Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins
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A icle
An i i al P ope ies o Chemical Inhibi o s o
Cellula An i-Apop o ic Bcl-2 P o eins
Da ia Bulano a 1,†, Aleksand Iane ski 1,†, And ii Bugai 2,† ID , Ye hen Akimo 1,
Su i Kui anen 3, Hen ik Paa ilainen 4, Lau a Kakkola 4, Ja in Nandania 1, Lau a Tu unen 1,
Tiina Ohman 5, Hanna Ala-Hongis o 6, Hanna M. Pesonen 6, Ma ika S. Kuisma 6,
Anni Honkimaa 7, Emma L. Wal on 8, Valen yn Oksenych 8, Ma ina B. Lo ey 9,
Dmi y Guschin 10, Jungmin Shim 10, Jinhee Kim 10, Thoa T. Than 10, So Young Chang 10,
Veijo Hukkanen 4, E geny Kulesskiy 1, Va pu S. Ma jomaki 11, Ilkka Julkunen 4,
Tuula A. Nyman 5,12, Sampsa Ma ikainen 9, Jani S. Saa ela 1, Fama a Sane 13, Didie Hobe 13,
Gülsah Gab iel 14, Je K. De B abande 15, Miika Ma ikainen 16, Ma c P. Windisch 10,
Ji-Young Min 10, Robe o B uzzone 10,17,18, Te o Ai okallio 1ID , Ma kus Vähä-Koskela 1,
Olli Vapalah i 19,20, A o Pulk 21, Vidya Velagapudi 1and Denis E. Kaino 1,8,10,21,*ID
1Ins i u e o Molecula Medicine Finland, FIMM, Uni e si y o Helsinki, Helsinki 00290, Finland;
[email p o ec ed] (D.B.); aleksand [email p o ec ed] (A.I.); [email p o ec ed] (Y.A.);
[email p o ec ed] (J.N.); [email p o ec ed] (L.T.); e geny[email p o ec ed] (E.K.);
[email p o ec ed] (J.S.S.); e o.ai [email p o ec ed] (T.A.); [email p o ec ed] (M.V.-K.);
[email p o ec ed] (V.V.)
2Depa men o Biochemis y and De elopmen al Biology, Uni e si y o Helsinki, Helsinki 00290, Finland;
[email p o ec ed]
3Depa men o Vi ology, Uni e si y o Helsinki, Helsinki 00290, Finland; [email p o ec ed]
4Depa men o Vi ology, Uni e si y o Tu ku, Tu ku 20520, Finland; [email p o ec ed] (H.P.);
[email p o ec ed] (L.K.); [email p o ec ed] (V.H.); [email p o ec ed] (I.J.)
5Ins i u e o Bio echnology, Uni e si y o Helsinki, Helsinki 00014, Finland; [email p o ec ed] (T.O.);
[email p o ec ed] (T.A.N.)
6Biomedicum Func ional Genomics Uni (FuGU), Helsinki, Helsinki 00290, Finland;
[email p o ec ed] (H.A.-H.); [email p o ec ed] (H.M.P.);
[email p o ec ed] (M.S.K.)
7Depa men o Vi ology, Uni e si y o Tampe e, Tampe e 33520, Finland; [email p o ec ed]
8Depa men o Clinical and Molecula Medicine, No wegian Uni e si y o Science and Technology,
T ondheim 7028, No way; [email p o ec ed] (E.L.W.); [email p o ec ed] (V.O.)
9Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Rheuma ology, Helsinki 00290, Finland;
[email p o ec ed] (M.B.L.); [email p o ec ed] (S.M.)
10 Ins i u Pas eu Ko ea, Gyeonggi-do 13488, Ko ea; [email p o ec ed] (D.G.);
[email p o ec ed]g (J.S.); [email p o ec ed]g (J.K.); hoa. han@ip-ko ea.o g (T.T.T.);
[email p o ec ed]g (S.Y.C.); ma c.windisch@ip-ko ea.o g (M.P.W.);
[email p o ec ed]g (J.-Y.M.); [email p o ec ed] (R.B.)
11 Depa men o Biological and En i onmen al Science/Nanoscience cen e , Uni e si y o Jy äskylä,
Jy äskylä 40500, Finland; [email p o ec ed]
12 Depa men o Immunology, Uni e si y o Oslo, Oslo 0424, No way
13 Uni e si y o Lille, CHU Lille labo a oi e de Vi ologie, EA3610, F-59037 Lille, F ance;
[email p o ec ed] (F.S.); didie [email p o ec ed] (D.H.)
