Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53.

1.9 MB·English

by  Wassman, Christopher D.

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Received6Jun 2012 |Accepted 6 Dec 2012 |Published 29 Jan 2013 DOI:10.1038/ncomms2361 OPEN
Computational identification of a transiently open
L1/S3 pocket for reactivation of mutant p53
Christopher D. Wassman1,2,*,w, Roberta Baronio2,3,*, O¨zlem Demir4,*,w, Brad D. Wallentine5,
Chiung-KuangChen5,LindaV.Hall2,3,FaezehSalehi1,2,Da-WeiLin3,BenjaminP.Chung3,G.WesleyHatfield2,6,7,
A. Richard Chamberlin4,8,9, Hartmut Luecke2,5,9,10,11, Richard H. Lathrop1,2,9,12, Peter Kaiser2,3,9
& Rommie E. Amaro1,4,8,w
The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reacti-
vation of mutant p53 by small molecules is an exciting potential cancer therapy. Although
several compounds restore wild-type function to mutant p53, their binding sites and
mechanisms of action are elusive. Here computational methods identify a transiently open
binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue
Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by
PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this
newlyrevealedpocketselectssticticacidasapotentialp53reactivationcompound.Inhuman
osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for
mutantR175HmorestronglythandoesPRIMA-1.TheseresultsindicatetheL1/S3pocketasa
target for pharmaceutical reactivation of p53 mutants.
1DepartmentofComputerScience,UniversityofCalifornia,Irvine,Irvine,California92697,USA.2InstituteforGenomicsandBioinformatics,Universityof
California,Irvine,Irvine,California92697,USA.3DepartmentofBiologicalChemistry,UniversityofCalifornia,Irvine,Irvine,California92697,USA.
4DepartmentofPharmaceuticalSciences,UniversityofCalifornia,Irvine,Irvine,California92697,USA.5DepartmentofMolecularBiologyandBiochemistry,
UniversityofCalifornia,Irvine,Irvine,California92697,USA.6DepartmentofMicrobiologyandMolecularGenetics,UniversityofCalifornia,Irvine,Irvine,
California92697,USA.7DepartmentofChemicalEngineeringandMaterialsScience,UniversityofCalifornia,Irvine,Irvine,California92697,USA.
8DepartmentofChemistry,UniversityofCalifornia,Irvine,Irvine,California92697,USA.9ChaoFamilyComprehensiveCancerCenter,Universityof
California,Irvine,Irvine,California92697,USA.10DepartmentofPhysiologyandBiophysics,UniversityofCalifornia,Irvine,Irvine,California92697,USA.
11CenterforBiomembraneSystems,UniversityofCalifornia,Irvine,Irvine,California92697,USA.12DepartmentofBiomedicalEngineering,Universityof
California,Irvine,Irvine,California92697,USA.*Theseauthorscontributedequallytothiswork.wPresentaddresses:GoogleInc.,1600Amphitheatre
ParkwayMountainView,California94043,USA(C.D.W.);DepartmentofChemistryandBiochemistry,UniversityofCalifornia,SanDiego;LaJolla,California
92093,USA(O¨.D.,R.E.A.).CorrespondenceandrequestsformaterialsshouldbeaddressedtoP.K.forbiology(email:[e-mail])ortoR.E.A.for
computation(email:[e-mail]).
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T
umoursuppressorproteinp53isatranscriptionfactorthat
has an important role in human cancers. It responds to
various cell stress conditions, such as DNA damage, and
controlspathwaysleadingtoapoptosis(programmedcelldeath),
cellular senescence and cell cycle arrest, among others. Tumour
initiation and maintenance depend upon inactivation of p53
C124 C124
pathways,whichotherwisewoulddeteruncontrolledcellgrowth.
Consequently,p53isthemostfrequentlymutatedgeneinhuman
cancers. Nearly half of all human tumours have mutant p53.
Approximatelythree-quartersofthosetumoursthathavemutant
p53 are found to express full-length p53 protein with a single-
residue missense mutation in the p53 DNA-binding core
domain1,2.
Theresultingpresenceoffull-lengthandsingle-point-mutated
p53 protein in about one-third of all human tumours holds the
promise of drugs that reactivate mutant p53 function, deter
cancer cell proliferation, and shrink or kill the tumour3. Several
p53-controlledpathwaysinhibitcellproliferation,andsoarevalid
pharmaceutical targets. Studies on transgenic mice demonstrate
that p53 reactivation enables tumour regression in vivo, even in
advancedtumourcases4–9.Reactivationofp53functionhasbeen
demonstratedinvariousp53cancermutantsbybothsecond-site
suppressormutations10–16andahandfulofsmallmolecules17–25.
Themostwell-studiedandpromisingofthesesmallmoleculesare
PRIMA-1and PRIMA-1Met,currently in phase I/IIclinical trials
(APR-246-01, http://www.aprea.com/home/). PRIMA-1 is
converted into several active products26, among them Figure1|Molecularvisualizationsofthep53coredomain.(a,b)All
methylene quinuclidinone (MQ), that react covalently to residuesofthep53coredomain(1TSR-B33)within10ÅofCys124are
alkylate p53 cysteine residues. This alkylation can reactivate p53 showninasurfacerepresentationcolouredbyatomtype:cyan,C;blue,N;
function26,althoughtheprecisereactivationmechanismremains red,O;yellow,S.(a)Cys124isinitiallyoccludedinthewtcrystalstructure.
