Nanodiscs for INPHARMA NMR Characterization of GPCRs: Ligand Binding to the Human A2A Adenosine Receptor.

G-protein-coupled-receptors (GPCRs) are of fundamental importance for signal transduction through cell membranes. This makes them important drug targets, but structure-based drug design (SBDD) is still hampered by the limitations for structure determination of unmodified GPCRs. We show that the interligand NOEs for pharmacophore mapping (INPHARMA) method can provide valuable information on ligand poses inside the binding site of the unmodified human A2A adenosine receptor reconstituted in nanodiscs. By comparing experimental INPHARMA spectra with back-calculated spectra based on ligand poses obtained from molecular dynamics simulations, a complex structure for A2A R with the low-affinity ligand 3-pyrrolidin-1-ylquinoxalin-2-amine was determined based on the X-ray structure of ligand ZM-241,358 in complex with a modified A2A R

Целевое взятие гистологического материала из зоны атипической мелкоацинарной пролиферации, выявленной по результатам повторной трансректальной биопсии простаты

Оbjective: to define the approach to the management of patients with the detected ASAP area.Materials and methods. In the time period from 2012 through 2015, 494 patients with previously negative biopsy and remaining suspicion of prostate cancer (PCa) were examined. The patients underwent repeat 24-core multifocal prostate biopsy with taking additional tissue samples from suspicious areas detected by multiparametric magnetic resonance imaging and transrectal ultrasound. An isolated ASAP area was found in 127 (25. 7 %) of the 494 examined men. All of them were offered to perform repeat target transrectal biopsy of this area. Targeted transrectal ultrasound guided biopsy of the ASAP area was performed in 56 (44.1 %) of the 127 patients, 53 of them being included in the final analysis.Results. PCa was diagnosed in 14 (26.4 %) of the 53 patients, their mean age being 64.4 ± 6.9 years. The average level of prostate-specific antigen (PSA) in PCa patients was 6.8 ± 3.0 ng/ml, in those with benign lesions – 9.3 ± 6.5 ng/ml; the percentage ratio of free/total PSA with PCa was 16.2 ± 7,8 %, with benign lesions – 23.3 ± 7.7 %; PSA density in PCa patients was 0.14 ± 0.07 ng/ml/cm3, in those with benign lesions – 0.15 ± 0.12 ng/ml/cm3. Therefore, with ASAP area being detected in repeat prostate biopsy samples, it is advisable that targeted extended biopsy of this area be performed. Введение. В статье оценена клинико-морфологическая значимость атипической мелкоацинарной пролиферации (АМАП), выявленной в материале повторной трансректальной мультифокальной биопсии предстательной железы (ПЖ).Цель исследования – определение тактики ведения пациентов с выявленной зоной АМАП.Материалы и методы. За период с 2012 по 2015 г. были обследованы 494 пациента с отрицательным результатом первичной систематической биопсии ПЖ и сохраняющимся подозрением на рак ПЖ (РПЖ). Пациентам проводили повторную 24-точечную мультифокальную биопсию ПЖ со взятием дополнительных образцов ткани из подозрительных зон по данным мультипараметрической магнитно-резонансной томографии и трансректального ультразвукового исследования. У 127 (25,7 %) пациентов была выявлена изолированная зона АМАП. Всем им предлагалось выполнение повторной прицельной трансректальной биопсии этой зоны. Прицельная биопсия под контролем трансректального ультразвукового исследования из зоны АМАП была проведена 56 (44,1 %) пациентам, из них в исследование вошли 53.Результаты. РПЖ был диагностирован у 14 (26,4 %) из 53 пациентов. Средний возраст – 64,4 ± 6,9 года. Средний уровень простатического специфического антигена (ПСА) в группе пациентов с РПЖ составил 6,8 ± 3,0 нг/мл, с доброкачественными изменениями – 9,3 ± 6,5 нг/мл; процентное содержание свободного ПСА по отношению к общему ПСА при РПЖ – 16,2 ± 7,8 %, с доброкачественными изменениями – 23,3 ± 7,7 %; плотность ПСА у пациентов с РПЖ – 0,14 ± 0,07 нг/мл/см3, с доброкачественными изменениями – 0,15 ± 0,12 нг/мл/см3. Таким образом, при выявлении зоны АМАП в материале повторной мультифокальной биопсии ПЖ целесообразно выполнение целенаправленной расширенной биопсии данной зоны.

