Astellas Research Institute of America

Skokie, IL, United States

Astellas Research Institute of America

Skokie, IL, United States
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Huang X.,University of Illinois at Chicago | Yan Y.,University of Cambridge | Tu Y.,University of Washington | Tu Y.,Astellas Research Institute of America | And 4 more authors.
Blood | Year: 2012

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z-protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop-cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction. © 2012 by The American Society of Hematology.


Snyder C.M.,Oregon Health And Science University | Snyder C.M.,Thomas Jefferson University | Cho K.S.,Oregon Health And Science University | Cho K.S.,Astellas Research Institute of America | And 3 more authors.
PLoS Pathogens | Year: 2011

Cytomegalovirus (CMV) is a β-herpesvirus that establishes a lifelong latent or persistent infection. A hallmark of chronic CMV infection is the lifelong persistence of large numbers of virus-specific CD8+ effector/effector memory T cells, a phenomenon called "memory inflation". How the virus continuously stimulates these T cells without being eradicated remains an enigma. The prevailing view is that CMV establishes a low grade "smoldering" infection characterized by tiny bursts of productive infection which are rapidly extinguished, leaving no detectable virus but replenishing the latent pool and leaving the immune system in a highly charged state. However, since abortive reactivation with limited viral gene expression is known to occur commonly, we investigated the necessity for virus reproduction in maintaining the inflationary T cell pool. We inhibited viral replication or spread in vivo using two different mutants of murine CMV (MCMV). First, famcyclovir blocked the replication of MCMV encoding the HSV Thymidine Kinase gene, but had no impact on the CD8+ T cell memory inflation once the infection was established. Second, MCMV that lacks the essential glycoprotein L, and thus is completely unable to spread from cell to cell, also drove memory inflation if the virus was administered systemically. Our data suggest that CMV which cannot spread from the cells it initially infects can repeatedly generate viral antigens to drive memory inflation without suffering eradication of the latent genome pool. © 2011 Snyder et al.


Hu E.,Amgen Inc. | Kunz R.K.,Amgen Inc. | Rumfelt S.,Amgen Inc. | Chen N.,Amgen Inc. | And 10 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2012

We report the discovery of 6,7-dimethoxy-4-(pyridin-3-yl)cinnolines as novel inhibitors of phosphodiesterase 10A (PDE10A). Systematic examination and analyses of structure-activity-relationships resulted in single digit nM potency against PDE10A. X-ray co-crystal structure revealed the mode of binding in the enzyme's catalytic domain and the source of selectivity against other PDEs. High in vivo clearance in rats was addressed with the help of metabolite identification (ID) studies. These findings combined resulted in compound 39, a promising potent inhibitor of PDE10A with good in vivo metabolic stability in rats and efficacy in a rodent behavioral model. © 2011 Elsevier Ltd. All rights reserved.


Yang W.,Astellas Research Institute of America | Kernstock R.,Astellas Research Institute of America | Simmons N.,Astellas Research Institute of America | Alak A.,Astellas Research Institute of America
Rapid Communications in Mass Spectrometry | Year: 2014

RATIONALEA protein internal standard (IS) is essential and superior to a peptide IS to achieve reproducible results in the quantitation of protein therapeutics using immunoaffinity-liquid chromatography/tandem mass spectrometry (LC/MS/MS). Guanidination has been used as a protein post-modification technique for more than half a century. A decade ago, the modification was applied to lysine-ending peptides to enhance their MALDI responses and peptide sequencing coverage. However, rarely has tryptic digestion of guanidinated proteins been investigated, likely due to the early conclusion that trypsin did not hydrolyze peptide bonds involving homoarginine in guanidinated proteins. In this study, the opposite was observed. Guanidinated lysine residues of proteins did not hinder the access of trypsin allowing for proteolytic digestion. Based on this observation, a new concept of internal standard, named Guanidinated Protein Internal Standard (GP-IS), was proposed for LC/MS/MS quantitation of protein therapeutics. METHODSThe GP-IS is prepared by treating a portion of the therapeutic protein (analyte) with guanidine to convert arginine residues in the protein into homoarginine residues. After tryptic digestion, the GP-IS produces a series of homoarginine-ending peptides plus another series of arginine-ending peptides. One of the homoarginine-ending peptides, which corresponds to the analyte surrogate (lysine-ending) peptide, was chosen as a peptide internal standard (GP-PIS) for LC/MS/MS quantitation. RESULTSUsing this GP-IS approach, a sensitive and robust immunoaffinity-LC/MS/MS assay was developed and fully validated with a linearity range from 10 to 1000 ng/mL using 200 μL of human serum for the quantitation of an Astellas protein drug in clinical development. CONCLUSIONSThe proposed strategy allows LC/MS/MS to play an ever-increasing role in bioanalytical support for protein therapeutics development because of its capability of completely tracking all variations from the beginning to the end of sample analysis, easier preparation compared to isotope-labeled protein-IS, and greater flexibility for changing to alternate analyte surrogate peptides. Copyright © 2014 John Wiley & Sons, Ltd.


