cells
Re iew
Ta ge ing he S ess-Induced P o ein NUPR1 o T ea
Panc ea ic Adenoca cinoma
Pa icia San o imia-Cas año 1,2, Yi Xia 3, Ling Peng 4, Ad ián Velázquez-Campoy 5,6,7,8,9 ,
Olga Abián5,6,7,8,10 , Wenjun Lan 1,2, Gwen Lombe k 11 , Raul U u ia 11, B uno Rizzu i 12 ,
Philippe Soubey an 1,2 , JoséLuis Nei a 5,13 and Juan Io anna 1,2,*
1Cen e de Reche che en Cancé ologie de Ma seille (CRCM), INSERM U1068, CNRS UMR 7258,
Aix-Ma seille Uni e si é, CEDEX, 13288 Ma seille, F ance; [email p o ec ed] (P.S.-C.);
[email p o ec ed] (W.L.); [email p o ec ed] (P.S.)
2Ins i u Paoli-Calme es, Pa c Scien i ique e Technologique de Luminy, CEDEX, 13288 Ma seille, F ance
3Chongqing Key Labo a o y o Na u al P oduc Syn hesis and D ug Resea ch, School o Pha maceu ical
Sciences, Chongqing Uni e si y, Chongqing 401331, China; [email p o ec ed]
4
Aix-Ma seille Uni e si
é
, CNRS, Cen e In e disciplinai e de Nanoscience de Ma seille, UMR 7325, «Equipe
Labellis
é
e Ligue Con e le Cance », Pa c Scien i ique e Technologique de Luminy, CEDEX, 13288 Ma seille,
F ance; [email p o ec ed]
5Ins i u o de Biocompu aciónyFísica de Sis emas Complejos, Join Uni s IQFR-CSIC-BIFI, and
GBsC-CSIC-BIFI, 50009 Uni e sidad de Za agoza, Spain; ad ian c@uniza .es (A.V.-C.);
oabi a@uniza .es (O.A.); [email p o ec ed] (J.L.N.)
6A agon Ins i u e o Heal h Resea ch (IIS A agon), Uni e sidad de Za agoza, 50009 Za agoza, Spain
7Cen o de In es igación Biomédica en Red en el Á ea Temá ica de En e medades Hepá icas y
Diges i as (CIBERehd), 28029 Mad id, Spain
8Depa amen o de Bioquímica y Biología Molecula y Celula , Uni e sidad de Za agoza,
50009 Za agoza, Spain
9Fundacion ARAID, Go e nmen o A agon, Uni e sidad de Za agoza, 50018 Za agoza, Spain
10 Ins i u o A agonés de Ciencias de la Salud (IACS), Uni e sidad de Za agoza, 50009 Za agoza, Spain
11 Di ision o Resea ch, Depa men o Su ge y and he Genomic Sciences and P ecision Medicine
Cen e (GSPMC), Medical College o Wisconsin, Milwaukee, WI 53226, USA; glombe [email protected] (G.L.);
u u [email protected] (R.U.)
12 CNR-NANOTEC, Lic yl-UOS Cosenza and CEMIF.Cal, Depa men o Physics, Uni e si y o Calab ia,
87036 Cosenza, I aly; [email p o ec ed]
13
Ins i u o de Biolog
í
a Molecula y Celula , Uni e sidad Miguel He n
á
ndez, Edi icio To egai
á
n, 03202 Elche,
Alican e, Spain
*Co espondence: [email p o ec ed]
Recei ed: 25 Sep embe 2019; Accep ed: 15 No embe 2019; Published: 17 No embe 2019


Abs ac :
Cance cells ac i a e s ess- esponse mechanisms o adap hemsel es o a a ie y o s ess ul
condi ions. Among hese p o ec i e mechanisms, hose con olled by he s ess-induced nuclea
p o ein 1 (NUPR1) belong o he mos conse ed ones. NUPR1 is an 82- esidue-long, monome ic, basic
and in insically diso de ed p o ein (IDP), which was ound o be in a iably o e exp essed in some,
i no all, cance issues. Rema kably, we and o he s ha e p e iously showed ha gene ic inac i a ion
o he Nup 1 gene an agonizes he g ow h o panc ea ic cance as well as se e al o he umo s.
Wi h he use o a mul idisciplina y s a egy by combining biophysical, biochemical, bioin o ma ic,
and biological app oaches, a i luope azine-de i ed compound, named ZZW-115, has been iden i ied
as an inhibi o o he NUPR1 unc ions. The an icance ac i i y o he ZZW-115 was i s alida ed on
a la ge panel o cance cells. Fu he mo e, ZZW-115 p oduced a dose-dependen umo eg ession o
he umo size in xenog a ed mice. Mechanis ically, we ha e demons a ed ha NUPR1 binds o
se e al impo ins. Because ZZW-115 binds NUPR1 h ough he egion a ound he amino acid Th 68,
which is loca ed in o he nuclea loca ion signal (NLS) egion o he p o ein, we demons a ed ha
Cells 2019,8, 1453; doi:10.3390/cells8111453 www.mdpi.com/jou nal/cells
Cells 2019,8, 1453 2 o 9
ea men wi h ZZW-115 inhibi s comple ely he ansloca ion o NUPR1 om he cy oplasm o he
nucleus by compe ing wi h impo ins.