14 Hein ich Pe e Ins i u e, Leibniz Ins i u e o Expe imen al Vi ology, Hambu g 20251, Ge many;
[email p o ec ed]
15
Depa men o Biochemis y, Uni e si y o Texas Sou hwes e n Medical Cen e , Dallas, TX 75390-9038, USA;
[email p o ec ed]
16 Depa men o Immunology, Gene ics and Pa hology, Science o Li e Labo a o y, Uppsala Uni e si y,
Uppsala 75237, Sweden; [email p o ec ed]
17 HKU-Pas eu Resea ch Pole, School o Public Heal h, Uni e si y o Hong Kong, Hong Kong, China
18 Depa men o Cell Biology and In ec ion, Ins i u Pas eu , Pa is 75015, F ance
Vi uses 2017,9, 271; doi:10.3390/ 9100271 www.mdpi.com/jou nal/ i uses
Vi uses 2017,9, 271 2 o 15
19 Depa men o Vi ology and Immunology, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al,
Helsinki 00014, Finland; [email p o ec ed]
20 Depa men o Ve e ina y Biosciences, Uni e si y o Helsinki, Helsinki 00014, Finland
21 Ins i u e o Technology, Uni e si y o Ta u, Ta u 50090, Es onia; [email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +358-50-415-5460
† These au ho s con ibu ed equally o his wo k.
Recei ed: 26 Augus 2017; Accep ed: 23 Sep embe 2017; Published: 25 Sep embe 2017
Abs ac :
Vi al diseases emain se ious h ea s o public heal h because o he sho age o e ec i e
means o con ol. To comba he su ge o i al diseases, new ea men s a e u gen ly needed. He e
we show ha small-molecules, which inhibi cellula an i-apop o ic Bcl-2 p o eins (Bcl-2i), induced
he p ema u e dea h o cells in ec ed wi h di e en RNA o DNA i uses, whe eas, a he same
concen a ions, no oxici y was obse ed in mock-in ec ed cells. Mo eo e , hese compounds limi ed
i al eplica ion and sp ead. Su p isingly, Bcl-2i also induced he p ema u e apop osis o cells
ans ec ed wi h i al RNA o plasmid DNA bu no o mock- ans ec ed cells. These esul s sugges
ha Bcl-2i sensi izes cells con aining o eign RNA o DNA o apop osis. A compa ison o he oxici y,
an i i al ac i i y, and side e ec s o six Bcl-2i allowed us o selec A-1155463 as an an i i al lead
candida e. Thus, ou esul s pa e he way o he u he de elopmen o Bcl-2i o he p e en ion
and ea men o i al diseases.
Keywo ds: apop osis; an i i al agen ; inna e immuni y; hos esponse
1. In oduc ion
Globaliza ion, en i onmen al changes, popula ion g ow h, and u baniza ion make i al diseases
(VDs) one o he majo causes o mo bidi y and mo ali y in he wo ld [
1
,
2
]. In pa icula , eme ging
and e-eme ging VDs pose a cons an h ea o public heal h [
3
]. To da e, 90 an i i al d ugs ha e
been app o ed o ea human immunode iciency i us (HIV) 1 and 2, hepa i is B and C i uses (HBV
and HCV), cy omegalo i us (CMV), he pes simplex i us (HSV) 1 and 2, human papilloma i us
(HPV), a icella zos e i us (VZV), accinia i us (VACV), Eps ein-Ba i us (EBV), espi a o y
syncy ial i us (RSV), o in luenza A and B i us (IAV and FLUBV) in ec ions bu no o he impo an
VDs [
4
]. Only aciclo i (HSV-1/-2, VZV), amiciclo i (HSV-1/-2, VZV), alaciclo i (HSV-1/-2, VZV,
EBV, CMV), ida abine (HSV-1/-2, VZV), b i udine (HSV-1, VZV), osca ne (HSV-1/-2, CMV),
lami udine (HBV, HIV-1/-2), pegin e e on
α
-2a (HBV, HCV), pegin e e on
α
-2b (HBV, HCV),
iba i ine (RSV, HCV), eno o i disop oxil (HBV, HIV-1/-2), and i lu idine (HSV-1/-2, VACV)
can a ge mo e han one VD [
4
]. Conside ing his, he e is an u gen need o he de elopmen o
no el an i i als, including d ugs agains eme ging and e-eme ging VDs.