Yang W.,Astellas Research Institute of America | Kernstock R.,Astellas Research Institute of America | Simmons N.,Astellas Research Institute of America | Alak A.,Astellas Research Institute of America
Bioanalysis | Year: 2015

Aim: Evaluate the performance of ELISA microplates versus commonly used magnetic beads for biological sample cleanup and/or enrichment in immunoaffinity-LC-MS/MS to reduce tedious beads washing procedures and a relatively high assay cost. Materials & Methods: ELISA microplates were used as immunicapture platform and compared with magnetic beads for sample cleanup for LC-MS/MS quantitation of protein therapeutics. Results: One unmodified and two surface-activated microplates provided comparable linear ranges and sensitivities for a therapeutic protein (mass 78 kDa) using a human serum sample of 100 μl with 1:1 dilution compared with Tosylactivated magnetic beads using 200 μl of human serum without sample dilution. The assays' precision and accuracy were all within acceptable ranges. No nonspecific binding or other selectivity issues were observed. Conclusion: The results suggested an ELISA microplate could be a viable immunocapture platform for immunoaffinity-LC-MS/MS quantitation of protein therapeutics. © 2015 Future Science Ltd.


Montalvo-Ortiz J.L.,Northwestern University | Keegan J.,Northwestern University | Gallardo C.,Northwestern University | Gerst N.,Astellas Research Institute of America | And 6 more authors.
Neuropsychopharmacology | Year: 2014

Antipsychotic drugs are widely prescribed to elderly patients for the treatment of a variety of psychopathological conditions, including psychosis and the behavioral disturbances associated with dementia. However, clinical experience suggests that these drugs may be less efficacious in the elderly individuals than in the young. Recent studies suggest that aging may be associated with epigenetic changes and that valproic acid (VPA), a histone deacetylase inhibitor, may reverse such changes. However, it is not yet known whether HDAC inhibitors can modulate age-related epigenetic changes that may impact antipsychotic drug action. In this study, we analyzed conditioned avoidance response (CAR) and c-Fos expression patterns to elucidate the effect of HDAC inhibitors VPA and entinostat (MS-275) on behavioral and molecular markers of the effects of haloperidol (HAL) in aged mice. Our results showed that HAL administration failed to suppress the avoidance response during the CAR test, suggesting an age-related decrease in drug efficacy. In addition, HAL-induced c-Fos expression in the nucleus accumbens shell and prefrontal cortex was significantly lower in aged mice as compared with young mice. Pretreatment with VPA and MS-275 significantly improved HAL effects on the CAR test in aged mice. Also, VPA and MS-275 pretreatment restored HAL-induced increases in c-Fos expression in the nucleus accumbens shell and prefrontal cortex of aged mice to levels comparable with those observed in young mice. Lastly, but most importantly, increases in c-Fos expression and HAL efficacy in the CAR test of the HAL+VPA and HAL+MS-275 groups were correlated with elevated histone acetylation at the c-fos promoter region in aged mice. These findings suggest that pretreatment with VPA or MS-275 increases the behavioral and molecular effects of HAL in aged mice and that these effects occur via modulation of age-related histone hypoacetylation in the nucleus accumbens shell and prefrontal cortex. © 2014 American College of Neuropsychopharmacology.


