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Strategies for the identification of novel inhibitors of deubiquitinating enzymes

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828BiochemicalSocietyTransactions(2008)Volume36,part5

Strategiesfortheidentificationofnovelinhibitors

ofdeubiquitinatingenzymes

SethJ.Goldenberg1,JeffreyL.McDermott,TauseefR.Butt,MichaelR.MatternandBenjaminNicholson

ProgenraInc.,271AGreatValleyParkway,Malvern,PA19355,U.S.A.

Abstract

DysregulationoftheUPS(ubiquitin–proteasomesystem)hasbeenimplicatedinawiderangeofpathologies

includingcancer,neurodegenerationandviralinfection.Inhibitingtheproteasomehasbeenshowntobean

effectivetherapeuticstrategyinhumans;yettoxicitywiththistargetremainshigh.DUBs(deubiquitinating

enzymes)representanalternativetargetintheUPSwithlowpredictedtoxicity.Currently,therearenoDUB

inhibitorsthathavebeenusedclinically.Toaddressthissituation,Progenrahasdevelopedanovelassay

tomeasuretheproteolyticcleavageofUb(ubiquitin)orUBL(Ub-likeprotein)conjugatessuchasSUMO

(smallUb-relatedmodifier),NEDD8(neural-precursor-cell-expressed,developmentallydown-regulated8)

orISG15(interferon-stimulatedgene15)byisopeptidases.Inthisreview,currentplatformsfordetecting

DUBinhibitorsarediscussedandtheadvantagesanddisadvantagesoftheapproachesareunderlined.

Introduction

TheconjugationofUb(ubiquitin)andUBLs(Ub-like

proteins)isanimportantregulatorymechanismthatis

widespreadinmanybiologicalprocesses[1].Theassorted

diseasesassociatedwiththesepathwaysmakethepathway

enzymesparticularlyinterestingfortherapeutictargets.The

UPS(Ub–proteasomesystem)hasbeenvalidatedfollow-

ingtheapprovaloftheproteasomeinhibitorBortezomib

forthetreatmentofmultiplemyeloma;however,significant

toxicitieswereseenduringclinicaltrials,suggestingtheneed

formoreselectivetargets[2].Ub-andUBL-isopeptidases

representauniquesetofdrugtargetsintheUPSthatare

responsibleforremovingUbandUBLs,suchasSUMO

(smallUb-relatedmodifier),NEDD8(neural-precursor-

cell-expressed,developmentallydown-regulated8)and

ISG15(interferon-stimulatedgene15),fromtargetproteins,

thusaffectingthetargets’fate[3].Todeveloptherapeutic

agentsthattargetisopeptidases,wedevelopedareadily

quantifiablenovelisopeptidaseassayplatformthatissuitable

forHTS(high-throughputscreening).Theassayplatform

consistsofUborUBLfusedtothereporterenzymePLA2(phospholipaseA2).IsopeptidaseactivityreleasesPLA2that

cleavesitssubstrate,generatingasignalthatislinearwith

isopeptidaseconcentrationandisabletodiscriminateDUB

(deubiquitinatingenzyme),deSUMOylase,deNEDDylase

Keywords:deISGylase,deNEDDylase,deSUMOylase,deubiquitinatingenzyme(DUB),high-throughputscreening(HTS),isopeptidase,smallubiquitin-relatedmodifier(SUMO).Abbreviationsused:DUB,deubiquitinatingenzyme;FRET,fluorescenceresonanceenergytransfer;HTS,high-throughputscreening;ISG15,interferon-stimulatedgene15;NEDD8,neural-precursor-cell-expressed,developmentallydown-regulated8;PLA2,phospholipaseA2;RING,reallyinterestingnewgene;SCF,Skp1/cullin/F-box;Skp,S-phasekinase-associatedprotein;Ub,ubiquitin;SUMO,smallUb-relatedmodifier;TRAF,tumour-necrosis-factor-receptor-associatedfactor;TR-FRET,time-resolvedFRET;Ub-AMC,Ub-7-amino-4-methylcoumarin;UBL,Ub-likeprotein;UBP/USP,Ub-specificproteases;ULP,UBL-specificprotease;UPS,Ub–proteasomesystem.1Towhomcorrespondenceshouldbeaddressed(emailGoldenberg@progenra.com).anddeISGylaseactivities.Theassaycanbesuccessfully

employedtoscreenforinhibitorsofisopeptidases.

TherapeutictargetsintheUPS

TheapprovaloftheproteasomeinhibitorBortezomib

(Velcade)forthetreatmentofmultiplemyelomavalidated

thetargetingoftheUPSforthetreatmentofcancer[4].