Apop osis is a cellula an i i al p ocess ha can be exploi ed in de elopmen o such d ugs [
5
,
6
].
Du ing his p ocess, pa e n ecogni ion ecep o s (PRRs) ecognize he in ading i uses and signal o
Bcl-2 p o eins [
7
]. The an i-apop o ic (Bcl-2, Bcl-xL, and Bcl-w) and p o-apop o ic (Bax, Bak, and Bad)
Bcl-2 p o eins associa e o dissocia e wi h each o he using Bcl-2-homology 3 (BH3) domains o s a a
cascade o e en s, which leads o mi ochond ia memb ane pe meabiliza ion (MoMP), cy och ome c
elease, and e en ually cell dea h [8–11].
An i-apop o ic Bcl-2 p o eins may ep esen cellula a ge s o no el an i i als [
12
–
14
]. Mo eo e ,
comme cially a ailable Bcl-2 inhibi o s (Bcl-2i) could be es ed as an i i als. In pa icula , he an i i al
p ope ies o wo s uc u ally dis inc classes o an icance agen s could be in es iga ed. The i s
class includes ABT-737 and i s de i a i es ABT-263 and ABT-199, whe eas he second class includes
WEHI-539 and i s de i a i es, A-1331852 and A-1155463 [
15
–
21
]. In e es ingly, ABT-263 is cu en ly
in clinical ials, and ABT-199 is app o ed o ea mul iple lymphoid malignancies. Howe e , only
A-1155463 causes e e sible h ombocy openia in animals [22,23].
Vi uses 2017,9, 271 3 o 15
He e, we showed ha ABT-263 limi ed he eplica ion o IAV, FLUBV, Middle Eas espi a o y
synd ome co ona i us (MERS-CoV), Zika i us (ZIKV), HBV, HSV-1 and -2, and echo i us 1 and 6
(EV1 and EV6) by inducing he p ema u e dea h o i us-in ec ed cells a concen a ions no oxic o
non-in ec ed cells. We u he showed ha ABT-263 induced apop osis in cells ans ec ed wi h i al
RNA o plasmid DNA bu no in mock- ans ec ed cells, sugges ing ha his compound accele a ed
apop osis in esponse o o eign nucleic acids. We also demons a ed ha ABT-737, ABT-199,
WEHI-539, A-1331852, and A-1155463 can speci ically induce he p ema u e dea h o IAV-in ec ed
cells, bu only ABT-737, ABT-263, A-1331852, and A-1155463 limi ed i al eplica ion. A compa ison
o he oxici y, an i i al ac i i y, and side e ec s o hese Bc2i allowed us o selec A-1155463 as an
an i i al lead candida e. Thus, we disco e ed a po en ial solu ion o he p e en ion and ea men o
a b oad-spec um o VDs.
2. Ma e ials and Me hods
2.1. Reagen s
ABT-199, ABT-263, ABT-737, WEHI-539, A-1331852, A-1155463, oba oclax, and gemci abine we e
pu chased om Selleck Chemicals (Hous on, TX, USA), Saliphenylhalamide (SaliPhe) was syn hesized
as desc ibed p e iously [
24
]. 10 mM solu ions o he compounds we e p epa ed in 100% dime hyl
sul oxide (Sigma-Ald ich, S . Louis, MO, USA) and s o ed a
−
80
◦
C. Lyophilized lipopolysaccha ide
(LPS, 10 mg/mL) was pu chased om Sigma-Ald ich (S . Louis, MO, USA). Plasmid pEGFP was om
Clon ech (Moun ain View, CA, USA) (ca #: HLP309). Hoechs 33342 (20 mM solu ion; ca #: 62249)
and ATP (10 mM solu ion; ca #: PV3227) we e om The mo Fishe Scien i ic (Wal ham, MA, USA).
Genomic RNA was isola ed om in luenza A/WSN/1933 s ain as desc ibed p e iously [11].