elusive27. (b)StructureextractedfromMDsimulationsofmutantR273Hrevealsnew
Here we describe computational analysis of p53 structural breathingstructuraltopographynearCys124withintherangeofnormal
models that suggests a transiently open L1/S3 binding pocket solutiondynamics.(c,d)Thep53coredomainisshowninmagenta,loopL1
around Cys124, Cys135 and Cys141 as a possible target for inblue,strandS3ingold.(c)Cys124(yellow)issurroundedbygenetic
reactivation of p53 mutants by small molecules. The pocket was mutationsitesthatcanreactivatep53function(green).(d)Druggable
identified through genetic mutations, all-atom molecular pockets(cyan)fromFTMAP44fortheR273Hmutant.TheopenL1/S3
dynamics (MD) simulations of the p53 core domain, computa- pocketsurroundsCys124(yellow).
tional solvent mapping of the resulting p53 structural ensemble
and ensemble-based virtual screening. Mutation of Cys124 to
alanineabolishedknownR175HreactivationeffectsofPRIMA-1 p53reactivationregion,andcovalentalkylationofacysteinethiol
in human Saos-2 cancer cells. Ensemble-based virtual screen- group had been suggested for p53 reactivation by PRIMA-1
ing28,29 was used to select 45 compounds with the most conversion products such as MQ and other p53 reactivation
favourable predicting binding affinity to the L1/S3 pocket. Of compounds26.Inthiscysteinetriad,Cys124wasthemostsolvent-
these, stictic acid emerged as a p53 reactivation compound in accessible, followed by Cys141 and then Cys135, in both the
cultured human osteosarcoma cells, with the ability to stabilize crystal structures and the MD simulations (Table 1). Initial
p53 in vitro. alkylation of Cys124 had been suggested to enable alkylation of
Cys141 and then Cys135 (ref. 32).
In the crystal structures of the human p53 core DNA-binding
Results domain(PDBIDs1TSR33,2OCJ34),Cys124ispartiallyoccluded
Computational identification of the L1/S3 binding pocket. andtheputativeL1/S3reactivationpocketisnotreadilyaccessible
Initial docking of known p53 reactivation compounds (MQ, 3- tosmallmoleculesmuchlargerthanwater.Thisocclusioninthe
methylene-2-norbornanone (NB), STIMA-1, MIRA-1, 2, 3 (refs crystal structure might seem to preclude alkylation of Cys124 in
17–22)) indicated preferential binding to a specific region in the humanp53.Blinddockingofknownp53reactivationcompounds
mousep53coredomaincrystalstructure(PDBID3EXJ30),which onto the entire protein surface of the human p53 core domain
has high sequence identity (B90%) to human p53. The crystalstructurePDBID1TSRchainB(1TSR-B)33indicatedthat
corresponding region in the human p53 core domain is flanked noneofthecompoundsboundincloseproximitytoanycysteine,
by loop L1 and sheet S3 (hence, the L1/S3 pocket). The region including those in the L1/S3 pocket (Supplementary Fig. S1).
had been labelled by genetic mutation studies10–16 as highly However, the L1 loop can undergo a conformational switch to
enriched for p53 reactivation mutations (Fig. 1; Phe113, Leu114, regulate DNA binding by an induced fit mechanism35,36. NMR
His115, Thr123, Leu137 and Asn235), indicating that subtle studies demonstrate that Cys124 is exposed to solvent B5% of
structural changes in this region can reactivate p53 cancer the time37. Mass spectrometry results indicate that Cys124 and
mutants. Four of those reactivation mutation sites (Phe113, Cys141 react preferentially with alkylating agents32 and with
Leu114, His115 and Thr123) plus Cys124 exhibit significant or glutathione38. Alkylating agents cause NMR chemical shifts of
moderate nuclear magnetic resonance (NMR) chemical shifts in several residues within the L1/S3 region32, including Leu114,
responsetobindingofthep53-stabilizingpeptideCDB3(ref.31). His115 and Thr123, already implicated above by genetic
Cys124, Cys135 and Cys141 are near the centre of this putative mutations and CDB3-induced NMR chemical shifts.
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Table1| Summaryof MDsimulation trajectories andopen pocket occurrences.
wtp53 R175H G245S R273H C124A
Totalnumberofclustersintrajectory 28 39 74 41 50
Openpocketas%oftotaltrajectorybygeometriccriteria 6.3 7.3 6.7 5.0 5.4
Openpocketas%oftotaltrajectorybyFpocket43 27.3 29.9 22.9 7.3 35.3
Avg.SASAÅ2±s.d.ofCys124sidechaininMD 10.93±4.81 9.58±4.45 6.94±4.71 8.38±5.84 (124Alavalue)4.45±2.99
Cys124exposureas%ofMDframeswithSASA45Å2 90.80 86.97 60.70 64.97 (124Alavalue)34.20
Avg.SASAÅ2±s.d.ofCys141sidechaininMD 0.51±0.84 1.03±1.27 0.56±0.86 0.78±1.32 1.56±1.54
Cys141exposureas%ofMDframeswithSASA45Å2 0.33 1.53 0.43 2.06 3.00
Avg.SASAÅ2±s.d.ofCys135sidechaininMD 0.17±0.48 0.41±0.91 0.09±0.27 0.09±0.34 0.20±0.44
Cys135exposureas%ofMDframeswithSASA45Å2 0.06 0.50 0.00 0.00 0.03
MD,moleculardynamics;SASA,solvent-accessiblesurfacearea.
Wild-type p53 is marginally stable at physiological
temperature. The DNA binding domain unfolds with a half-life
of around 9min3 and has a low melting temperature (T ) of
m
about 36–461C depending on experimental conditions39–41. All-
atom explicit solvent MD simulations for 30ns of wild-type p53
and three frequent cancer mutants, R175H, G245S and R273H,
revealed a high degree of flexibility for the core domain42 and
particularly the L1/S3 region surrounding Cys124
(Supplementary Movie 1). Clustering the MD ensembles with
respect to the L1/S3 pocket conformation further demonstrated
the flexibility of the region (Table 1). In the MD ensembles of
different p53 variants, average solvent-accessible surface area
(SASA) values of the Cys124 side chain were calculated to be in
therangeof6–11Å2(Table1).Insharpcontrast,inthep53core
domaincrystalstructure1TSR-BtheCys124sidechainSASAwas
calculated to be only 0.43Å2.