PubMed | Astellas Research Institute of America and Northwestern University
Type: Journal Article | Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology | Year: 2014

Antipsychotic drugs are widely prescribed to elderly patients for the treatment of a variety of psychopathological conditions, including psychosis and the behavioral disturbances associated with dementia. However, clinical experience suggests that these drugs may be less efficacious in the elderly individuals than in the young. Recent studies suggest that aging may be associated with epigenetic changes and that valproic acid (VPA), a histone deacetylase inhibitor, may reverse such changes. However, it is not yet known whether HDAC inhibitors can modulate age-related epigenetic changes that may impact antipsychotic drug action. In this study, we analyzed conditioned avoidance response (CAR) and c-Fos expression patterns to elucidate the effect of HDAC inhibitors VPA and entinostat (MS-275) on behavioral and molecular markers of the effects of haloperidol (HAL) in aged mice. Our results showed that HAL administration failed to suppress the avoidance response during the CAR test, suggesting an age-related decrease in drug efficacy. In addition, HAL-induced c-Fos expression in the nucleus accumbens shell and prefrontal cortex was significantly lower in aged mice as compared with young mice. Pretreatment with VPA and MS-275 significantly improved HAL effects on the CAR test in aged mice. Also, VPA and MS-275 pretreatment restored HAL-induced increases in c-Fos expression in the nucleus accumbens shell and prefrontal cortex of aged mice to levels comparable with those observed in young mice. Lastly, but most importantly, increases in c-Fos expression and HAL efficacy in the CAR test of the HAL+VPA and HAL+MS-275 groups were correlated with elevated histone acetylation at the c-fos promoter region in aged mice. These findings suggest that pretreatment with VPA or MS-275 increases the behavioral and molecular effects of HAL in aged mice and that these effects occur via modulation of age-related histone hypoacetylation in the nucleus accumbens shell and prefrontal cortex.


PubMed | Astellas Pharma Inc. and Astellas Research Institute of America
Type: Journal Article | Journal: Drug metabolism and pharmacokinetics | Year: 2016

Mirabegron, the first selective


PubMed | Astellas Research Institute of America
Type: Journal Article | Journal: Bioanalysis | Year: 2015

Evaluate the performance of ELISA microplates versus commonly used magnetic beads for biological sample cleanup and/or enrichment in immunoaffinity-LC-MS/MS to reduce tedious beads washing procedures and a relatively high assay cost.ELISA microplates were used as immunicapture platform and compared with magnetic beads for sample cleanup for LC-MS/MS quantitation of protein therapeutics.One unmodified and two surface-activated microplates provided comparable linear ranges and sensitivities for a therapeutic protein (mass 78 kDa) using a human serum sample of 100 l with 1:1 dilution compared with Tosylactivated magnetic beads using 200 l of human serum without sample dilution. The assays precision and accuracy were all within acceptable ranges. No nonspecific binding or other selectivity issues were observed.The results suggested an ELISA microplate could be a viable immunocapture platform for immunoaffinity-LC-MS/MS quantitation of protein therapeutics.


PubMed | Randstad Pharma, Astellas Pharma Inc. and Astellas Research Institute of America
Type: | Journal: Journal of nuclear cardiology : official publication of the American Society of Nuclear Cardiology | Year: 2015

Regadenoson is a selective AHealthy males and females were randomized to receive intravenous regadenoson [100g (3 doses), 200g (3 doses), or 400g (2 doses)], or placebo (2 or 3 doses; 0.9% sodium chloride); all doses 10minutes apart. The primary endpoint was vital sign measurements (blood pressure and heart rate). Secondary endpoints included 12-lead electrocardiogram measurements, clinical laboratory evaluations (hematology, chemistry, and urinalysis), and adverse events. Thirty-six subjects were randomized and completed the study. Plasma concentrations of regadenoson increased in a dose-related manner and with successive doses. No consistent effect was observed for systolic blood pressure, although diastolic blood pressure was slightly lower than placebo for all regadenoson groups. Transient, dose-dependent increases in heart rate were observed in all regadenoson groups. There were no serious adverse events; 27 adverse events occurred in 14 regadenoson-treated subjects vs two events in two placebo-treated subjects.Repeated doses of regadenoson appeared to be safe and well tolerated in healthy subjects.

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