2.2. Vi uses
Human in luenza A/Udo n/307/1972 (H3N2) and B/Shandong/7/97 i uses we e g own in
emb yona ed hen eggs as desc ibed p e iously [
25
,
26
]. EV1 and EV6 s ains we e p opaga ed in
a monolaye o a ican g een monkey kidney (Ve o) and adenoca cinomic human al eola basal
epi helial A549 cells, espec i ely, as desc ibed ea lie [
27
,
28
]. Ve o cells we e used o p epa e HSV-1
and -2 s ocks as desc ibed p e iously [
29
]. ZIKV FB-GWUH-2016 s ain was cul u ed in Ve o E6 cells
as desc ibed ea lie [
30
]. Semliki Fo es i us (SFV) exp essing VA7-mChe y was gene a ed by he
in i o
ansc ip ion o pSP6-SFV4 cDNA and he elec opo a ion o mRNA in baby hams e kidney
(BHK) 21 cells as p e iously desc ibed [
31
]. HBV pa icles we e p epa ed om he cul u e supe na an
o HepAD38 cells as desc ibed p e iously [
32
]. MERS-CoV was p opaga ed in a monolaye o Ve o
cells o h ee days a 37
◦
C as p e iously desc ibed [
33
]. The i us s ocks we e s o ed a
−
80
◦
C.
All he expe imen s wi h i uses we e pe o med in compliance wi h he guidelines o he na ional
au ho i ies using app op ia e Biosa e y labo a o ies unde app op ia e e hical and sa e y app o als.
2.3. Cells
All cells we e p opaga ed a 37
◦
C in 5% CO
2
. Human e inal pigmen epi helial (RPE) cells
we e g own in Dulbecco
'
s modi ied Eagle
'
s medium (DMEM) F12 supplemen ed wi h 50 U/mL
penicillin-s ep omycin (PenS ep), 2mM L-glu amine, 10% e al bo ine se um (FBS), and 0.25%
sodium bica bona e (Sigma-Ald ich). Vi us g ow h medium (VGM) o RPE con ained 0.2% BSA,
2 mM L-glu amine, 0.348% NaHCO
3
, and 1
µ
g/mL l-1- osylamido-2-phenyle hyl chlo ome hyl
ke one- ypsin (TPCK)- ypsin (Sigma-Ald ich) in DMEM-F12. Human A549 cells we e g own in
DMEM medium, supplemen ed wi h 50 U/mL PenS ep, 2 mM L-glu amine, and 10% FBS. VGM o
A549 con ained 0.2% BSA, 2 mM L-glu amine, and 1
µ
g/mL 1- osylamido-2-phenyle hyl chlo ome hyl
ke one- ypsin (TPCK)- ypsin (Sigma-Ald ich) in DMEM. Pa02C we e g own in egula DMEM
supplemen ed wi h 10% FBS. HepG2-NTCP2 cells we e main ained in DMEM supplemen ed wi h 10%
FBS, 2 mM L-glu amine, 50 U/mL penicillin, and 50
µ
g/mL s ep omycin as p e iously desc ibed [
32
].
Vi uses 2017,9, 271 4 o 15
Ve o cells we e g own in high glucose DMEM supplemen ed wi h 10% hea -inac i a ed FBS and
1×An ibio ic-An imyco ic solu ion (Gibco/Li e Technologies, Ca lsbad, CA, USA).
2.4. Compound Toxici y and E icacy Assays
App oxima ely 4
×
10
4
cells we e seeded in each well o a 96-well pla e. The cells we e g own
o 24 h in app op ia e cell g ow h medium. The media was eplaced wi h VGM con aining CellTox
G een Exp ess cy o oxici y eagen (CTxG, 1:2000 dilu ion in he assay well, P omega, Madison, WI,
USA). The compounds we e added o he cells in h ee- old dilu ions a se en di e en concen a ions
s a ing om 10
µ
M. No compounds we e added o he con ol wells. The cells we e mock- o
i us- in ec ed. The mul iplici y o in ec ion (moi) was 0.1 o 3 depending on he i us s ain. When
Bcl-2i induced a cy opa hic e ec in i us-in ec ed cells ( ypically on 12–24 h), he cells we e imaged
using a Cy a ion 5 Imaging eade (BioTek Ins umen s Inc., Winooski, VT, USA) in a b igh - ield
o luo escen mode (485–500 nm
Ex
/520–530 nm
Em
) and he luo escence was measu ed wi h a
PHERAs a FS pla e eade (BMG Lab ech, O enbe g, Ge many). The media was emo ed om
he cells. CellTi e -Glo iabili y (CTG, P omega, Madison, WI, USA) eagen was added (30
µ
L
pe well). The luminescence was measu ed wi h a PHERAs a FS pla e eade . The aw da a was
no malized agains calib a ion (s anda d) cu es. The cu es we e gene a ed using pu i ied human
genomic DNA o ATP (Sigma Ald ich). Each o he cu es was i ed using logis ic eg ession analysis,
wi h R
2PRESS
= 0.997 and R
2PRESS
= 0.995 o he dsDNA and ATP calib a ion cu es, whe e R
2PRESS
is
a p edic ed coe icien o de e mina ion R-squa ed calcula ed om he p edic ed esidual e o sum o
squa es (PRESS) s a is ic [
34
]. This allowed accu a e es ima ion o unknown concen a ions by inding
in e sec s wi h known luo escence and luminescence measu emen s on he calib a ion cu e.