DockingintotheL1/S3pocket.AtransientlyopenL1/S3pocket
formed around Cys124 due to positional and conformational
changesofSer116,Thr123,Cys124,Thr140andespeciallyLeu114
(Fig. 1). Three of these residues, Leu114, Thr123 and Cys124,
already were implicated above. We determined five distance/
dihedral angle criteria (Supplementary Methods) that indicated
formation of an open L1/S3 pocket in about 5–8% of each
simulation (Table 1). Fpocket43, a program to measure the
volume of a defined binding pocket (Fig. 1), also identified an
open L1/S3 pocket around Cys124 across several p53 variants in
5–30% of the 3,000 frames of each MD trajectory (Table 1). For
evaluatingFpocketresults,theL1/S3pocketwasconsideredopen
if its volume was 4150Å3, a lower bound chosen based on
cancer mutant R273H, which exhibited thelowest average L1/S3
pocket volumes. FTMap44, a program to identify ligand-binding
hot spots in proteins using organic solvent probes, predicted the
L1/S3 pocket to be among the most highly occupied by the
solventprobeswhenusedtoassessdruggabilityofMD-generated
Figure2|Knownp53reactivationcompoundsdockedintotheopenL1/
conformations (Fig. 1). For wt p53 and mutant R273H MD S3pocketofR175H.(a)MQ(fromPRIMA-1)26,(b)NB21,(c)STIMA-1(ref.
trajectories,FTMappredictedadruggableL1/S3pockettooccur,
22),(d)MIRA-1(ref.19),(e)MIRA-2(ref.19),(f)MIRA-3(ref.19).Known
respectively, in 11 and 7 of the 15 most-populated cluster
p53reactioncompoundscoulddockfavourablyintothemost-populated
centroids. In contrast, neither Cys124 nor any other cysteine in
R175HMDtrajectoryclustercentroidthathadanopenL1/S3pocket.Allof
the original PDB crystal structure 1TSR-B33 was identified as a
theninedockingposesgeneratedbyAutodockVinaforeachcompound
druggable site by FTMap.
abovescoredwithinthepublishedstandarderrorofAutodockVinadocking
Several previously identified alkylating p53 reactivation scores60(SupplementaryTableS1).Ineachcase,thebindingposedepicted
compounds, MQ26, NB21, STIMA-1(ref. 22) and MIRA-1, 2,
abovehadthesmallestdistancebetweenthereactivemethyleneofthesmall
3 (ref. 19), subsequently were docked into the most-populated
moleculeandtheCys124sulphydrylgroup.Theproteinsurface
MD trajectory cluster representative that had an open L1/S3
representationispurple,exceptCys124atomsarecolouredbyatomtypeas
bindingpocketasdefined(Table1,SupplementaryMethods).All
inFig.1a,b.
of these known active compounds presented low-energy
predicted docking poses with the reactive methylene oriented in corresponding ligand interaction diagrams, and Supplementary
close proximity to the Cys124 sulphydryl group for p53 cancer TableS1providesthecorrespondingdockingstatistics.Reliability
mutant R175H (Fig. 2). Supplementary Fig. S2 depicts the of the molecular docking strategy was supported by a positive
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control, PhiKan083 (ref. 23), which docked within 1.0Å root- L1/S3pocketidentifiesanovelp53reactivationcompound.An
mean-square-deviation(RMSD)ofthereferencecrystalstructure ensemble-basedvirtualscreeningapproach,knownastherelaxed
(Supplementary Fig. S1). The transiently open L1/S3 pocket was complex scheme28,29, was performed against the mutant R273H.
sharedacrosswild-typeandthethreep53cancermutantsstudied Initially, 1,324 compounds from the NCI/DTP Open Chemical
(SupplementaryFig.S3).Thus,severallinesofevidencesuggested RepositoryDiversitySetIIwerevirtuallyscreened(TheNCI/DTP
a transiently open and druggable binding pocket in the L1/S3 Open Chemical Repository, http://dtp.cancer.gov). They were
region of the human p53 protein. docked into each of the most-populated 15 cluster centroids—
altogether representing 92.5% of the entire trajectory—obtained
by clustering the MD trajectory of the R273H cancer mutant
Biological validation of the L1/S3 binding pocket hypothesis. according to the L1/S3 pocket conformation. Compounds with
Weexamined theroleof Cys124intheL1/S3bindingpocketby predicted binding affinities of (cid:2)6.5kcalmol(cid:2)1 or better to any
of the 15 centroids were chosen with a limit of 50 compounds
site-directed mutagenesis of Cys124 to alanine (C124A). If
Cys124were acritical residuefor p53reactivation, then removal fromeachcentroid.Theresulting84compounds(corresponding
to B6% of the entire NCIDS2 database) were ranked according
of its thiol group by this otherwise innocuous mutation would
abolish the PRIMA-1 reactivation effect. For example, Cys124 to their population-weighted average binding affinities. The
absorption, distribution, metabolism, and excretion (ADME)
might be a key attachment residue for PRIMA-1 conversion
products such as MQ26, it might initiate a Cys124-Cys141- properties of the compounds, as predicted by Schrodinger
Cys135 alkylation cascade32, or it might form a disulphide bond (QikProp, version 3.3, Schro¨dinger, LLC, New York, NY, 2010),
that stabilized p53 in an active conformation induced by were used for further filtering. Lipinski’s Rule of Five and
Jorgensen’s Rule of Three were followed strictly, resulting in 24
compound binding. The literature suggests that a reducing
environment leads to correct p53 folding and disulphide bond compounds.Similarly,compoundsfromtheNCIMechanisticSet,
formationmayleadtoaggregation27.Thereisnoapriori reason NCINaturalProductsSet,NCICOMBOSet,andNCIOncology-
Approved Set (consisting of 974 compounds in total) were
to rule out any of these hypotheses.
