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Structure-BasedDesignofanOrganorutheniumPhosphatidyl-inositol-3-kinaseInhibitorRevealsaSwitchGoverning LipidKinasePotencyandSelectivity
PengXie
†,‡
,DouglasS.Williams
‡
,G.EkinAtilla-Gokcumen
‡,¶
,LeslieMilk
§
,MinXiao
†
,KeiranS.M.Smalley
†
,MeenhardHerlyn
†
,EricMeggers
‡,¶
,andRonenMarmorstein
†,‡,§,
*
†
The Wistar Institute,
‡
Department of Chemistry, University of Pennsylvania, and
§
Graduate Group in Biochemistry andMolecular Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania,
¶
Current address:Department of Chemistry, Philipps-University Marburg, Marburg, Germany.
P
hosphatidyl-inositol-3-kinases(PI3Ks)areafamilyofheterodimericdual-specificlipidki-nasestypicallyconsistingofbothregulatoryandcatalyticsubunits.Differentcombinationsofregulatoryandcatalyticsubunitsdistinguishthethreemajor classesofPI3Ksandconferdiversesubstratespecific-ityandmechanismofupstreamsignaling(
1–3
).ClassIPI3Ksincludefourisoformsconsistingoftwosubdivi-sions,classIA(PI3K
,
,
)andclassIB(PI3K
).Uponactivationbyupstreamsignalingevents,theclassIPI3Ksphosphorylatethe3
=
positionofthemembrane-embeddedphosphatidylinositol4,5-biphosphate(PIP
2
)toconvertittophosphatidylinositol3,4,5-triphosphate(PIP
3
)(
1,4
).PIP
3
servesasanimportantsecondmes-sengermoleculetorecruitdownstreamPHdomaincon-tainingeffectorssuchas3-phosphoinositide-dependentkinase(PDK)andAKT(alsocalledproteinkinaseB),whichinturnactivateavarietyofdownstreameffectorsthatturnonthesignalingcascadesleadingtocellprolif-eration,survival,andcellgrowth(
5
).DisruptionofthePI3Ksignalingpathwayfavoringpro-growthsignalingdirectlyleadstoandisexploitedbyavariety of diseases, most notably cancer. Indeed, PI3Kenzymes have been known to have oncogenic proper-tiesfordecades(
6,7
).Inparticular,30%ofhumancan-cersamplesincludingmalignantmelanomacontainso-maticmutationsinthePIK3CAgene,whichencodesthePI3Kp110
catalyticalsubunit.Ofthesemutations,80%consistofatleastoneofthetwohotspotmuta-tionsconferringmarkedincreaseinPI3K
kinaseactiv-
*Corresponding author,marmor@wistar.org.
Received for review February 25, 2008and accepted April 15, 2008.Published online May 16, 200810.1021/cb800039y CCC: $40.75
© 2008AmericanChemicalSociety
ABSTRACT
Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, arefound in a variety of human cancers, implicating the PI3K lipid kinase as an attrac-tive target for the development of therapeutic agents to treat cancer and other re-lated diseases. In this study, we report on the combination of a novel organome-tallic kinase inhibitor scaffold with structure-based design to develop a PI3Kinhibitor,calledE5E2,withanIC
50
potencyinthemid-low-nanomolarrangeandse-lectivity against a panel of protein kinases. We also show that E5E2 inhibitsphospho-AKT in human melanoma cells and leads to growth inhibition. Consis-tentwitharoleforthePI3Kpathwayintumorcellinvasion,E5E2treatmentalsoin-hibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3K
/E5E2 complex reveals the molecular features that give rise to this po-tencyandselectivitytowardlipidkinaseswithimplicationsforthedesignofasub-sequent generation of PI3K-isoform-specific organometallic inhibitors.
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ity(
8,9
).AlthoughPI3KisoformsoutsideofPI3K
haveaweakercorrelationwithsomaticactivatingmutationsinprimarytumorsamples,overexpressionofthesefunc-tionallynonredundantisoformsofPI3Kalsohavetrans-formingabilitiesincancersofspecifictissuetypes(
10
).Forexample,PI3K
andPI3K
isoformsshowincreasedexpressionlevelsincolonandbladdercancersandglio-blastoma,respectively(
10–12
),andthePI3K
isoformplaysanimportantroleintheprogressionofchronicmy-eloidleukemia(
10,13
).Inadditiontohumancancers,PI3Kisoforms
and
havealsobeenimplicatedinrheumatoidarthritisandotherinflammatoryimmunedisorders(
14
).TheaccumulatingevidenceimplicatingPI3K
andotherisoformsasmajoroncoproteinshaspointedtoPI3Kasanimportanttargetforthedevelopmentofsmallmoleculeinhibitors.Tothisend,WortmanninandLY294002weredevelopedandarenowwidelyusedasPI3Kinhibitorsforcellularstudiestoelucidatethemo-lecularmechanismofPI3Ksignaling(
15–18
).However,theseinhibitorssufferfrompoorperformanceintermsof potency,stability,andisoformselectivityandarethere-forenotusefulfortherapeuticpurposes.Recentim-provementshavebeenmadeusingsmall,organicPI3K-specificinhibitors(
14,19
).Nonetheless,therationaldevelopmentofpotentandspecificsmallmoleculein-hibitorsagainstthePI3Klipidkinasesremainsamajor challenge.Theuseoforganometalliccompoundsasscaffoldsfordevelopingproteinkinaseinhibitorsoriginatedfrommimickingstaurosporine,whichisanonselectivekinaseinhibitor.Thisnovelapproachtodevelopkinaseinhibi-tors has a number of advantages (
20
). Most notably, itfacilitates the exploration of a large unexplored area of chemical space with relatively less synthetic effort thanconventionalsyntheticorganicchemistryapproaches(
21–23
).Inaddition,themetalcoordinationbondstorutheniumhavebeenshowntobekineticallystablewithinabiologicalenvironmentwithnometal-relatedcytotoxicities(
24–26
).TheefficacyofthismethodishighlightedbyitsuseindevelopingthemostpotentandselectivekinaseinhibitorsknownforGSK3(
25
)andPIM1(
27
).Herewereportanextensionofthisorgano-metallicinhibitorscaffolddesignapproachtothedevel-opmentofapotentandselectivePI3Klipidkinasein-hibitor,calledE5E2,usingcombinedeffortsofmedicinalchemistry,structure
activityrelationships,andstructure-baseddesign.Wealsoreportthecrystalstruc-tureofthePI3K
/E5E2complextorevealthestructuralfeatures underlying lipid kinase potency and selectivitywithimplicationsforthedesignofasubsequentgenera-tionofPI3K-isoform-specificorganometallicinhibitors.