2.5. Sco ing D ug Response P o iles
The dose- esponse cu es we e i ed wi h ou -pa ame e logis ic unc ions (Equa ion (1)), whe e
Amin
and
Amax
a e he uppe and lowe asymp o es (minimal and maximal d ug e ec s),
m
is he dose
ha p oduces he hal -maximal e ec , and λis he s eepness (slope) o he cu e.
(x)=Amin +Amax −Amin
1+x
mλ(1)
We used a composi e apezoidal ule o es ima e he a ea unde he dose- esponse cu e
(
AUC
), which is a commonly used me ic o he quan i ica ion o d ug esponses h ough mul iple
dose-le els [
35
–
37
]. This allowed us o calcula e di e en ial AUC
(dAUC)
be ween wo dose- esponse
cu es ( o example, plus minus i us).
The hal maximal cy o oxic concen a ion (CC
50
), he hal maximal e ec i e concen a ion (EC
50
)
o each compound was de e mined as desc ibed p e iously [
38
]. The ela i e e ec i eness o he d ug
was de ined as he he apeu ic o selec i i y index (SI = CC50/EC50).
2.6. Vi us Ti a ion
The cells we e ea ed wi h 1
µ
M Bcl-2i o emained non- ea ed and in ec ed wi h a i us. A e
24 h, supe na an s we e collec ed, se ially dilu ed in PBS, and added o Ve o-E6, MDCK, o A549
cells. The media was changed, and he cells we e o e laid wi h plaque assay media. The cells we e
ixed, and i al i e s we e calcula ed. The i e s we e exp essed as plaque- o ming uni s (PFU), 50%
issue cul u e in ec i e dose (TCID50), o luo escence- o ming uni s (FFU)/mL. The EC
50
alues as
well as he a ios be ween i us i e s in non- ea ed and compound- ea ed cells a ce ain Bcl-2i
concen a ions we e calcula ed.
IAV and FLUBV we e i e ed using plaque assay on MDCK cells, as desc ibed ea lie [
25
]. EV1
and EV6 i e s we e also de e mined by plaque assay on A549 cells [
39
]. HSV-1 and HSV-2 i e s we e
Vi uses 2017,9, 271 5 o 15
de e mined by plaque i a ion in Ve o cells in he p esence o human immunoglobulin G (
20 µg/mL
) as
desc ibed ea lie [
29
]. SFV4 was i e ed by plaque assay on BHK-21 cells, as p e iously desc ibed [
31
].
2.7. T ans ec ions o RPE Cells wi h RNA o Plasmid DNA
RPE cells we e cul u ed o 80% con luence in 96 well pla es and ans ec ed wi h 160 ng i al
genomic RNA using Lipo ec amine RNAiMAX (The mo Fishe Scien i ic, Wal ham, MA, USA) o
wi h 30, 100, o 300 ng o plasmid DNA (pEGFP) using Lipo ec amine 3000 (The mo Fishe Scien i ic,
Wal ham, MA, USA).
2.8. Li e Mic oscopy o Semliki Fo es Vi us In ec ion Du ing Bcl-2-Inhibi ion
One housand Pa02C cells we e seeded pe well in 384-well pla es in duplica e in he p esence
o 1:2000 dilu ion o CellToxG een eagen . Cells we e in ec ed wi h 10 PFUs pe well o SFV ec o
VA7-mChe y [
31
]. Pla es we e placed in an Incucy e Zoom li e mic oscope (Essen Bioscience,
Ann A bo
, MI, USA) and se o image e e y 30 min. Mo ies we e assembled using all ames,
while s ill images show he cells e e y 2 h.