virtually screened and filtered for ADME properties. A more
p53 null human osteosarcoma Saos-2 cells were engineered to
havedoxycycline-inducible expression of eitherwild-typep53 or stringent ADME filter (octanol/water partition coefficient o3)
was enforced to improve compound solubility. A similar
various p53 mutants. Single cloneswith similar expression levels
predicted binding affinity cutoff of (cid:2)6.5kcalmol(cid:2)1 was used,
of the different p53 variants were selected for direct comparison
resultingin24additionalcompounds.Threeselectedcompounds
of small-molecule effects (Supplementary Fig. S4). As expected,
were discarded because they were unavailable (NSC-653010,
expression of wild-type p53 in Saos-2 cells halted cell
NSC-92339 and NSC-653016). In total, 45 compounds were
proliferation, whereas expression of mutant R175H had no
selected for biological assay (Supplementary Tables S2 and S3).
effectoncellproliferation(Fig.3).MutationofCys124toalanine
One of these compounds, stictic acid (NSC-87511), proved to
had little effect on otherwise wild-type p53 (C124A) nor did it
be a p53 reactivation compound. Figure 4 illustrates the best-
restore activity of R175H (R175H_C124A), indicating that the
scoringsticticacidposewhendockedintotheL1/S3pocketsofwt
C124A mutation is largely neutral for general p53 function.
Consistent with previous results26, addition of PRIMA-1 to cells p53 and mutants R175H, R273H and G245S. Supplementary
expressing inactive mutant R175H led to p53 reactivation and
subsequent inhibition of cell cycle progression. Importantly,
PRIMA-1reactivationofp53functionwasblockedinthedouble
mutantR175H_C124A(Fig.3),demonstratingtheimportanceof
Cys124 in pharmacological reactivation of mutant R175H by
PRIMA-1. These experimental results supported the
computational identification of the L1/S3 binding pocket
surrounding Cys124 as significant for p53 reactivation.
20
n
se i2 h 15
a7
e increumber 10
vn
elaticell 5
R
0
PRIMA-1 + – + – + – + – + –
p w C R CR
5 t 1 1 11
3 2 7 27
null 4A 5H 4A5H
Figure3|PRIMA-1actsthroughcysteine124inp53.p53nullSaos-2cells Figure4|SticticaciddockedintoopenL1/S3pocketofp53variants.
expressingwild-typep53,ortheindicatedp53mutants,weretreatedwith SticticacidcoulddockfavourablyintotheopenL1/S3pocketof
50mMpreheatedPRIMA-1(bluebar)orthemediumwithoutPRIMA-1 MD-generatedsnapshotsforallfourp53variantsstudied:(a)wtp53;
(DMSO,greenbar)for72h.Theincreaseincellnumberduring72hof (b)R175H;(c)R273H;(d)G245S.Onlypolarhydrogensofsticticacidare
growthisshown.Thes.d.representfourindependentsamples. depictedhere.ColoursareasinFig.2.
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Figure S5 shows how stictic acid ranked on a histogram of best specificmannerasexpected25(Fig.6a).Activationofp21reporter
dockingscoresofthescreenedcompoundsusingmutantR273H, transcription was more pronounced and confirmed that stictic
and Supplementary Fig. S6 compares docking scores between acid is a p53 mutant reactivation compound comparable to
mutants R273H and R175H (correlation coefficient¼0.80). The previously identified compounds.
predicted binding affinities, z-scores, mean and standard Differential scanning fluorimetry (DSF) provides the ability to
deviation for the nine docked poses of stictic acid with mutant detect molecular interactions between a small molecule and a
R175H are presented in Supplementary Table S1. To gauge the proteinbasedonchangesinthemeltingtemperature(T )ofthe
m
stability of stictic acid in the L1/S3 active site, a MD simulation protein46. These T changes are taken to reflect changes in
m
was performed starting from the best-scoring docking pose of protein stability upon binding of thesmall molecule. Variants of
stictic acid in mutant R175H. The initial docking score was the p53 core domain (wild type, G245S and R175H) were tested
(cid:2)6.97 and the final docking score was (cid:2)7.11. Supplementary in DSF assays with a range of concentrations of PRIMA-1, MQ
FiguresS7andS8showthatthep53coredomainwasstableand and stictic acid (Fig. 7). The T values in the absence of
m
stictic acid persisted in the L1/S3 pocket throughout the entire compounds for wild type, G245S and R175H, at 4mM, were
60ns MD simulation. Supplementary Figure S9 compares the 36.54±0.11, 33.51±0.09 and 27.89±0.111C, respectively.
posesanddetailedmolecularinteractionsofsticticaciddockedto PRIMA-1 and MQ stabilized each of the p53 variants by
mutantR175HinthedockedposeofFig.4bandagainafter60ns Br11 for concentrations ranging from 10 to 80mM, 2.5(cid:3) to
of MD simulation. 20(cid:3) stoichiometric ratio of compound to protein (Fig. 7). The
greatestthermalstabilizationwasobservedwithsticticacid,upto
4.60±0.391C at 80mM in mutant R175H. All three of these
Experimental support for stictic acid and p53 reactivation. compounds demonstrated a relative dose dependence in their
Stictic acid partially restored p53 activity in human Saos-2 cells abilitytostabilizep53.Noneofthesecompoundshadstabilizing
expressing cancer mutant R175H (Fig. 5), as demonstrated by effectsonthreecontrolproteinstested,annexinA2,S100A4and
induction of the cell cycle inhibitor p21. p21 is a canonical gene S100A10, suggesting that their interactions are specific to p53.