RESULTSANDDISCUSSIONCocrystalStructureofPI3K
inComplexwithanInitialOrganorutheniumInhibitorLeadCompound.
InordertoidentifyinitialleadinhibitorsforPI3Kfromapyr-idocarbazoleorganometallicruthenium-basedscaffold,alibraryof75compounds(
22,27
)withadiverseconfig-urationofligandsaroundtherutheniummetal(Figure1,panela)werescreenedagainstthehumanPI3K
iso-formusingafluorescencepolarization-basedkinaseas-say(EchelonBiosciences).Fromthislibrary,weidenti-fiedtheracemiccompoundDW12,whichbearsanadditionalhydroxylgroupontheindolegroupasthemostpotentcompoundwithanIC
50
valueof
1
Mus-ingaKinaseGloassay(Figure1,panela;Supplemen-taryFigureS1).DW12isalsoknowntobeahighlypo-tentinhibitorfortheproteinkinasesGSK3(
26
)andPIM1(
23
).Inordertounderstanditsmodeofinhibitionandtoprovideastructuralplatformforstructure-basedin-hibitoroptimizationtoimprovepotencyaswellasselec-tivity,wecocrystallizedoneenantiomer,namedDW2,withhumanPI3K
.ThePI3K
/DW2cocrystalsformedinthespacegroup
C
2withoneprotein/inhibitorcom-plexperasymmetricunitcell,andthestructurewasde-terminedbymolecularreplacementusingthehumanunligandedPI3K
structureasasearchmodel.ThestrongelectrondensitysignaloftherutheniumatomandtheproximaloutlineoftheinhibitorallowedfortheunambiguousplacementoftheDW2inhibitorintothePI3K
activesite(Figure1,panelb).Thestructurewasrefinedto2.8Åresolutiontoexcellentrefinementstatis-ticsandgeometricalparameters(Table1).Overall,theproteincomponentofthePI3K
/DW2structureadoptsthesameconformationastheunligandedPI3K
pro-tein,andtheconservationoftheoverallstructuresug-geststhattheorganorutheniuminhibitorbindstothePI3K
ATPbindingpocketwithoutsignificantalterationofthenativePI3K
conformation(Figure1,panelc).
DetailedViewoftheInteractionsbetweenthePI3K
KinaseDomainandDW2.
Asdesigned,theDW2inhibi-toroccupiestheATPbindingpocketofthekinasedo-mainwitha
2.4Ådisplacementtowardsolventex-posedregionrelativetoATP(Figure1,paneld).ThemaleimidemoietyoftheDW2inhibitorlargelyoverlaps
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XIE
ET AL.
N-lobeC-lobeDW2
NNHNOORuR
1
R
3
R
2
WZYX
Compounds library
NNHNOORuCHOO
DW12
ScreeningN NNOONHHO
Staurosporine
Design
α
2
β
5
β
4
β
7
β
6
α
3
α
9
α
10
α
6
β
10
β
8
α
4
α
12
β
9
DW2 ATPP-loop ActivationloopDW2M804I831
P-loopCpActivationloop
O4O2CO
a bc de f
I881E880V882F961D884M953I963T887K890S806K802W812D964H
2
OO1N12O2O4RuCOCp
Figure 1. Initial lead inhibitor identification and crystal structure of PI3K
in complex with DW2. a) Chemical structures of staurosporine, the organoruthenium scaffold, and the initial lead inhibitor DW12 (racemate) identified from this library.b) Electron density map corresponding to DW2 of the PI3K
/DW2 cocrystal structure, the map is contoured at 4
from asimulated annealing
F
o
F
c
omit map without contribution from the DW2 inhibitor model. c) Overall structure of PI3K
p110 catalytic subunit in complex with DW2; N-lobe, C-lobe, P-loop and activation loop of the kinase domain are color coded blue, red, cyan and yellow, respectively. Inhibitor DW2 is colored white with the ruthenium atom highlighted inmagenta and coordination bonds displayed as magenta dashes. The same color coding scheme is preserved in all panels of this figure. d) Superposition of the DW2-bound PI3K
p110 structure with the ATP-bound PI3K
p110 complex (PDB1E8X). ATP is colored orange, and secondary structure elements are assigned according to the numbering in the ATP com-plex structure (
37
). e) Details of DW2 interactions with PI3K
. Hydrogen bonding interactions are represented with bluedashed lines. Atoms and ligands of interest are labeled and color coded blue. f) Surface representation of DW2 bound tothe PI3K
p110 active site.
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