However,extendedtreatmentwithBortezomibisassociated

withtoxicityanddrugresistance,limitingitsefficacy[2].

Incontrast,therapeuticstrategiesthattargetspecificaspects

oftheUPSupstreamoftheproteasomewouldbepredicted

tohavelowertoxicity.Whileactivatingenzymes(E1)and

conjugatingenzymes(E2)areupstreamoftheproteasome,

onemustbeawareoftheconsequencesoftargetingthem,as

disruptionoftheE1leadstocell-cyclearrest[5]andE2shave

beenshowntoberequiredfordevelopment[6].Targeting

theUb-activatingenzymemaybepredictedtoaffecttoo

manycellularfunctionsforittobetoleratedbynormalcells;

yettargetingtheNEDD8-activatingenzymeforinhibition

hasbeenreportedtobesuccessfulinpreclinicalstudies[7].

Themechanismofactionismostlikelythroughinactivation

ofthecullin-basedE3ligases,manyofwhichplayacrucial

roleincell-cyclecheckpointswhosedisruptionwouldhave

amoreimmediateeffectonrapidlydividingcancercells.

E3ligases,withonlyalimitednumberofsubstrates,

representattractivedrugtargetsintheUPS.Oneofthemost

interestingE3targetsisSCF[Skp(S-phasekinase-associated

protein)1/cullin/F-box].TheSCFcomplexconsistsofmany

variableF-boxadaptorproteinseachofwhichtargetonlya

fewsubstratesforubiquitination[8].Twotherapeuticallyrel-

evantF-boxproteinsareSkp2[9]andβ-TRCP(β-transducin

repeat-containingprotein)[10],whichplaykeyrolesin

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制定策略英英文表达

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Developing Strategies: A Comprehensive Guide.

Introduction.

In the realm of business and beyond, the importance of

strategic planning cannot be overstated. It's the backbone

of any successful operation, providing a roadmap for

decision-making, resource allocation, and overall direction.

This article aims to provide a comprehensive understanding

of strategy development, its key components, and how it can

be effectively implemented.

1. Defining Strategy.

Strategy, at its core, is a plan of action designed to

achieve a specific goal or set of goals. It involves the

identification of opportunities and threats, the allocation

of resources, and the development of actions that align

with the organization's vision and mission.

2. The Strategy Development Process.

The strategy development process typically involves

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Planning Strategies, Monitoring Strategies, and

Adaptive Strategies.

In the realm of strategic management, three key

concepts stand out as crucial for the success of any

organization: planning strategies, monitoring strategies,

and adaptive strategies. These strategies, when effectively

implemented, can lead to enhanced performance, increased

efficiency, and a more responsive and resilient

organization.

Planning Strategies.

Planning strategies refer to the process of setting

goals, developing action plans, and allocating resources to

achieve those goals. These strategies are typically

formulated based on an organization's mission, vision, and

values, taking into account its internal strengths and

weaknesses as well as external opportunities and threats. Effective planning strategies are comprehensive, well-researched, and designed to guide the organization towards

一代测序的操作流程

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Next-generation sequencing (NGS), also known as next-generation

sequencing, is a popular method for DNA sequencing.

下一代测序（NGS），也称为下一代测序，是一种常用的DNA测序方法。

NGS has revolutionized the field of genomics with its high-throughput capability, cost-effectiveness, and rapid turnaround time.

NGS以其高通量能力，成本效益和快速周转时间，彻底改变了基因组学领域。

The NGS workflow typically includes library preparation, cluster

generation, sequencing, and data analysis.

NGS工作流程通常包括文库准备，簇生成，测序和数据分析。

First, the DNA or RNA sample is processed to create a library of

fragments that can be sequenced.

首先，对DNA或RNA样本进行处理，以创建可以进行测序的片段文库。

This library is then amplified and attached to a solid surface to create

clusters, where each cluster contains multiple copies of the same

DNA fragment.

然后，将这个文库进行扩增，并将其附着在固体表面上形成簇，其中每个簇包含同一DNA片段的多个复制。

During sequencing, a sequencer reads each cluster and identifies the

流行病与卫生统计学英语

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English:

Epidemiology, the study of the distribution and determinants of

health-related states or events in specified populations, plays a

critical role in understanding and combating infectious diseases. By

examining patterns of disease occurrence and identifying risk factors,

epidemiologists can develop strategies for prevention and control.

Key to this effort is the use of statistical methods to analyze data on

disease incidence, prevalence, and mortality rates, allowing for the

identification of trends and the assessment of the effectiveness of

interventions. Furthermore, epidemiology provides valuable insights

into the social, environmental, and behavioral factors that influence

disease transmission, helping to inform public health policies and

interventions. Through surveillance systems and research studies,