLR6,</i> (C) <i>IFNα</i>, (D) <i>IFNα7,</i> (E) <i>NFkB,</i> (F) <i>IFNγ</i>, (G) <i>MMD</i> and (H) <i>MCP1</i> in mixed (DC, DF), neutrophilic (TF, FD) and lymphocytic (FFA, LPP, CCCA) PCA. These genes are significantly upregulated in affected tissue compared to unaffected tissue from the same patients (*p<0.05, **p<0.01). The unpaired <i>t-</i>test was used for statistical analysis. Differences in the pattern of expression of these genes were observed in the different PCA subtypes. (I) IPA identified the interferon signaling pathway or the “interferon-responsive signature” in the gene expression profiles of LPP. The intensity of the node color red indicates the degree of upregulation, and the intensity of the color green indicates the degree of downregulation. Genes shown as uncolored nodes were not identified as differentially expressed in our experiment and were integrated into the computationally generated networks based on the evidence stored in the IPA knowledge base, which indicated a relevance to this network. The node shapes denote enzymes, phosphatases, kinases, peptidases, transmembrane receptors, cytokines, transporters, translation factors, nuclear receptors and transcription factors. The interferon target genes <i>IRF1, IRF8, IFNA5, IFNAR2, IFIT3</i>, <i>IFITM1, MX1, OAS1</i> and <i>IFI35</i> are significantly upregulated in LPP. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038449#pone.0038449.s006" target="_blank">Table S2</a>.</p

Inflammatory pathways and networks activated in C57BL/6J mouse skin after topical treatment with 7-DHC and BM15766.

<p>(A) The most significant signaling pathways altered by 7-DHC treatment participated in the inflammatory and immune responses and were identified using IPA. Fisher’s exact test was used to calculate p values to determine the probability that the association between the genes in the dataset and the pathway could be explained by chance alone. The yellow line indicates the threshold of significance (p<0.05) and represents the ratio of the number of molecules from the data set that map to the pathway to the total number of molecules that map to the pathway. (B) The top differentially regulated pathways in BM15766-treated mouse skin. The majority of the upregulated pathways participated in the inflammatory and immune responses. (C, D) The top two predicted networks in 7DHC-treated mouse skin, determined using IPA. The <i>TLR4</i> and <i>IFN</i> gene networks are significantly upregulated by 7-DHC. Solid lines denote direct relationships between genes. Dotted lines denote an indirect relationship between two genes. A red node denotes an upregulated gene, and a green node denotes a downregulated gene. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038449#pone.0038449.s008" target="_blank">Table S4</a>. Real-time PCR validation of (E) <i>TLR4</i>, (F) <i>TLR6</i>, (G) <i>IFNα</i>, (H) <i>IFNα7</i>, (I) <i>NFkB</i>, (J) <i>IFNγ,</i> (K) <i>MMD</i> and (L) <i>MCP1</i> gene expression in mouse skin treated with 7-DHC or BM15766 compared with vehicle-treated (ethanol or DMSO) controls (n = 3; *p<0.05, **p<0.01). The unpaired <i>t-</i>test was used for the statistical analysis. Treatment with 7-DHC and BM15766 can induce the expression of some or all of these genes.</p

Ingenuity Pathways Analysis of the top toxic pathways in cicatricial alopecia.

<p>IPA-Tox®, a data analysis capability tool within the Ingenuity Pathways Analysis, was used to analyze the microarray data and to determine the toxicity associated with the observed gene expression changes in PCA. The figure shows the top toxicity lists (Toxlists) associated with gene expression changes in samples from unaffected and affected scalp areas in patients with LPP, CCCA, FFA and TF. Cholesterol biosynthesis appears to be the most significant toxicity-related pathway associated with the lymphocytic PCA subtypes.</p