Keywo ds:
d ug design; in insically diso de ed p o ein; panc ea ic duc al adenoca cinoma;
molecula dynamics; NUPR1; s ess esponse; spec oscopy
1. In oduc ion
In his sho e iew, we summa ize ou 20-yea -long wo k on NUPR1, om i s cloning o he
de elopmen o d ugs capable o in e e ing wi h he unc ions o he p o ein. We i s desc ibe how
NUPR1 is a s ess-p o ein exp essed in se e al issues, and which is diso de ed; ha is, i does no
ha e a well- ixed seconda y and e ia y s uc u e. Thus, designing d ugs agains such a p o ein is
challenging. In he second sec ion o his e iew we shall desc ibe how we ha e su moun ed hose
di icul ies by using a mul idisciplina y app oach in ol ing se e al ields o expe ise o epu pose a
d ug a ge ing NUPR1. Finally, we desc ibe he mechanism o unc ion o such a d ug a he molecula
le el: hampe ing he ansi o NUPR1 in o he nucleus by compe i ion wi h impo ins.
2. Why Is he S ess Response Essen ial o Cance Cells and Why Could I Be an Exploi able
The apeu ic Rou e?
Simila o plan s, bac e ia, yeas , and o he uni- o mul i-cellula o ganisms ha ha e de eloped
pa hways o espond o en i onmen al ha sh condi ions, cance cells ha e de eloped molecula
mechanisms o acili a e adap a ion o a a ie y o s ess ul condi ions. Cellula s ess esponses
ep esen a ange o molecula changes ac i a ed when a cell aces hos ile en i onmen al and cellula
condi ions. Cance cells in gene al, and panc ea ic duc al adenoca cinoma (PDAC) in pa icula ,
g ow unde ex emely ha sh ci cums ances. These s esses o igina es om: (1) he en i onmen al
condi ions (i.e., hypo- ascula iza ion wi h hypoxia and low con ibu ion o nu ien s, mechanical
p essu e, DNA-damaging agen s); o (2) he cance iza ion p ocesses hemsel es as a consequence o
he al e ed me abolism o he ans o med cells (i.e., high eac i e oxygen species (ROS ) p oduc ion,
endoplasmic e iculum (ER) s ess) [
1
,
2
], limi ing hei no mal de elopmen and p omo ing a d ama ic
ep og amming o hei pheno ype [
3
–
5
]. These si ua ions ac i a e he exp ession o some s ess
p o eins [
6
–
8
] o allow he cells o su i e, g ow, and ul ima ely p og ess as a umo . Thus, cance
cells become highly dependen on he s ess p o ein unc ions, and hen we can assume ha a ge ing
speci ically hese s ess ac o s and unde s anding hei egula ion mechanisms could be an e icien
(and al e na i e) he apeu ic cance s a egy, as al eady sugges ed yea s ago [
9
]. Among hese su i al
mechanisms, hose con olled by he s ess induced p o ein NUPR1 seem o be he some o he mos
common, and he e o e, p omising o a ge (Figu e 1).
Cells 2019,8, 1453 3 o 9
Cells 2019, 8, 1453 3 o 10
Figu e 1. Ta ge ing he s ess esponse p o ein NUPR1 is a p omising he apy o ea ing cance .
Panc ea ic cance cells a e ex emely challenged by a s ess ul en i onmen due o a poo
concen a ion o nu ien and oxygen, among o he ac o s. In o de o adap hemsel es and su i e,
umo cells ac i a e s ess esponse pa hways o e exp essing s ess p o eins such as NUPR1, which
is cons an ly p esen in he umo issue. Ou s a egy is ocused on a ge ing NUPR1 o induce cance
cell dea h as a he apeu ic ea men o cance .
3. The In insically Diso de ed S ess P o ein NUPR1 in PDAC
NUPR1 was i s desc ibed as being ac i a ed du ing he acu e phase o he panc ea i is [10].
