Discovery of Novel Checkpoint Kinase 1 Inhibitors by Virtual Screening Based on Multiple Crystal Structures

Incorporating receptor flexibility is considered crucial for improvement of docking-based virtual screening. With an abundance of crystallographic structures freely available, docking with multiple crystal structures is believed to be a practical approach to cope with protein flexibility. Here we describe a successful application of the docking of multiple structures to discover novel and potent Chk1 inhibitors. Forty-six Chk1 structures were first compared in single structure docking by predicting the binding mode and recovering known ligands. Combinations of different protein structures were then compared by recovery of known ligands and an optimal ensemble of Chk1 structures were selected. The chosen structures were used in the virtual screening of over 60 000 diverse compounds for Chk1 inhibitors. Six novel compounds ranked at the top of the hits list were tested experimentally, and two of these compounds inhibited Chk1 activity–the best with an IC₅₀ value of 9.6 μM. Further study indicated that achieving a better enrichment and identifying more diverse compounds was more likely using multiple structures than using only a single structure even when protein structures were randomly selected. Taking into account conformational energy difference did not help to improve enrichment in the top ranked list

Multivariate analysis for favorable clinical outcome (mRS ≤2) at 3 months.

<p>ASPECTS indicates Alberta Stroke Program Early CT Score; AUC, area under the curve; ICA, internal carotid artery; and NIHSS, National Institutes of Health Stroke Scale.</p><p>Multivariate analysis for favorable clinical outcome (mRS ≤2) at 3 months.</p

Effects of Rottlerin on LPS-injected MCD mice.

<p>(A) Representative liver tissue sections from LPS-treated MCD mice injected with either saline or Rottlerin (10 mg/kg) were stained with Masson’s trichrome (Masson’s) and α-SMA and phospho-PKCδ antibody (original magnification 200×). (B) Liver tissue extracts were prepared from mice fed the MCD diet for 3 weeks and subsequently injected with 2.5 mg/kg LPS and 10 mg/kg Rottlerin. α-SMA, vimentin and phospho-PKCδ protein content were quantified by Western blot using equal amounts of total liver proteins. The levels of α-SMA and vimentin were observed to significantly decrease in the Rottlerin treatment group. For statistical significance, three liver extracts from each individual were used for each group. Expression levels were normalized relative to GAPDH. (C) mRNA levels of hepatic collagen were determined by qRT-PCR analysis. Data plots represent the mean ± SD of three independent experiments. *<i>P</i><0.05 versus saline group. (D) Indicated LPS-injected MCD diet mice were pretreated with TAT or δV1-1 peptide (0.2 mg/kg) for 1 hour. Representative liver tissue sections from LPS-treated MCD mice injected with either TAT or δV1-1 peptide were stained α-SMA with antibody (original magnification 200×).</p

LPS-stimulated PKCδ activation in MCD diet mice.

<p>(A) Liver sections from ND mice and LPS-injected MCD mice were analyzed for E-cadherin, α-SMA and phospho-PKCδ expression using immunohistochemistry staining (original magnification 200×). (B) Liver tissue extracts were prepared from mice fed MCD diet for 1 weeks or 3 weeks and subsequently injected with 2.5 mg/kg LPS. Liver phospho-PKCδ protein contents were quantified by Western blot using an equal amount of total liver proteins. Expression levels were normalized relative to GAPDH. Liver extracts from a pool of three mice in each group were analyzed by Western blot using specific antibodies.</p

Clinical outcome according to collateral status.

<p>All patients with incomplete collaterals showed unfavorable outcome at 3 months. Patients with complete collaterals had favorable clinical outcome more frequently than did patients with incomplete collaterals. * <i>P</i> = 0.003; ** <i>P</i><0.001</p

The proportion of favorable outcome at 90 days according to temporary opening in patients with good collaterals (a) and with poor collaterals (b).

<p>The proportion of favorable outcome at 90 days according to temporary opening in patients with good collaterals (a) and with poor collaterals (b).</p

Involvement of PKCδ in TGFβ1-induced α-SMA expression.