Targeted histology sampling from atypical small acinar proliferation area detected by repeat transrectal prostate biopsy

Оbjective: to define the approach to the management of patients with the detected ASAP area.Materials and methods. In the time period from 2012 through 2015, 494 patients with previously negative biopsy and remaining suspicion of prostate cancer (PCa) were examined. The patients underwent repeat 24-core multifocal prostate biopsy with taking additional tissue samples from suspicious areas detected by multiparametric magnetic resonance imaging and transrectal ultrasound. An isolated ASAP area was found in 127 (25. 7 %) of the 494 examined men. All of them were offered to perform repeat target transrectal biopsy of this area. Targeted transrectal ultrasound guided biopsy of the ASAP area was performed in 56 (44.1 %) of the 127 patients, 53 of them being included in the final analysis.Results. PCa was diagnosed in 14 (26.4 %) of the 53 patients, their mean age being 64.4 ± 6.9 years. The average level of prostate-specific antigen (PSA) in PCa patients was 6.8 ± 3.0 ng/ml, in those with benign lesions – 9.3 ± 6.5 ng/ml; the percentage ratio of free/total PSA with PCa was 16.2 ± 7,8 %, with benign lesions – 23.3 ± 7.7 %; PSA density in PCa patients was 0.14 ± 0.07 ng/ml/cm3, in those with benign lesions – 0.15 ± 0.12 ng/ml/cm3. Therefore, with ASAP area being detected in repeat prostate biopsy samples, it is advisable that targeted extended biopsy of this area be performed

Characterization of a tumor-associated activating mutation of the p110β PI 3-kinase.

The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β

The hinge region of the scaffolding protein of cell contacts, zonula occludens protein 1, regulates interacting with various signaling proteins

Zonula occludens protein 1 (ZO-1) is a ubiquitous scaffolding protein, but it is unknown why it functions in very different cellular contacts. We hypothesized that a specific segment, the unique hinge region, can be bound by very different regulatory proteins. Using surface plasmon resonance spectroscopy and binding assays to peptide libraries, we show, for the first time, that the hinge region directly interacts with disparate signal elements such as G-proteins alpha 12 and alpha i2, the regulator of G-protein signaling 5, multifunctional signaling protein ahnak1, and L-type Ca2+-channel beta-2-subunit. The novel binding proteins specifically bound to a coiled coil-helix predicted in the hinge region of ZO-. The interactions were modulated by phosphorylation in the hinge helix. Activation of the G-proteins influenced their association to ZO-1. In colon cells, G alpha i2 and ZO-1 were associated, as shown by coimmunoprecipitation. After cotransfection in kidney cells, G alpha i2 barely colocalized with ZO-1; the colocalization coefficient was significantly increased when epinephrine activated G-protein signaling. In conclusion, proteins with different regulatory potential adhere to and influence cellular functions of ZO-proteins, and the interactions can be modulated via its hinge region and/or the binding proteins

Better understanding of phosphoinositide 3-Kinase (PI3K) pathways in vasculature: towards precision therapy targeting angiogenesis and tumor blood supply

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3K{alpha} isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3K{alpha} and other class I isoforms, i.e. PI3K{beta}, {gamma}, {delta}, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3K{gamma} and PI3K{delta} (Pik3cg(-/-)/Pik3cd(-/-)) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 {my}M). These vasoconstrictions were also inhibited by the PI3K{alpha} isoform inhibitors A66 (10 {my}M) and PI-103 (1 {my}M), but not by the PI3K{beta} isoform inhibitor TGX 221 (100 nM). Pik3cg(-/-)/Pik3cd(-/-)-arteries showed normal vasoconstriction. We conclude that PI3K{alpha} is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via {alpha}1-adrenergic receptors. Our results highlight a regulatory role of PI3K{alpha} in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3K{alpha} in cancer cells and tumor angiogenesis and regional blood flow

Role of kinase activity on the increased proliferation and migration by the p110β mutant.

<p>(A) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in 10% NCS, with or without 200 nM TGX-221, and assayed using the MTT assay. (B) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated, with or without 200 nM TGX-221 in both chambers as indicated. Data are mean ± SEM of at least duplicate samples from two separate experiments.</p

Effect of p110β mutant on transformation and chemotaxis.

<p>(A) NIH 3T3 cells stably expressing wild type or E633K p110β were plated in soft agar and colonies were counted after 3 weeks. Colony counts are normalized to the number of colonies produced by cells expressing p110β alone. (B) Equal number of NIH 3T3 cells stably expressing wild type or E633K p110β were plated and left to grow to confluence for 10 days. Foci were counted and normalized to cells expressing wild-type p110β. (C) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated. Data are mean ± SEM of triplicate samples from two experiments.</p

Akt signaling, proliferation and survival of cells expressing mutant p110β.

<p>(A) Expression level of wild-type or E633K myc-p110β in stably-transfected cells. (B) Cells stably expressing wild type or E633K p10β were incubated overnight in 10%, 0.5% or 0% NCS media. Whole cell lysates were analyzed by western blotting with anti-pT308 Akt, anti-pT389 S6K, and anti-β-actin antibodies. (C-E) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in (C) 10% NCS medium, (D) 0.5% NCS medium, or (E) 0% NCS medium, and assayed using the MTT assay. (F) Cells stably expressing wild type or E633K p110β were incubated for 24 hours in 10%, 0.5%, or 0% NCS medium. Cell viability was assayed by Trypan blue staining. Dead cells are displayed as percent of total number of cells. Data are mean ± SEM of triplicate samples from two separate experiments.</p