A e wa ds, he ansi o y exp ession o NUPR1 was disco e ed o be a su oga e o he s ess
esponse caused by many s imuli (including minimal s imuli) in mos cell ypes, cha ac e izing
NUPR1 as a ypical s ess-associa ed p o ein [11,12]. Then, NUPR1 was ound o be sys ema ically in
mos o cance issues. A cellula le el, NUPR1 was desc ibed o pa icipa e in many cance -
associa ed p ocesses including cell-cycle egula ion, apop osis [13,14], senescence [15], cell mig a ion
and in asion [16], and de elopmen o me as ases [17]. Indeed, NUPR1 has ecen ly elici ed
signi ican a en ion due o i s ole in p omo ing cance de elopmen and p og ession in panc eas
[18,19]. NUPR1-dependen e ec s also media e esis ance o an icance d ugs [20–22]. Rema kably,
we ha e p e iously shown ha gene ic inac i a ion o Nup 1 an agonizes he g ow h o panc ea ic
cance [16,23], and o he labo a o ies ha e also demons a ed ha gene ic inac i a ion o NUPR1
s ops he g ow h o hepa oca cinoma [24], non-small lung cance [25], cholangioca cinoma [26],
glioblas oma [27], mul iple myeloma [28,29], and os eosa coma [30], he eby suppo ing NUPR1 as a
p omising he apeu ic a ge o he de elopmen o new he apies agains cance s. Howe e , gene ic
app oaches, such as an isense oligonucleo ide (ASOs)- o siRNA-based inac i a ion, a e s ill a away
om being used in clinic in he nex ew yea s. The e o e, we ha e de eloped an o iginal s a egy o
selec small compounds agains NUPR1 o be used o ea ing PDAC.
4. Sc eening Small Compounds as An icance Agen s agains NUPR1
S uc u ally, NUPR1 is an 82- esidue-long in insically diso de ed p o ein (IDP) [31–35].
The e o e, he cu en a ge -based high- h oughpu sc eening o d ug-selec ion, used o well-
olded p o eins, is challenging o NUPR1. In gene al, d ug- a ge ing IDPs is di icul due o hei
ex emely dynamic na u e [36], he ypically weak binding a ini ies owa ds hei na u al pa ne s,
and he ac ha many o hem ha e usually se e al binding ho spo s (all o which a e ea u es
occu ing in NUPR1). We ha e ecen ly de eloped a bo om-up app oach by using biophysical,
biochemical, bioin o ma ic, and biological echniques o a molecula sc eening in i o, in i o, in
silico, and in cellulo o selec po en ial d ug candida es agains NUPR1 [37]. We ha e i s
cha ac e ized he in e ac ions be ween NUPR1 and se e al po en ial ligands by sc eening a collec ion
o 1120 compounds app o ed by he Food D ug Adminis a ion (P es wick Chemical Lib a y,
Figu e 1.
Ta ge ing he s ess esponse p o ein NUPR1 is a p omising he apy o ea ing cance .
Panc ea ic cance cells a e ex emely challenged by a s ess ul en i onmen due o a poo concen a ion
o nu ien and oxygen, among o he ac o s. In o de o adap hemsel es and su i e, umo cells
ac i a e s ess esponse pa hways o e exp essing s ess p o eins such as NUPR1, which is cons an ly
p esen in he umo issue. Ou s a egy is ocused on a ge ing NUPR1 o induce cance cell dea h as
a he apeu ic ea men o cance .
3. The In insically Diso de ed S ess P o ein NUPR1 in PDAC
NUPR1 was i s desc ibed as being ac i a ed du ing he acu e phase o he panc ea i is [
10
].
A e wa ds, he ansi o y exp ession o NUPR1 was disco e ed o be a su oga e o he s ess esponse
caused by many s imuli (including minimal s imuli) in mos cell ypes, cha ac e izing NUPR1 as a
ypical s ess-associa ed p o ein [
11
,
12
]. Then, NUPR1 was ound o be sys ema ically in mos o cance
issues. A cellula le el, NUPR1 was desc ibed o pa icipa e in many cance -associa ed p ocesses
including cell-cycle egula ion, apop osis [
13
,
14
], senescence [
15
], cell mig a ion and in asion [
16
], and
de elopmen o me as ases [
17
]. Indeed, NUPR1 has ecen ly elici ed signi ican a en ion due o i s ole
in p omo ing cance de elopmen and p og ession in panc eas [
18
,
19
]. NUPR1-dependen e ec s also
media e esis ance o an icance d ugs [
20
–
22
]. Rema kably, we ha e p e iously shown ha gene ic
inac i a ion o Nup 1 an agonizes he g ow h o panc ea ic cance [
16
,
23
], and o he labo a o ies
ha e also demons a ed ha gene ic inac i a ion o NUPR1 s ops he g ow h o hepa oca cinoma [
24
],
non-small lung cance [
25
], cholangioca cinoma [
26
], glioblas oma [
27
], mul iple myeloma [
28
,
29
], and
os eosa coma [
30
], he eby suppo ing NUPR1 as a p omising he apeu ic a ge o he de elopmen
o new he apies agains cance s. Howe e , gene ic app oaches, such as an isense oligonucleo ide
(ASOs)- o siRNA-based inac i a ion, a e s ill a away om being used in clinic in he nex ew yea s.
