10 research outputs found

    (A) Paraformaldehyde-fixed livers of CYP2D6 mice infected with either Ad-2D6 or Ad-GFP at week 8 after infection

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    Bars, 0.5 cm. (B) LDS for evaluation of the morphological appearance of mouse liver after adenovirus infection. Pictures of representative livers of LDS 0 (Ad-GFP infected) and LDS 5 (Ad-2D6 infected) are shown in A. (C) Livers of CYP2D6 and FVB/N mice infected with either Ad-2D6 or Ad-GFP were harvested at several times after infection, and LDS was determined by morphological examination. The mean LDS for each group is indicated by horizontal lines. Note that Ad-2D6–infected mice only develop persistent liver damage. Statistical analysis (Mann-Whitney test) revealed significant differences between Ad-2D6 and Ad-GFP mice at the indicated times. *, P < 0.01; **, P < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1409-1422.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413037.</p><p></p

    (A) Pictures of representative end results of total IgG or CYP2D6-specific spot-forming assays

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    Colored spots indicate the reaction of enzyme-labeled secondary antibodies against secreted IgG and represent one antibody-secreting cell. (B) Quantification of total IgG (top) and CYP2D6-specific IgG (bottom)–secreting cells per 10 splenocytes in FVB/N and CYP2D6 mice at the time points indicated. Note that B cells secreting CYP2D6 antibody–specific antibodies were only detectable after Ad-2D6 infection. ND, not detectable. (C) IgM and IgG type anti-CYP2D6 antibody generation in FVB/N and CYP2D6 mice infected with either Ad-2D6 or Ad-GFP over time. Data are mean ± SEM. (D) Representative pictures of rat liver sections stained with sera from Ad-2D6–infected CYP2D6 mice, Ad-2D6–infected FVB/N mice, AIH-1 patients (ANA), AIH-2 patients (LKM-1), and PBC patients (antimitochondrial antibodies), followed by Alexa Fluor 488–conjugated anti–mouse IgG or FITC-conjugated anti–human IgG secondary antibody. Bars, 50 μm.<p><b>Copyright information:</b></p><p>Taken from "Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1409-1422.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413037.</p><p></p

    (A) SPOTs membrane containing staggered peptides of 12 aa in length covering the entire 497 aa of the human CYP2D6 molecule

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    The membrane was developed with serum of an Ad-2D6–infected CYP2D6 mouse. Numbers indicate the position within the CYP2D6 sequence of the first aa of the first peptide spot in each horizontal lane. (B) Quantification of the serum reactivity profile to SPOTs peptides. Representative serum samples of an Ad-2D6–infected CYP2D6 mouse, an Ad-2D6–infected FVB/N mouse, an AIH-2 patient, and an AIH-1 patient. Numbers indicate the aa position within the human CYP2D6 sequence. (C) For an overview of the epitope profile, the human CYP2D6 sequence was divided into 20 25-aa-long sections, as indicated at the top of each column. Signal intensities of sera from Ad-2D6–infected CYP2D6 and FVB/N mice, patients with AIH-2, and patients with other autoimmune liver diseases, such as AIH-1, AIH-3, or PBC, are indicated in red (strong), orange (medium), gray (low), and white (no reactivity). Note that the region containing the immunodominant WDPAQPPRD epitope (region 251–275) is recognized by all sera of Ad-2D6–infected CYP2D6 and FVB/N mice, as well as by all tested sera of AIH-2 patients, but not by sera from patients with other autoimmune liver diseases.<p><b>Copyright information:</b></p><p>Taken from "Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1409-1422.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413037.</p><p></p

    (A) Expression of GFP in liver sections at several times after infection of CYP2D6 mice with Ad-GFP

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    Bars, 50 μm. (B) Serum levels of ALT and AST in humanized CYP2D6 and wild-type FVB/N mice infected with Ad-2D6 or Ad-GFP at several times after infection. Note that elevations in serum ALT and AST were transient and not persistent. Statistical evaluation ( test) revealed no significant differences in AST and ALT augmentation curves over time between Ad-2D6– and Ad-GFP–infected mice (FVB, P = 0.7 [ALT] and 0.79 [AST]; CYP2D6, P = 0.5 [ALT] and 0.13 [AST]) and between FVB/N and CYP2D6 mice (Ad-2D6, P = 0.64 [ALT] and 0.59 [AST]; Ad-GFP, P = 0.31 [ALT] and 0.82 [AST]). (C) Serum levels of AP in humanized CYP2D6 and wild-type FVB/N mice infected with Ad-2D6 or Ad-GFP at several times after infection ( = 5 per group). Transient AP elevation was detected in CYP2D6 and FVB/N mice at weeks 1 and 2 after infection with Ad-2D6 but not Ad-GFP. Statistical evaluation ( test) revealed significant differences in serum AP compared with preinfection levels (week 1, P = 0.035 [FVB/N Ad-2D6]; week 2, P = 0.044 [FVB/N Ad-2D6] 0.029 [CYP2D6 Ad-2D6]) and between Ad-2D6– and Ad-GFP–infected mice (week 1, P = 0.03 [FVB] and 0.063 [CYP2D6]; week 2, P = 0.055 [FVB] and 0.029 [CYP2D6]). Data are mean ± SD.<p><b>Copyright information:</b></p><p>Taken from "Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1409-1422.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413037.</p><p></p

    (A) Sirius red staining of collagen fibers in the subcapsular region of the liver at several times after infection of CYP2D6 and FVB/N mice with either Ad-2D6 or Ad-GFP