2.9. Dynamic BH3 Pep ide P o iling
Dynamic BH3 p o iling was pe o med as desc ibed p e iously [
40
]. B ie ly, 4
×
10
4
RPE cells
we e pla ed pe well in 96-well pla es (Co ning, Co ning, NY, USA). A e 16 h, he cells we e in ec ed
wi h IAV o ans ec ed wi h pEGFP plasmid using Lipo ec amine 3000 ollowing he manu ac u e ’s
ins uc ions. Th ee hou s pos in ec ion and one hou pos ans ec ion, he medium was eplaced wi h
100
µ
L o De i ed om T ehalose Expe imen al Bu e (DTEB). The DTEB con ained 135 mM ehalose,
50 mM KCl, 20
µ
M EDTA, 20
µ
M EGTA, 0.1% BSA, 5 mM succina e, 10 mM HEPES-KOH, 0.005%
digi onin, 10
µ
g/mL Oligomycin A, 5
µ
M
β
-me cap oe hanol, 1
µ
M JC-1 luo escen p obe (all om
Sigma), and BH3-domain pep ides BIM (1
µ
M), BID (5
µ
M), PUMA (5
µ
M), NOXAA (5
µ
M), BAD
(
10 µM
), HRK (5
µ
M; KJ Ross-Pe e ssen, ApS, Copenhagen, Denma k), o DMSO (Sigma-Ald ich).
The luo escen
signal was measu ed a
λ
= 593 nm o 150 min wi h 10 min in e als using a Cy a ion
5 Imaging eade (BioTek Ins umen s Inc., Winooski, VT, USA). The kine ic cu es we e plo ed
and analyzed using G aphPad P ism7 (G aphPad So wa e Inc., La Jolla, CA, USA). Mi ochond ial
depola iza ion and del a apop o ic p iming we e calcula ed as desc ibed [40].
2.10. Me abolomics
Me abolomics analysis was pe o med as desc ibed p e iously [
41
]. B ie ly, 10
µ
L o labeled
in e nal s anda d mix u e was added o 100
µ
L o he sample (cell cul u e media), and 0.4 mL o
sol en (99% ACN and 1% FA) was added o each sample. The samples we e il e ed. Sample analysis
was pe o med on an Acqui y UPLC-MS/MS sys em (Wa e s Co po a ion, Mil o d, MA, USA). The
de ec ion sys em, a Xe o TQ-S andem iple quad upole mass spec ome e (Wa e s, Mil o d, MA,
USA), was ope a ed in bo h posi i e and nega i e pola i ies wi h a pola i y swi ching ime o 20 msec.
Elec o sp ay ioniza ion was chosen as he ioniza ion mode wi h a capilla y ol age a 0.6 kV in bo h
pola i ies. The sou ce empe a u e and desol a ion empe a u e o 120
◦
C and 650
◦
C, espec i ely,
we e main ained cons an ly h oughou he expe imen . The declus e ing po en ial and collision
ene gy we e op imized o each compound. The Mul iple Reac ion Moni o ing (MRM) acquisi ion
mode was selec ed o he quan i ica ion o he me aboli es wi h an indi idual span ime o 0.1 s
gi en in hei indi idual MRM channels. The dwell ime was calcula ed au oma ically by he so wa e
based on he egion o he e en ion ime window, he numbe o MRM unc ions, and also depending
on he numbe o da a poin s equi ed o o m he peak. MassLynx so wa e ( e sion 4.1, Wa e s
Co po a ion, Mil o d, MA, USA) was used o da a acquisi ion, da a handling, and ins umen con ol.
Da a p ocessing was done using Ta ge Lynx so wa e (Wa e s Co po a ion, Mil o d, MA, USA), and he
me aboli es we e quan i ied by calcula ing he cu e a ea a io using labeled in e nal s anda ds and
ex e nal calib a ion cu es. The me abolomics da a was log2 ans o med o linea modeling and
Vi uses 2017,9, 271 6 o 15
empi ical-Bayes-mode a ed - es s using he R/Bioconduc o so wa e package limma [
42
]. To analyse
he di e ences in me aboli es le els, a linea model was i o each me aboli e. The Benjamini-Hochbe g
me hod was used o co ec o mul iple es ing. The signi ican me aboli es we e de e mined a a
Benjamini-Hochbe g alse disco e y a e (FDR) con olled a 10%. The hea map was gene a ed
using he phea map package based on log ans o med p o iling da a. Me aboAnalys ( e sion 3.0,
McGill Uni e si y, S e. Ann de Belle ue, QC, Canada) was used o iden i y he me abolic pa hways
associa ed wi h i us in ec ion o a ec ed by Bcl-2i ea men [43].