target of active p53 and a sensitive and selective output for p53
activity. Induction of p21 by stictic acid was dose-responsive, as
shown in Fig. 5. It was also dependent upon p53, because it had Discussion
onlyaminoreffectonp21expressionincellslackingp53(Fig.5, Despite continuous progress in cancer research and care, mil-
Supplementary Fig. S4). lions of people die annually from this disease. Therapeutic reac-
p53 primarily functions as a transcriptional activator. To test tivationofp53couldpreventordelaymanyofthesedeaths.The
moredirectlytheeffectofsticticacidonrestoringtranscriptional p53Y220Creactivationpocketalreadyhasbeenusedforrational
activator function of p53 mutants we generated stable cell lines drugdesignanddiscoveryinthatspecificmutation23,24.Herewe
harbouring reporter constructs driven by two different p53- reportcomputer-guidedidentificationofatransientlyopenL1/S3
dependentpromoterelements.Theseelementswerederivedfrom
thepromotersofthecellcycleinhibitorp21andtheproapoptotic
gene PUMA and have previously been utilized to monitor p53 a 3 PUMA
transactivationactivity45.Wecomparedtheeffectofsticticacidto
that of PRIMA-1 and the recently identified allele-specific p53 nt 2.5 DMSO
e
rcpreeo5apm3cotprimtvoeauurtntiPoadnUnstMsmcAoRodm,1ee7psx5tocHlyuepnbtadunNtdSNsCiGgS-nC23i4-1fi359c1S7a92n75tto2l,y5werhex(siprctreohefr.saesdc2ttet5hdh;eeinFapibragion.laitpmy6o)uop.tftaotnAthitlce-l p53-dependactivation1.152 SBNtoSiciCltei3cd 1 ap9cr7iimd25a-1
Rel. 0.5
0
0.8
R175H G245S
R175H mutant
p53null cells b p21
0.6 10
els ent 8 DMSO
p21 Lev00..42 p53-dependactivation 46 SBNtoSiciCltei3cd 1 ap9cr7iimd25a-1
el. 2
R
0
0 R175H G245S
0 5 10 40 50
Stictic acid (µM) Figure6|p53-dependentactivationofPUMAandp21transcription
reporterbyreactivationcompounds.Preheated26PRIMA-1(25mM),
Figure5|Dose-dependentp21activationinresponsetosticticacid. NSC-319425(25mM)andsticticacid(10mM)wereaddedtostableSoas-2
Saos-2cells(p53null)andSaos-2cellsstablyexpressingthep53cancer (p53null)celllinesharbouring(a)PUMA-or(b)p21-dependentluciferase
mutantR175Hweretreatedwithdoxycyclinefor8htoinduceexpressionof reporterandexpressingthep53mutantsR175HorG245Sasindicated.
mutantR175H.Cellswerethengrownfor48hinthepresenceofsticticacid ActivationofPUMAandp21transcriptionalreporterwasmeasuredby
attheconcentrationsindicated.p21expressionwasquantifiedusingaFuji luciferaseactivityassays.Reportercelllineswithoutp53expressionwere
LAS-4000imagingsystemandplottednormalizedtotheexpressionof processedinparallelandusedfornormalizationtovisualizep53-dependent
tubulinineachsample. effects.Dataarerepresentedasmean±s.d.(n¼4).
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Wild Type G245S R175H
43.0 40.0 34.0
42.0 39.0 33.0
C)
e (° 41.0 38.0 32.0
ur 40.0 37.0 31.0
at
er 39.0 36.0 30.0
p
m 38.0 35.0 29.0
e
T
37.0 34.0 28.0
36.0 33.0 27.0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
(0:1) (5:1) (10:1) (15:1) (20:1) (0:1) (5:1) (10:1) (15:1) (20:1) (0:1) (5:1) (10:1) (15:1) (20:1)
Compound concentration (µM)
(Stoichiometric ratio of compound:protein concentrations)
Figure7|Thermalstabilizationofp53mutantsbyreactivationcompounds.SelectedconcentrationsofPRIMA-1(blue,square),MQ(green,circle)and
sticticacid(red,triangle)wereaddedto4mMofwildtype,G245SandR175Hcoredomainprotein.ThermalstabilizationwasmeasuredusingDSF.Data
pointsareplottedasthemeanofsextupletmeasurements,withcorresponding95%confidenceintervals.Confidenceintervalbarsnotvisiblearesmaller
thanthesymbolusedtoplotthedatapoint.PRIMA-1andMQstabilizedeachp53variantabout11Corlessoverthisrangeofconcentrations.Sticticacid
resultedinthegreateststabilizationforbothp53mutantsstudied.
reactivation pocket in p53 that may present a more broadly crucialLeu114residue.AsuitableLeu114conformationoccurred
applicable druggable region for such cancer therapies. The in only three or four frames of the entire 3,000-frame 30-ns
coordinates of the human p53 core domain structures with the trajectory (Methods, RMSD-based clustering). The sampling in
open binding pocket conformations shown in Fig. 4 appear as MDislimited,sothefactthatanysuitablepocket-openingevents
Supplementary Data S1, and also have been deposited in the were seen at all is indication that such pockets exist at thermal
Protein Model DataBase (PMDB)47 (http://mi.caspur.it/PMDB/; equilibriumforthismutant.Indeed,threeoccurrencesin30nsis
PMDBID(a)PM0078199forwtp53;(b)PM0078200forR175H; equivalent to 108 occurrences per second, whereas three
(c) PM0078201 for G245S; and (d) PM0078202 for R273H) and occurrences in 3,000 frames is equivalent to a probability of
inSourceForge (http://sourceforge.net/projects/p53cancerrescue/, 10(cid:2)3,andsotheeventcannotbeconsideredrare.Instead,wesee
in folder ‘Coordinates of p53 core domain’). We anticipate that here a limitation of our current computational methods for
theL1/S3reactivationregionreportedherewillbeusefultoother modelling complex molecular processes.