The e o e, we ha e de eloped an o iginal s a egy o selec small compounds agains NUPR1 o be
used o ea ing PDAC.
4. Sc eening Small Compounds as An icance Agen s agains NUPR1
S uc u ally, NUPR1 is an 82- esidue-long in insically diso de ed p o ein (IDP) [
31
–
35
]. The e o e,
he cu en a ge -based high- h oughpu sc eening o d ug-selec ion, used o well- olded p o eins,
is challenging o NUPR1. In gene al, d ug- a ge ing IDPs is di icul due o hei ex emely dynamic
na u e [
36
], he ypically weak binding a ini ies owa ds hei na u al pa ne s, and he ac ha
many o hem ha e usually se e al binding ho spo s (all o which a e ea u es occu ing in NUPR1).
We ha e ecen ly de eloped a bo om-up app oach by using biophysical, biochemical, bioin o ma ic,
and biological echniques o a molecula sc eening
in i o
,
in i o
, in silico, and in cellulo o selec
po en ial d ug candida es agains NUPR1 [
37
]. We ha e i s cha ac e ized he in e ac ions be ween
NUPR1 and se e al po en ial ligands by sc eening a collec ion o 1120 compounds app o ed by
he Food D ug Adminis a ion (P es wick Chemical Lib a y, h p://www.p es wickchemical.com/
Cells 2019,8, 1453 4 o 9
lib a ies-sc eening-lib-pcl.h ml). We ha e used luo escence he mal dena u a ion, on he basis o he
la ges shi s in he mal dena u a ion midpoin s o he he mal cu es o NUPR1 wi h an ex e nal
dye in he p esence and absence o he compounds o he lib a y. In pa allel, we ha e ca ied ou a
ou -pa s a egy based on expe imen al and compu a ional me hods: (1) we ha e de e mined he
he modynamic pa ame e s o he binding eac ion wi h NUPR1 o he mos p omising compounds
(i.e., hose showing he la ges changes in he mal shi s when compa ed o isola ed NUPR1) by using
iso he mal i a ion calo ime y (ITC); (2) we ha e pe o med molecula dynamics (MD) simula ions
o ob ain an ensemble o NUPR1 con o ma ions in isola ion; (3) we ha e used his ensemble o dock
he mos p omising sc eened compounds; and (4) we ha e de e mined s uc u e-ac i i y ela ionships
(SAR) by NMR wi h he complexes o NUPR1 and he selec ed compounds (i.e., de ec ing NUPR1
esidues a ec ed by he p esence o he co esponding ligand). The blind s a egy combining SAR-NMR
and MD simula ions alida ed ou app oach, as we ha e essen ially obse ed a close analogy be ween
he esidues in con ac wi h he compound and hose esidues whose signals in NMR spec a we e
a ec ed by he p esence o he compound (and hen, when binding was happening). The dissocia ion
cons an s o he compounds measu ed by ITC we e in he same o de (mic omola ange) as hose
ound o he na u al binding pa ne s o NUPR1 [14,31,32,34,35].
A he end o his p ocedu e, we iden i ied i luope azine (TFP), and i s s uc u ally ela ed
luphenazine hyd ochlo ide, as he compounds wi h he la ges a ini y o NUPR1. Pheno ypic assays
ha e been ca ied ou o assess he po en ial bioac i i y o TFP. Cell iabili y assays in he p esence
o TFP ha e led o hal -maximal inhibi o y concen a ion (IC
50
alues) o ~10
µ
M. Mos impo an ly,
es s pe o med wi h TFP
in i o
, wi h human panc ea ic cance cell-de i ed xenog a s implan ed
in o immunocomp omised mice, ha e shown an a es o he umo g ow h in a dose-dependen
manne [
37
]. The e o e, we ha e success ully epu posed TFP as a possible cance d ug o ea ing
PDAC. Un o una ely, high doses o TFP as necessa y o ea ing PDAC ha e also led o unwan ed
neu ological e ec s such as s ong le ha gy and hunched pos u e in he ea ed mice [
37
]. Thus, al hough
ela i ely e icien as an an icance agen , he neu ological e ec s obse ed p eclude he use o TFP in
clinic o ea cance s.
5. ZZW-115 Is a S ongly Imp o ed T i luope azine-De i ed Compound wi h a New Mechanism
o Ac ion
We ha e de eloped a mul idisciplina y app oach o imp o e he e iciency o TFP by: (1) inc easing
i s an icance e ec as much as possible wi h he aim o dec easing he doses o ea pa ien s;
and (2) educing i s undesi able neu ological side-e ec s.
A a ional, in silico ligand-based design elying on a combina ion o MD and docking guided
he i s s eps o he o ganic syn heses o TFP-de i ed compounds. These new molecules show:
(1) a s onge a ini y
in i o
o NUPR1 han TFP, as indica ed by a combina ion o spec oscopic
( luo escence, NMR, and ci cula dich oism (CD)) and biophysical s udies (ITC); and (2) he same
NUPR1 binding egions as TFP.