2.11. Immuno-P ecipi a ion and Mass-Spec ome y
The Bcl-xL-, Bcl-2-, o Mcl-1-associa ed ac o s we e immuno-p ecipi a ed om IAV-in ec ed and
non-in ec ed RPE cells using abbi an i-Bcl-xL, Bcl-2, o Mcl-1 an ibodies (1:200; Cell Signalling
Technology, Dan e s, MA, USA), sepa a ed wi h sodium dodecyl sul a e polyac ylamide gel
elec opho esis (SDS-PAGE) and isualized by Coomassie s aining. The en i e lanes o speci ic p o ein
bands we e cu . The p o eins we e in-gel diges ed wi h ypsin. The esul ing pep ides we e analyzed
using liquid ch oma og aphy– andem mass spec ome y, as desc ibed p e iously [
11
,
44
]. The mass
spec ome y da a we e sea ched using in-house Masco and he P o einPilo in e ace agains he
SwissP o da abase. Only s a is ically signi ican da a (p< 0.05) we e selec ed.
3. Resul s
Ou dynamic BH3 pep ide p o iling e ealed ha Bad, Bim, Bid, Puma, and Noxa enhanced
MoMP in IAV- bu no in mock-in ec ed human non-malignan RPE cells, which ep esen na u al
a ge s o IAV in ec ion (Figu e S1) [
45
–
50
]. A co-immunop ecipi a ion expe imen using an ibodies
agains p o-su i al Bcl-xL, Bcl-2, o Mcl-1 ollowed by mass spec ome y showed ha se e al cellula
p o eins, including Bad, Bax, Bak, UACA, PAWR, FLII, T im21, IMMT, 14-3-3, EFHD2, DHX9, DDX3,
NLRP3, and LRRFIP2, as well as i al ac o s M1, NS1, HA, and NP we e p esen in he complexes
(Figu e S2). Thus, hese expe imen s demons a ed ha p o-apop o ic Bcl-2 p o eins (Bad, Bax, Bak),
PRRs (DHX9, DDX3, LRRFIP2), and o he ac o s can be in ol ed in he p og ammed dea h o
IAV-in ec ed cells.
I was shown ha ABT-263 a ge s Bcl-xL and Bcl-2 and al e s hei in e ac ion wi h p o-apop o ic
Bax, Bad, and Bak [
19
,
20
]. We es ed he e ec o ABT-263 on he iabili y o RPE cells in ec ed wi h
IAV o mock by ca ying ou dose esponse s udies. As eadou s, we used luo escen mic oscopy,
which isualizes dead (g een) and li ing (blue) cells. Fluo escen mic oscopy e ealed ha ABT-263
induced he p ema u e dea h o IAV-in ec ed cells a concen a ions no oxic o non-in ec ed cells
(Figu e 1A).
We alida ed he esul s wi h he CTxG and CTG assays. The CTxG assay uses luo escen
asymme ic cyanine dye ha s ains he DNA o dead cells, whe eas CTG assay quan i ies ATP,
an indica o o me abolically ac i e li ing cells. We calcula ed he alues o he di e en ial a ea unde
he dose- esponse cu es be ween he mock- and IAV-in ec ed cells o bo h assays (
∆
AUC
CTG
and
∆
AUC
CTG
; Figu e 1B,C,E). High alues indica e ha ABT-263 was an e ec i e igge o he p ema u e
dea h o in ec ed cells. In in ec ed cells, he e ec was obse ed al eady a 12 h pos in ec ion, whe eas
no oxici y was de ec ed o a leas 36 h a 0.4
µ
M ABT-263 in he mock-in ec ed cells (Figu e 1E).
Mo eo e , he e ec was dependen no only on he dose o ABT-263 bu also on he i al load
(Figu e 1D).
In addi ion, we e alua ed he e ec o ABT-263 on IAV eplica ion by i e ing i uses p oduced
in cells ea ed and non- ea ed wi h ABT-263. ABT-263 ea men lowe ed IAV p oduc ion in RPE
cells, as indica ed by he old change be ween i us i e s (Figu e 1D,E). We ob ained simila esul s in
A549 cells (Figu e S3). Fu he mo e, ABT-263 ac i a ed caspase 3 in IAV-in ec ed bu no in non- ea ed
o ABT-263- ea ed mock-in ec ed RPE cells a 12 h pos in ec ion (Figu e S4). These esul s collec i ely
sugges ha ABT-263 induced p ema u e apop osis in IAV-in ec ed cells and lowe ed IAV p oduc ion.