researchers carrying out structure-based drug discovery and AsseeninSupplementaryFigsS7andS9,sticticacidremained
development of p53 reactivation drug leads. dockedafter60nsofstableMDsimulation.Therotamerstateof
AnotableadvantageoftheL1/S3pocketisthatitoccursacross Leu114 was critical toopening upa binding site largeenoughto
severalp53variants,asseeninFig.4andSupplementaryFigsS3 accommodate large molecules such as stictic acid. In the initial
and S6, giving hope that p53 reactivation compounds can be dockedposeofsticticacid(SupplementaryFig.S9a),thereisonly
developedtotargetseveralp53cancermutantssimultaneously.In one direct H-bond interaction between stictic acid and the
contrast, for example, known compounds that target the p53 Cys124 backbone amino group, in addition to various
Y220C reactivation pocket23,24 may be specific to the Y220C hydrophobic interactions including Pro142 and Leu114
mutant,becausespecificstructuraldetailsoftheYtoCmutation (Supplementary Fig. S9c). The hydrophobic interactions with
were used in their design or discovery. None of the R175H, Pro142 and Leu114 were conserved throughout the simulation.
G245SorR273HmutationslieintheL1/S3pocketorhavedirect The final pose (Supplementary Fig. S9b) provides an elegant
effects on its conformation. Its generalizability is illustrated here illustrationofinducedfituponbinding.Sticticacidmoveddeeper
by the result that virtual screening against one mutant, R273H, into the pocket, achieved a very nice shape complementarity,
produced a compound that resulted in biological validation substantially expanded the initial network of favourable direct
againstadifferentmutant,R175H,andinducedDSFtemperature and water-mediated interactions, and shifted the ligand and
shifts in all p53 variants studied. receptor exposure patterns (Supplementary Fig. S9d).
Protein conformational flexibility is well-recognized as an PRIMA-1hasconversionproductsthataremoreactivethanit
important factor in ligand binding. A few computational drug is itself26, and this may be the case for stictic acid as well. The
discovery approaches attempt to accommodate protein-induced existence of a highly active and bioavailable stictic acid
fitonligandbindingbyallowingsidechainstomovetoimprove decomposition product was suggested by initial experiments
the fit of the ligand into the binding pocket. Very few such carried out with a stictic acid solution that had a long storage
approachesallowtheproteinbackbonetomovetoaccommodate time. Suchhypothetical conversion products werenotpresent in
the ligand. Nevertheless, the results above provide a cautionary thefreshlyorderednewlydissolvedpurecompound,fromwhich
tale: a ligand-binding pocket of medical importance may be the results presented here were derived.
hiddenanddifficulttorecognizeinstructuraldatabases,yetother It is unclear whether or not stictic acid requires covalent
virtual structures extracted from its conformational ensemble linkage to p53 for reactivation according to the general cysteine
may support successful structure-based drug discovery. With alkylation mechanism proposed by Lambert et al.26 for several
appropriate protein conformations in hand for computational otherp53reactivationcompounds.Sticticacidmay,inprinciple,
analysis, we screened only 45 compounds through biological bindcovalentlytonucleophilicproteinsidechainsbyacylatingor
assays to find a novel p53 reactivation compound, stictic acid. alkylating nucleophilic residues such as cysteine. Likely reaction
Our relaxed complex scheme28,29 clusters are based on sitesandmodesofreactivity(SupplementaryFig.S10)includethe
conformations of all residues surrounding the L1/S3 pocket, not direct addition of a nucleophile (a cysteine thiol group of the
on specific conformations of particular residues. No cluster protein, as represented in the figure) to either the seven-
centroidproducedasuitabledockingposeforsticticaciddocked membered ring lactone carbonyl group (A) or to the carbonyl
to G245S (Fig. 4d), due to the position and rotamer state of the group of the five-membered ring lactone (B), resulting in
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NATURECOMMUNICATIONS|DOI:10.1038/ncomms2361
acylation of the thiol and covalent attachment to the protein as druggable protein regions, and highlight the feasibility of using
the respective thioester. Alkylation is another possible MD simulations to expose druggable protein regions that
mechanism, occurring via a more circuitous mechanism (C) in otherwise may not be evident.
which dehydration generates a reactive quinone methide-type
speciesthatcanalkylatenucleophiles,asillustratedschematically
for a p53 cysteine side chain thiol group. An alternative Methods
dehydration pathway (loss of a proton on the para-methyl MDsimulations.Wild-typep53MDsimulationswereperformedusingcrystal
group,notshown)couldproduceadifferentmethidethatwould structure1TSR-B33fortheinitialcoordinates.ModelsformutantsR273H,R175H,
G245SandC124Awerepreparedbyvirtualmutationofwild-typep53usingthe
resultinalkylationatthecorrespondingbenzylicposition.There AMBER10suite48.Thecrystallographicwatermoleculeswereretainedandeach
is no a priori reason to rule out any of these pathways. systemwassolvatedinaTIP3Pwaterbox49.Zinc-coordinatingresidueswere
InDSFexperiments,preheatedPRIMA-1(toinduceformation modelledusingthecationicdummyatommodel50.Protonationstatesoftitratable
of conversion products26) induced thermal shifts (T increases) residuesweredeterminedusingtheWhatifWebInterface51.Histidine,asparagine
m andglutaminesidechainorientationswerecorrectedasneededusingthe
that were slightly higher than that for MQ. Though slight, the Molprobitywebserver52.Eachsystemwasneutralizedbyaddingchlorideionsas
differences were statistically significant at higher concentrations, needed.Thestructurefilesofeachsystemconsistedofabout27,260atoms,which
whichisconsistentwiththepreviousevidencethatMQisnotthe werepreparedusingtheAmberFF99SBforcefield53.