The an icance ac i i y o he ZZW-115, one o he syn hesized compounds, was es ed on a panel
o 11 p ima y PDAC-de i ed cells and i was ound o be e icien o kill he cance cells wi h IC
50
in he
ange om 0.84
µ
M (ANOR cells) o 4.93
µ
M (HN14 cells); hese
in i o
esul s a e in good ag eemen
wi h he esul s om ITC measu emen s. Mos impo an ly, ZZW-115 shows a dose-dependen umo
eg ession in xenog a ed mice leading o almos a disappea ance a e 30 days o ea men wi h
5 mg/kg/day
, in ou independen PDAC models [
38
]. No ewo hy, his happened wi h no appa en
neu ological e ec s.
To u he show ha ZZW-115 displays i s an icance ac i i y ia a ge ing NUPR1, we ha e
ob ained a ew clones in which Nup 1 has been inac i a ed by a CRISPR/Cas9 app oach. As expec ed,
we ha e ound ha h ee Nup 1 KO clones a e signi ican ly mo e esis an o ZZW-115- ea men han
wo Nup 1 WT clones. These esul s indica e ha ZZW-115 is ce ainly exe ing i s e ec by binding o
NUPR1. Howe e , hese indings do no unambiguously p o e ha NUPR1 is he sole p o ein a ge ed
Cells 2019,8, 1453 5 o 9
by ZZW-115; a he , ou esul s show ha a ge ing NUPR1 seems o be he main mode o ac ion o
ZZW-115, and i s binding o he p o ein is mainly esponsible o i s an i umo e ec [38].
Since esis ance o chemo he apy is a common issue ha oncologis s mus ace in he ea men o
pa ien s wi h PDAC, we ha e used he MiaPaCa-2 cell line, which has become esis an o he wo mos
equen ly used chemo he apeu ic agen s, oxalipla in o gemci abine, o assess whe he esis ance o
hem is also con e ing esis ance o ZZW-115. Rema kably, ZZW-115- ea men o esis an MiaPaCa-2
cells shows he same sensi i i y as he pa en al cells, sugges ing ha he an i umo e ec o he
ZZW-115 is no a ec ed by he esis ance o o he s d ugs and may ac on he umo by ollowing some
o he di e en in acellula pa hways [38].
6. ZZW-115 Induces Tumo Cell Dea h by Nec op osis and Apop osis
A he cellula le el, we ha e demons a ed ha ZZW-115 induces cell dea h by bo h nec op o ic
(as measu ed by l-lac a e dehyd ogenase (LDH) elease) and apop o ic (as measu ed by caspase 3/7
ac i i y) mechanisms. Mo eo e , we ha e pe o med escue expe imen s by using Nec os a in-1 and
Z-VAD-FMK, ei he alone o in combina ion. Bo h inhibi o s imp o ed cell iabili y when adminis e ed
alone, wi h a g ea e e ec when hey we e used in combina ion. F om he he apeu ic poin o iew,
he ac ha ZZW-115 os e s di e en cell dea h pa hways is an ad an age, compa ed wi h o he
d ugs commonly used in clinic. In ac , by using concen a ions o ZZW-115 o pacli axel (a classical
p o-apop o ic d ug) ha induced simila caspase ac i a ion le el, ZZW-115 demons a ed s onge
an icance ac i i y. In addi ion, he use o a compound like ZZW-115 ha is capable o p omo ing cell
dea h by apop osis, and concomi an ly also nec op osis, ep esen s he bes s a egy agains cance s
wi h in insic o acqui ed esis ance o apop osis (unpublished esul s).
I is well-known ha ATP plays an impo an ole in cell dea h a e. In e es ingly, ZZW-115
induced a d ama ic dec ease o ATP con en in ea ed cells. To be e unde s and he causes ha
led o his dec ease o he ATP le el, we ca e ully s udied he kine ics o he main sou ces o i s
p oduc ion: oxida i e phospho yla ion (OXPHOS) and anae obic glycolysis. On he one hand,
OXPHOS me abolism su e ed a ime-dependen dec ease a e ZZW-115 ea men , wi h a g ea
ailu e in mi ochond ial espi a ion and ATP p oduc ion. On he o he hand, he glycoly ic pa hway
shi ed a ea lie ime (4 h), as an a emp o compensa e he mi ochond ial collapse. Howe e , his
swi ch o a highe glycoly ic me abolism was ansi o y and he ea ed cell apidly consumed he
glycoly ic ese e. As a consequence, o al ATP p oduc ion and con en apidly d opped a e 24 h o
ea men . I is well-known ha dis up ion o mi ochond ial unc ion is a key e en ha igge s cell
dea h, in which mi ochond ial ROS o ma ion has an ac i e ole. In his ega d, ZZW-115 was also
capable o inc easing he o ma ion o supe oxide ions in he mi ochond ia, con ibu ing in his way
o he mi ochond ial ailu e and cell dea h [
38
] (Figu e 2). Impo an ly, he molecula consequences
desc ibed abo e, which led o nec op o ic and apop o ic cell dea h, we e simila o hose obse ed
in NUPR1-de icien cells [
39
]. Consequen ly, ZZW-115 cons i u es a p omising d ug candida e o
panc ea ic cance wi h an o iginal molecula mechanism, since i combines he concomi an induc ion
o nec op osis and apop osis wi h a concomi an mi ochond ial ailu e.