<p>(A) Hepatic levels of <i>TGF β1</i> mRNA from ND, MCD and LPS-injected MCD mice in WT or Tlr4^Lps-d (CH3/HeJ) were measured by qRT-PCR. Genes were normalized to18S rRNA as an internal standard. *P<0.05 versus the ND group. (B) Mice were injected with 2.5 mg/kg LPS for 6 hours in Tlr4^Lps-d (CH3/HeJ) and WT. Liver α-SMA, phospho-PKCδ, phospho-PKCα and phospho-IκBα protein contents were quantified by Western blot. Expression levels were normalized relative to GAPDH. Liver extracts from a pool of three mice in each group were analyzed by Western blot using specific antibodies. (C) Liver tissue extracts were prepared from mice fed MCD diet for 3 weeks and subsequently injected with 2.5 mg/kg LPS and TGFβ1 inhibitor (SB431542). Liver α-SMA, phospho-PKCδ and PKCδ protein contents were quantified by Western blot using an equal amount of total liver proteins. Liver extracts from a pool of three mice in each group were used. (D) Representative liver tissue sections from LPS-treated MCD mice injected with either saline or SB431542 (10 mg/kg) were stained with phospho-PKCδ antibody (original magnification 200×).</p

Multivariate analysis of the determinants for favorable outcome at 90 day in patients with acute ischemic stroke.

<p>NIHSS, National Institutes of Health Stroke Scale; ASPECTS, Alberta Stroke Program Early Computed Tomography Score; M1, First branch of middle cerebral artery; M2, Second branch of middle cerebral artery.</p><p>Multivariate analysis of the determinants for favorable outcome at 90 day in patients with acute ischemic stroke.</p

Baseline characteristics and outcomes of patients with and those without temporary opening.

<p>Data are number (%) or mean (±standard deviation).</p><p>ASPECTS, Alberta Stroke Program Early Computed Tomography Score; IV t-PA, Intravenous tissue plasminogen activator; TOAST, Trial of Org 10172 in Acute Stroke Treatment; NIHSS, National Institutes of Health Stroke Scale.</p><p>Baseline characteristics and outcomes of patients with and those without temporary opening.</p

Clinical Risk Scoring Models for Prediction of Acute Kidney Injury after Living Donor Liver Transplantation: A Retrospective Observational Study

<div><p>Acute kidney injury (AKI) is a frequent complication of liver transplantation and is associated with increased mortality. We identified the incidence and modifiable risk factors for AKI after living-donor liver transplantation (LDLT) and constructed risk scoring models for AKI prediction. We retrospectively reviewed 538 cases of LDLT. Multivariate logistic regression analysis was used to evaluate risk factors for the prediction of AKI as defined by the RIFLE criteria (RIFLE = <i>r</i>isk, <i>i</i>njury, <i>f</i>ailure, <i>l</i>oss, <i>e</i>nd stage). Three risk scoring models were developed in the retrospective cohort by including all variables that were significant in univariate analysis, or variables that were significant in multivariate analysis by backward or forward stepwise variable selection. The risk models were validated by way of cross-validation. The incidence of AKI was 27.3% (147/538) and 6.3% (34/538) required postoperative renal replacement therapy. Independent risk factors for AKI by multivariate analysis of forward stepwise variable selection included: body-mass index >27.5 kg/m² [odds ratio (OR) 2.46, 95% confidence interval (CI) 1.32–4.55], serum albumin <3.5 mg/dl (OR 1.76, 95%CI 1.05–2.94), MELD (model for end-stage liver disease) score >20 (OR 2.01, 95%CI 1.17–3.44), operation time >600 min (OR 1.81, 95%CI 1.07–3.06), warm ischemic time >40 min (OR 2.61, 95%CI 1.55–4.38), postreperfusion syndrome (OR 2.96, 95%CI 1.55–4.38), mean blood glucose during the day of surgery >150 mg/dl (OR 1.66, 95%CI 1.01–2.70), cryoprecipitate > 6 units (OR 4.96, 95%CI 2.84–8.64), blood loss/body weight >60 ml/kg (OR 4.05, 95%CI 2.28–7.21), and calcineurin inhibitor use without combined mycophenolate mofetil (OR 1.87, 95%CI 1.14–3.06). Our risk models performed better than did a previously reported score by Utsumi et al. in our study cohort. Doses of calcineurin inhibitor should be reduced by combined use of mycophenolate mofetil to decrease postoperative AKI. Prospective randomized trials are required to address whether artificial modification of hypoalbuminemia, hyperglycemia and postreperfusion syndrome would decrease postoperative AKI in LDLT.</p></div