mostactiveconversionproductofPRIMA-1(ref.26).Thesetwo Eachofthep53variantsystemswasfirstrelaxedusing36,000stepsof
minimizationfollowedbyfourconsecutiverestrained250psMDsimulations.
known reactivation compounds showed significantly lower
Thereafter,30nsunrestrainedMDsimulationwasperformedat310Kand1atm
stabilizing effects on the p53 mutants tested than did stictic foreachsystemusingtheNAMD2.7suite54.DetailsoftheminimizationandMD
acid. Stictic acid is a much larger molecule than MQ. It has the simulationprotocolcanbefoundinDemiretal.42
potential to make more interactions within the binding pocket Togaugethestabilityofthesticticacidinthebindingpocket,a60nsMD
simulationwasperformed.Thebest-scoringdockedposeofthemoleculeinthe
(compareFigs2with4),perhapsgivingitagreatercapacitythan
R175HL1/S3open-pocketconformationwasusedasthestartinggeometry
PRIMA-1 or MQ for stabilizing p53 cancer mutants. We cannot
(Fig.4b,SupplementaryTableS1).Sticticacidparametersweregeneratedwiththe
rule out other effects, for example, the possibility that the low AntechambermoduleofAmber10usingtheGeneralizedAmberForceField55,56
thermal shifts for PRIMA-1 may be due to its incomplete withRESPHF6-31G*charges.
conversion into more active products, or that the low thermal Foreachp53variantstudied,thesolvent-accessiblesurfaceareaoftheCys124
sidechainovertheMDtrajectorywascalculatedusingacustomizedvisual
shiftsforMQmaybeduetoitshighlyreactivenatureleadingto moleculardynamics57(VMD)script.
rapid decomposition on contact with water. Thecoordinatesofthehumanp53coredomainstructureswiththeopen-
Sticticaciddisplayedpromisingresultsinreactivatingseverely bindingpocketconformationsshowninFig.4appearasSupplementaryDataS1,
destabilized mutant R175H in human cancer cells. Reactivation andalsohavebeendepositedinthePMDB47(http://mi.caspur.it/PMDB/;PMDB
ID(a)PM0078199forwtp53;(b)PM0078200forR175H;(c)PM0078201for
was evident from dose-dependent and p53-dependent induction
G245S;and(d)PM0078202forR273H)andinSourceForge(http://
of p21 expression. Cell death was not activated by stictic acid, sourceforge.net/projects/p53cancerrescue/,infolder‘Coordinatesofp53core
which may be due to insufficient reactivation of cancer mutant domain’).
R175H or may indicate pathway-selective restoration of p53
activity. Such probes with pathway discriminatory activity will
become valuable tools for the study of p53 biology. PUMA RMSD-basedclustering.Proteinsnapshotsextractedfromthesimulationswere
reporter assays demonstrated comparable p53 reactivation by clusteredwithrespecttoallheavyatomsoftheL1/S3site.Foreachvariant,atomic
coordinateswereextractedat10psintervalsovertheir30nsMDtrajectory,
stictic acid and PRIMA-1 (Fig. 6), yet PRIMA-1 is a potent
resultingin3,000simulatedstructurespertrajectory.These3,000structureswere
inducer of apoptosis in cells with mutant p53. It is possible that superimposedwithrespecttoallCaatomstoremoveoveralltranslationand
additional, p53-independent cytotoxic functions of PRIMA-1 or rotation.Theywerethenclusteredusingthegromosalgorithm58intheGROMACS
one of its degradation products26 act synergistically with p53 4.0.5program59atanRMSDcutoffvalueof1.65Å,basedonatomiccoordinatesof
allatomsofthoseresiduesthathadatleastoneatomwithin10ÅofCys124inthe
reactivationtoinduceapoptosis.Suchadditiveeffectsmaynotbe
minimizedandequilibratedformofeachp53variant.Ineachcluster,thestructure
induced by stictic acid. withthesmallestaverageRMSDtoalltheotherstructuresintheclusterwas
MutantR175Hshowedagreaterdegreeofthermalstabilization designatedasthe‘centroid’or‘representative’structureofthecluster.
inDSFbysticticacidthandidwildtypeorG245S.Perhaps,thisis ClustercentroidsexhibitingopenL1/S3pocketswereidentifiedforeachp53
variantusingthegeometriccriteria(SupplementaryMethods).Theseopen
because R175H is initially highly destabilized (a Zn region
centroidswereusedfordockingsticticacidandtheknownp53reactivatingsmall
mutant, 30% folded at 371C40) while wild type and G245S (a molecules.InthecaseofG245S,theknownsmallmoleculesweresuccessfully
DNAregionmutant,95%foldedat371C40)arenot.Theseresults dockedintotheclustercentroidwithanidentifiedopenL1/S3pocket.However,it
indicate that stabilization effects and reactivation outcome wasnotpossibletoidentifyaclustercentroidforG245Sthatproducedabinding
poseforsticticacid(arelativelymuchlargermolecule)inwhichapossible
depend on the p53 mutation, and that thermal stabilization of
alkylationreactionwiththeCys124sidechainseemedreasonable.Acloserlookat
the p53 core domain may reactivate some p53-controlled
theG245SMDtrajectoryrevealedthatthepositionandrotamerstateofLeu114
pathways and not others in a way that depends on the ligand. wascriticalforL1/S3pocketopening.Leu114,alreadyimplicatedabove,exhibiteda
DSFinvitroexperimentsindicateddirectbindingofsticticacid favourableconformationinonlythreeorfourframesoftheentire3,000-frame
to all p53 variants tested. Induction of p21 expression by stictic trajectory.Thus,asnapshotwithanopenL1/S3pocketwasextractedmanually
fromtheG245SMDtrajectoryandsubsequentlyusedtodocksticticacid.