Cells 2019,8, 1453 6 o 9
Cells 2019, 8, 1453 6 o 10
Figu e 2. ZZW-115 is a p omising he apeu ic agen inducing umo cell dea h by nec op osis and
apop osis. T ea men wi h ZZW-115, a a concen a ion o 3 o 5 µM o 24 h, o panc ea ic cance cells
(MiaPaCa-2 cells) demons a ed ha ou compound was able o induce cell dea h by apop osis and
nec op osis on a dose-dependen manne , by measu ing caspase 3/7 ac i i y o LDH elease,
espec i ely.
7. ZZW-115 Is Ac i e in Some Type o Cance s
Since NUPR1 is o e exp essed in se e al (i no all) umo s, we ha e e alua ed he e ec o
ea ing cellula lines de i ed om se e al umo s wi h inc easing concen a ions o ZZW-115.
T ea men o cells such as U87 (glioblas oma), A375 and B16 (melanoma), U2OS and SaOS-2
(os eosa coma), HT29, SK-CO-1, and LS174T (colon cance ), H1299 and H358 (lung cance ), HepG2
(hepa oca cinoma), PC-3 (p os a e), THP-1 (acu e monocy ic leukemia), Daudi (lymphoma), Ju ka
(acu e T cell leukemia), and MDA-MB-231 (b eas cance ), demons a ed ha ZZW-115 was e icien
o kill hese umo cells wi h IC50 alues in he ange om 0.42 µM (Hep2G cells) o 7.75 µM (SaOS-2
cells). These da a sugges ha ZZW-115 could be po en ially ac i e o ea ing cance s om a ious
issues by a ge ing NUPR1. Impo an ly, we ha e also alida ed he an icance e ec o ZZW-115 in
i o in bo h hepa oca cinoma and glioma xenog a ed umo s (unpublished da a).
8. ZZW-115 Inhibi s he Nuclea T ansloca ion o NUPR1 by Compe ing wi h Impo ins
NUPR1 is a nuclea p o ein ha con ains a canonical bipa i e domain o posi i ely cha ged
amino acids, ypical o NLS, in ol ing p o ein esidues 63–78, as es ed by heo e ical p edic ions and
si e di ec ed mu agenesis [40]. The in e ac ome analysis o NUPR1 e ealed ha i may bind o 30
componen s o he nuclea po e including se e al impo ins o ka yophe ins (KPNA1, KPNA2,
KPNA3, KPNA4, and KPNA6) and 17 NUP p o eins (unpublished da a). In ou p e ious wo k, we
ha e shown ha NUPR1 binds ZZW-115 by using esidues a ound Ala33 and Th 68 [38], he wo
ho -spo egions o NUPR1 [35,37]. Because Th 68 belongs o he NLS egion o NUPR1, i is p obable
ha ZZW-115 can hinde he in e ac ion be ween NUPR1 ( h ough i s NLS) and impo ins, and hen
i can block he NUPR1 nuclea ansloca ion. The e o e, by using NUPR1 immuno luo escence
s aining, we ha e s udied he po en ial impac o ZZW-115 on he in acellula loca ion o NUPR1,
and we ha e ound ha ea men wi h ZZW-115 inhibi s almos comple ely he ansloca ion o
NUPR1 om he cy oplasm o he nucleus (Figu e 3) (unpublished esul s). This esul has led us o
he conclusion ha ZZW-115 can inac i a e NUPR1 by p e en ing i s ansloca ion in o he nucleus,
whe e i is p esumed o play i s essen ial oles ega ding cell su i al.
Figu e 2.
ZZW-115 is a p omising he apeu ic agen inducing umo cell dea h by nec op osis and
apop osis. T ea men wi h ZZW-115, a a concen a ion o 3 o 5
µ
M o 24 h, o panc ea ic cance cells
(MiaPaCa-2 cells) demons a ed ha ou compound was able o induce cell dea h by apop osis and
nec op osis on a dose-dependen manne , by measu ing caspase 3/7 ac i i y o LDH elease, espec i ely.