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    Tissue sections covering entire livers from three to five mice per experimental group were analyzed, and a representative picture of the subcapsular region is displayed for each time point. Note the increase in magnitude of collagen deposition in Ad-2D6–infected mice over time and the accelerated subcapsular fibrosis in FVB/N compared with CYP2D6 mice. Bars, 20 μm. (B) Reconstruction of a large section of an Ad-2D6–infected CYP2D6 mouse liver (week 8 after infection), including the left medial (LM) and right medial (RM) lobes and the left lateral (LL) lobe, stained for collagen I (green). Note the subcapsular fibrosis and the individual lobes that are glued together by collagen I deposit. (inset) Reconstruction of the left lateral lobe of an Ad-GFP–infected CYP2D6 mouse liver (week 8 after infection). Bars, 0.1 cm. Regions 1–4 are shown at a higher magnification. In addition to staining for collagen I (green), staining for B cells (B220; red) was overlaid. Regions 1 and 2 show B cells entrapped in pockets within the collagen I deposits in the subcapsular region of the liver. Regions 3 and 4 show areas where two or three individual liver lobes are joined by collagen I fibers. Bars, 50 μm.<p><b>Copyright information:</b></p><p>Taken from "Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1409-1422.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413037.</p><p></p

    (A) Hematoxylin and eosin staining of liver sections obtained from CYP2D6 and FVB/N mice infected with either Ad-2D6 or Ad-GFP at weeks 1, 4, and 8 after infection

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    Note that persistent hepatic damage (massive cellular infiltrations and significant necrosis) is present in Ad-2D6–infected mice only. Infection of FVB/N mice with Ad-GFP results in a similar histology as Ad-GFP infection of CYP2D6 mice (not depicted). Bars, 50 μm. (B) The following scoring system was used to assess liver infiltration: 0, no visible infiltration; 1, few focal infiltrates; 2, numerous scattered focal infiltrates; 3, few large clusters of infiltrating cells (perivascular or subcapsular); 4, numerous large clusters of infiltrating cells occupying >50% of portal tracts or central veins; and 5, confluent periportal infiltrates. The presented data are mean infiltration scores ± SEM. Statistical analysis (Mann-Whitney test) revealed significant differences between mice infected with Ad-2D6 or Ad-GFP at the indicated times after infection. *, P < 0.05. The number of individual mouse livers analyzed per experimental group is indicated at the bottom of each data column. (C) Immunohistochemistry of liver sections of wild-type FVB/N and humanized CYP2D6 mice at week 2 after infection with Ad-2D6. Cellular infiltrations consisted predominantly of B cells (B220), CD4 T cells, and macrophages (F4/80). Bars, 50 μm.<p><b>Copyright information:</b></p><p>Taken from "Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1409-1422.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413037.</p><p></p

    Azaindole-Based Inhibitors of Cdc7 Kinase: Impact of the Pre-DFG Residue, Val 195

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    To investigate the role played by the unique pre-DFG residue Val 195 of Cdc7 kinase on the potency of azaindole-chloropyridines (<b>1</b>), a series of novel analogues with various chloro replacements were synthesized and evaluated for their inhibitory activity against Cdc7. X-ray cocrystallization using a surrogate protein, GSK3β, and modeling studies confirmed the azaindole motif as the hinge binder. Weaker hydrophobic interactions with Met 134 and Val 195 by certain chloro replacements (e.g., H, methyl) led to reduced Cdc7 inhibition. Meanwhile, data from other replacements (e.g., F, O) indicated that loss of such hydrophobic interaction could be compensated by enhanced hydrogen bonding to Lys 90. Our findings not only provide an in-depth understanding of the pre-DFG residue as another viable position impacting kinase inhibition, they also expand the existing knowledge of ligand-Cdc7 binding

    Pyrimidine-Based Tricyclic Molecules as Potent and Orally Efficacious Inhibitors of Wee1 Kinase

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    Aided by molecular modeling, compounds with a pyrimidine-based tricyclic scaffold were designed and confirmed to inhibit Wee1 kinase. Structure–activity studies identified key pharmacophores at the aminoaryl and halo-benzene regions responsible for binding affinity with sub-nM <i>K</i><sub>i</sub> values. The potent inhibitors demonstrated sub-μM activities in both functional and mechanism-based cellular assays and also possessed desirable pharmacokinetic profiles. The lead molecule, <b>31</b>, showed oral efficacy in potentiating the antiproliferative activity of irinotecan, a cytotoxic agent, in a NCI-H1299 mouse xenograft model

    Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality

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    A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer’s disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug–drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound <b>16</b>, which exhibits robust in vivo efficacy as a BACE1 inhibitor

    Utilizing Structures of CYP2D6 and BACE1 Complexes To Reduce Risk of Drug–Drug Interactions with a Novel Series of Centrally Efficacious BACE1 Inhibitors

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    In recent years, the first generation of β-secretase (BACE1) inhibitors advanced into clinical development for the treatment of Alzheimer’s disease (AD). However, the alignment of drug-like properties and selectivity remains a major challenge. Herein, we describe the discovery of a novel class of potent, low clearance, CNS penetrant BACE1 inhibitors represented by thioamidine <b>5</b>. Further profiling suggested that a high fraction of the metabolism (>95%) was due to CYP2D6, increasing the potential risk for victim-based drug–drug interactions (DDI) and variable exposure in the clinic due to the polymorphic nature of this enzyme. To guide future design, we solved crystal structures of CYP2D6 complexes with substrate <b>5</b> and its corresponding metabolic product pyrazole <b>6</b>, which provided insight into the binding mode and movements between substrate/inhibitor complexes. Guided by the BACE1 and CYP2D6 crystal structures, we designed and synthesized analogues with reduced risk for DDI, central efficacy, and improved hERG therapeutic margins
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