acid showeddependence on p53-mediated pathways, because no
such induction was observed in the Saos-2 parent cell lines
lacking p53. Taken together, these in vitro and in cellulo results
Insilicodocking.InitialdockingstudieswereperformedusingAutoDock4.2.2
indicate that stictic acid acts directly on p53. (SMILESstringsforallsmallmoleculesconsideredexplicitlyinthispaperaregiven
Recently, two compounds from the thiosemicarbazone family inSupplementaryTableS4).AllsubsequentdockingstudiesusedAutoDockVina60
(for example, NSC-319725) were identified as allele-selective withanexhaustivenessvalueof100anda20(cid:3)20(cid:3)20Åsearchspacecentredon
reactivation compounds for p53 cancer mutant R175H25. thesulphuratomofCys124.Ninedifferentdockingposeswerecalculatedforeach
consideredcombinationofacompoundandap53variant.Forinitialcompound
Whether these compounds directly bind to p53 and target the
scoring,population-weightedaveragebindingaffinitiesofthemost-populated15
L1/S3 reactivation pocket identified in this study is currently clustercentroidsfromtheR273HMDtrajectorywerecalculatedusingthebest
unclear, but will be interesting questions to address in future bindingaffinityfoundbyAutodockVinaforeachcompound.The48compounds
experiments. selectedwerere-scoredusingthepopulation-weightedaveragebindingaffinitiesof
themost-populated30clustercentroids.Ligandinteractiondiagramswere
Our results demonstrate the value of integrated strategies
preparedusingtheMolecularOperatingEnvironmentprogram(Chemical
combining genetics and computation for identification of ComputingGroupInc.,Montreal,Canada).
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&2013MacmillanPublishersLimited.Allrightsreserved.
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NATURECOMMUNICATIONS|DOI:10.1038/ncomms2361
ComputationalfragmentmappingusingFTMap.TheFTMapprogram44utilizes 16,530cm(cid:2)1M(cid:2)1at280nm.Allproteinpurificationstepsweremonitored
16probemoleculestoidentifyandrankthemostenergeticallyfavourablebinding by4–12%Tris-GlycineSDS–PAGE(Lonza,Basel,Switzerland)toensuretheywere
pocketsonaproteinsurface.Forthispurpose,FTMapfirstperformsrigidbody virtuallyhomogeneous.AllchemicalsandsolventswereanalyticalandHPLC
dockingofprobemoleculeswithafastFouriertransformmethod,thenminimizes grade,respectively.
andrescoresthedockedposes.Next,itclustersthemforeachprobeusingasimple
greedyalgorithm,andfinallylocatestheconsensussitesofbindingpockets
identifiedbyallprobes. Differentialscanningfluorimetry(DSF).Purifiedp53protein(0.22mMin
50mMHEPESatpH7.5,150mMNaCl,0.1mMdithiothreitol)wasdilutedto
4mMfinalconcentrationin20mMHEPESatpH7.5.Compoundsweredissolved
Celllines.Soas-2cellswereobtainedfromtheAmericanTypeCultureCollection inDMSOtovariousconcentrationsandaddedtodilutedproteinforovernight
(Manassas,VA,USA).Theyweremaintainedat371CinDMEMhighglucose incubationat41C.AnequivalentvolumeofDMSOwasusedasacontrolandalso
supplementedwith10%FBS,penicillinGsodium(100unitsml(cid:2)1)andstrepto- incubatedovernightat41C.PRIMA-1inDMSOwaspreheatedat901Cfor20min
mycin(100mgml(cid:2)1). andcooledtoroomtemperaturebeforeuse26.AnnexinA2,S100A4andS100A10
StableSoas-2celllinesharbouringdoxycycline-induciblep53wildtypeor wereusedascontrolproteinstoruleoutnonspecificinteractionsbetween
mutantswereestablishedbyalentivirus-basedstrategy.Humanp53geneswere compoundsandp53.SyproOrangestain(Bio-Rad,Hercules,CA)at5,000(cid:3) stock
clonedasSacI/PstIfragmentsintopEN_TmiRc3,replacingGFPandccdBgenesin concentrationwasdilutedin20mMHEPESatpH7.5andaddedtoeachwellfora
theplasmid.Recombinationofp53-pEN_TmiRc3andthelentivirusvectorpSlik final5(cid:3) concentration.Eachproteinandcompoundconditionwastestedas
wasperformedusingtheLRClonaseEnzymeMix(Invitrogen,Carlsbad,CA) replicatesof6with20mlperwellofa96-wellplate.DSFwasperformedonwild-
accordingtomanufacturer’sinstructions.Lentivirusesweregeneratedbyco- typep53andmutantG245SusingtheCFX96TouchRT–PCRsystem(Bio-Rad)by
transfecting5mgoflentiviralvectorpSlikand5mgofeachpackagingvector increasingthetemperatureoftheplate0.51Cperminfrom25to52.51C.DSF
(codingforGag,Pol,Tat,RevandVSVG)in90%confluent293Tcellsin10-cm performedonmutantR175Hwasidenticalexceptthatthestartingtemperature
platesusingLipofectamine2000(Invitrogen,Carlsbad,CA).Supernatantswere was101C.Fluorescencemeasurementsweretakenonceeveryminuteduringall
collected48haftertransfectionanduseddirectlytoinfectSoas-2cells.Forty-eight experiments.T valuesforeachwell,meanT values,and95%confidence
m m
hoursafterthisinfection,theSoas-2cultureswereswitchedtohygromycin intervalswerecalculatedwiththeprogramPrism5(GraphPadSoftware,LaJolla,
B-containingmedia.Toisolatesinglep53-expressingclones,cellswereinduced CA)byfittingtherawfluorescencedatatoaBoltzmannsigmoidalcurve.
withdoxycyclineandtestedbyimmunoblotting.Doxycyclinewasusedata
concentrationof1mgml(cid:2)1,hygromycinBat50mgml(cid:2)1.
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