7. ZZW-115 Is Ac i e in Some Type o Cance s
Since NUPR1 is o e exp essed in se e al (i no all) umo s, we ha e e alua ed he e ec o ea ing
cellula lines de i ed om se e al umo s wi h inc easing concen a ions o ZZW-115. T ea men o
cells such as U87 (glioblas oma), A375 and B16 (melanoma), U2OS and SaOS-2 (os eosa coma), HT29,
SK-CO-1, and LS174T (colon cance ), H1299 and H358 (lung cance ), HepG2 (hepa oca cinoma), PC-3
(p os a e), THP-1 (acu e monocy ic leukemia), Daudi (lymphoma), Ju ka (acu e T cell leukemia), and
MDA-MB-231 (b eas cance ), demons a ed ha ZZW-115 was e icien o kill hese umo cells wi h
IC
50
alues in he ange om 0.42
µ
M (Hep2G cells) o 7.75
µ
M (SaOS-2 cells). These da a sugges ha
ZZW-115 could be po en ially ac i e o ea ing cance s om a ious issues by a ge ing NUPR1.
Impo an ly, we ha e also alida ed he an icance e ec o ZZW-115
in i o
in bo h hepa oca cinoma
and glioma xenog a ed umo s (unpublished da a).
8. ZZW-115 Inhibi s he Nuclea T ansloca ion o NUPR1 by Compe ing wi h Impo ins
NUPR1 is a nuclea p o ein ha con ains a canonical bipa i e domain o posi i ely cha ged
amino acids, ypical o NLS, in ol ing p o ein esidues 63–78, as es ed by heo e ical p edic ions
and si e di ec ed mu agenesis [
40
]. The in e ac ome analysis o NUPR1 e ealed ha i may bind o
30 componen s o he nuclea po e including se e al impo ins o ka yophe ins (KPNA1, KPNA2,
KPNA3, KPNA4, and KPNA6) and 17 NUP p o eins (unpublished da a). In ou p e ious wo k, we
ha e shown ha NUPR1 binds ZZW-115 by using esidues a ound Ala33 and Th 68 [
38
], he wo
ho -spo egions o NUPR1 [
35
,
37
]. Because Th 68 belongs o he NLS egion o NUPR1, i is p obable
ha ZZW-115 can hinde he in e ac ion be ween NUPR1 ( h ough i s NLS) and impo ins, and hen i
can block he NUPR1 nuclea ansloca ion. The e o e, by using NUPR1 immuno luo escence s aining,
we ha e s udied he po en ial impac o ZZW-115 on he in acellula loca ion o NUPR1, and we ha e
ound ha ea men wi h ZZW-115 inhibi s almos comple ely he ansloca ion o NUPR1 om he
cy oplasm o he nucleus (Figu e 3) (unpublished esul s). This esul has led us o he conclusion ha
ZZW-115 can inac i a e NUPR1 by p e en ing i s ansloca ion in o he nucleus, whe e i is p esumed
o play i s essen ial oles ega ding cell su i al.
Cells 2019,8, 1453 7 o 9
Cells 2019, 8, 1453 7 o 10
Figu e 3. ZZW-115 hampe ed he nuclea ansloca ion o NUPR1. (Le panel) NUPR1 is a nuclea
p o ein wi h a p edic ed NLS. This pa o he p o ein binds o impo ins and acili a es i s
ansloca ion om he cy oplasm o he nucleus, h ough he nuclea po e complex. Thus, NUPR1
exhibi s a nuclea localiza ion (as showed in his immuno luo escence), whe e i can de elop i s
ac i i y and p omo e umo p og ession. (Righ panel) Nuclea magne ic esonance da a indica ed
ha ZZW-115 binds o NUPR1 in he esidue Th 68 [35,37], loca ed wi hin he NLS. Thus,
pha macological inhibi ion o NUPR1 hampe ed he in e ac ion wi h impo in and i s ansloca ion
o he nucleus (as showed in his immuno luo escence). A e ZZW-115 ea men , NUPR1 was
loca ed in he pe inuclea and cy oplasmic a ea, and inhibi ion o he nuclea ac i i y o NUPR1
induced umo g ow h a es .
9. Conclusions
Ou wo k p o ides a p oo -o -concep ha s ess p o eins a e e ec i e he apeu ic a ge s o
ea ing cance s. In ou wo k, we ha e shown how epu posing a d ug can be used as a s a ing poin
o imp o e he design and he e iciency o be e molecules agains cance , e en o challenging
a ge s such as IDPs. Ou s udies cons i u e an inno a i e example o success ul ligand-based design
(as opposed o s uc u e-based design, employed in he d ug-design o well- olded p o eins) o an
inhibi o o an en i ely un olded p o ein.
Au ho Con ibu ions: Concep ualiza ion, P.S.-C., Y.X., A.V.-C., O.A., W.L., G.L., R.U., B.R., P.S., J.L.N. and J.I.;
W i ing—o iginal d a p epa a ion, J.I.; W i ing— e iew and edi ing, P.S.-C., Y.X., A.V.-C., O.A., W.L., G.L.,
R.U., B.R., P.S., J.L.N. and J.I.
Funding: This wo k in he au ho s’ labo a o ies was suppo ed by La Ligue Con e le Cance , INCa, Fonda ion
de F ance, and INSERM ( o JI); Fonda ion ARC ( o PS); he Miguel Se e P og am om Ins i u o de Salud Ca los
III (CPII13/00017 o OA); he Fondo de In es igaciones Sani a ias (PI15/00663 and PI18/00343 o OA); he Spanish
Minis y o Economy and Compe i i eness (BFU2016-78232-P o AVC, RTI2018 097991-BIO o JLN); he
Dipu ación Gene al de A agón (P o ein Ta ge s G oup B89 o AVC, and Diges i e Pa hology G oup B01 o OA);
he Cen o de In es igación Biomédica en Red en En e medades Hepá icas y Diges i as (CIBERehd); he
Fundación Al onso Ma ín-Escude o and Fonda ion de F ance ( o PSC); he China Schola ship Council ( o WL
and CH); P og amme XU GUANGQI and CAI YUANPEI ( o YX and JI); and he Na ional Na u al Science
Founda ion o China (81502920 o YX).
Acknowledgmen s: BR acknowledges he kind hospi ali y o he Eu opean Magne ic Resonance Cen e (CERM),
Ses o Fio en ino (Flo ence), I aly.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Figu e 3.
ZZW-115 hampe ed he nuclea ansloca ion o NUPR1. (Le panel) NUPR1 is a nuclea
p o ein wi h a p edic ed NLS. This pa o he p o ein binds o impo ins and acili a es i s ansloca ion
om he cy oplasm o he nucleus, h ough he nuclea po e complex. Thus, NUPR1 exhibi s a nuclea
localiza ion (as showed in his immuno luo escence), whe e i can de elop i s ac i i y and p omo e
umo p og ession. (Righ panel) Nuclea magne ic esonance da a indica ed ha ZZW-115 binds
o NUPR1 in he esidue Th 68 [
35
,
37
], loca ed wi hin he NLS. Thus, pha macological inhibi ion
o NUPR1 hampe ed he in e ac ion wi h impo in and i s ansloca ion o he nucleus (as showed
in his immuno luo escence). A e ZZW-115 ea men , NUPR1 was loca ed in he pe inuclea and
cy oplasmic a ea, and inhibi ion o he nuclea ac i i y o NUPR1 induced umo g ow h a es .
9. Conclusions
Ou wo k p o ides a p oo -o -concep ha s ess p o eins a e e ec i e he apeu ic a ge s o
ea ing cance s. In ou wo k, we ha e shown how epu posing a d ug can be used as a s a ing
poin o imp o e he design and he e iciency o be e molecules agains cance , e en o challenging
a ge s such as IDPs. Ou s udies cons i u e an inno a i e example o success ul ligand-based design
(as opposed o s uc u e-based design, employed in he d ug-design o well- olded p o eins) o an
inhibi o o an en i ely un olded p o ein.
Au ho Con ibu ions:
Concep ualiza ion, P.S.-C., Y.X., L.P., A.V.-C., O.A., W.L., G.L., R.U., B.R., P.S., J.L.N. and
J.I.; W i ing—o iginal d a p epa a ion, J.I.; W i ing— e iew and edi ing, P.S.-C., Y.X., L.P., A.V.-C., O.A., W.L.,
G.L., R.U., B.R., P.S., J.L.N. and J.I.
Funding:
This wo k in he au ho s’ labo a o ies was suppo ed by La Ligue Con e le Cance , INCa, Fonda ion de
F ance, and INSERM ( o JI); Fonda ion ARC ( o PS); he Miguel Se e P og am om Ins i u o de Salud Ca los III
(CPII13/00017 o OA); he Fondo de In es igaciones Sani a ias (PI15/00663 and PI18/00343 o OA); he Spanish Minis y
o Economy and Compe i i eness (BFU2016-78232-P o AVC, RTI2018 097991-BIO o JLN); he Dipu aci
ó
n Gene al de
A ag
ó
n (P o ein Ta ge s G oup B89 o AVC, and Diges i e Pa hology G oup B01 o OA); he Cen o de In es igaci
ó
n
Biom
é
dica en Red en En e medades Hep
á
icas y Diges i as (CIBERehd); he Fundaci
ó
n Al onso Ma
í
n-Escude o and
Fonda ion de F ance ( o PSC); he China Schola ship Council ( o WL and CH); P og amme XU GUANGQI and CAI
YUANPEI ( o YX and JI); and he Na ional Na u al Science Founda ion o China (81502920 o YX).
Acknowledgmen s:
BR acknowledges he kind hospi ali y o he Eu opean Magne ic Resonance Cen e (CERM),
Ses o Fio en ino (Flo ence), I aly.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Cells 2019,8, 1453 8 o 9
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