Vi uses 2017,9, 271 7 o 15
Vi uses 2017, 9, 271 7 o 15
Figu e 1. A 24 h pos in ec ion, ABT-263 kills in luenza A (IAV)-in ec ed bu no mock-in ec ed RPE
cells and lowe s he p oduc ion o in ec ious i al pa icles. (A) Fluo escen mic oscopy images
showing ha inc easing concen a ions o ABT-263 kill IAV-in ec ed (moi 3) bu no mock-in ec ed
e inal pigmen epi helium (RPE) cells a 24 h. Asymme ic cyanine dye s ains he dsDNA o dead
cells. Hoechs s ains DNA in li ing cells; (B) quan i ica ion o dsDNA in dead cells using
CellToxG een cy o oxici y (CTxG) assay. Mean ± s anda d de ia ion (SD), n = 3; (C) quan i ica ion o
in acellula ATP in li ing cells using CellTi e -Glo luminescen cell iabili y (CTG) assay. Mean ±
s anda d de ia ion (SD), n = 3; (D) RPE cells we e non- o ABT-263- ea ed (0.4 μM) and in ec ed wi h
IAV a moi 0.08, 0.4, 2, and 10. Cell iabili y was measu ed using a CTG assay 24 h a e in ec ion.
Mean ± SD, n = 3; (E) RPE cells we e non- o ABT-263- ea ed (0.4 μM) and mock- o IAV-in ec ed
(moi 3), and cell iabili y was measu ed using a CTG assay a he indica ed ime poin s. Mean ± SD,
n = 3; (F) example o plaque assay measu ing i us p oduc ion in Bcl-2i- (3 μM) and DMSO- ea ed
RPE cells a 24 hpi; (G) able summa izing he di e en ial e ec o ABT-263 on he iabili y o i us-
and mock-in ec ed cells, exp essed as ΔAUCCxTG and ΔAUCCTG. I also shows he e ec o ABT-263 on
i us p oduc ion in d ug- (3 μM) and DMSO- ea ed RPE cells, which is exp essed in log10 old change
(FC). Mean ± SD, n = 3.
Figu e 1.
A 24 h pos in ec ion, ABT-263 kills in luenza A (IAV)-in ec ed bu no mock-in ec ed
RPE cells and lowe s he p oduc ion o in ec ious i al pa icles. (
A
) Fluo escen mic oscopy images
showing ha inc easing concen a ions o ABT-263 kill IAV-in ec ed (moi 3) bu no mock-in ec ed
e inal pigmen epi helium (RPE) cells a 24 h. Asymme ic cyanine dye s ains he dsDNA o dead
cells. Hoechs s ains DNA in li ing cells; (
B
) quan i ica ion o dsDNA in dead cells using CellToxG een
cy o oxici y (CTxG) assay. Mean
±
s anda d de ia ion (SD), n= 3; (
C
) quan i ica ion o in acellula
ATP in li ing cells using CellTi e -Glo luminescen cell iabili y (CTG) assay. Mean
±
s anda d
de ia ion (SD), n= 3; (
D
) RPE cells we e non- o ABT-263- ea ed (0.4
µ
M) and in ec ed wi h IAV a
moi 0.08, 0.4, 2, and 10. Cell iabili y was measu ed using a CTG assay 24 h a e in ec ion. Mean
±
SD,
n= 3
; (
E
) RPE cells we e non- o ABT-263- ea ed (0.4
µ
M) and mock- o IAV-in ec ed (moi 3), and cell
iabili y was measu ed using a CTG assay a he indica ed ime poin s. Mean
±
SD, n= 3; (
F
) example
o plaque assay measu ing i us p oduc ion in Bcl-2i- (3
µ
M) and DMSO- ea ed RPE cells a 24 hpi;
(
G
) able summa izing he di e en ial e ec o ABT-263 on he iabili y o i us- and mock-in ec ed
cells, exp essed as
∆
AUC
CxTG
and
∆
AUC
CTG
. I also shows he e ec o ABT-263 on i us p oduc ion
in d ug- (3
µ
M) and DMSO- ea ed RPE cells, which is exp essed in log
10
old change (FC).
Mean ±SD
,
n= 3.
Impo an ly, ABT-263 induced he p ema u e dea h o RPE cells in ec ed wi h FLUBV, SFV,
o HSV-1 and lowe ed i us p oduc ion, all a concen a ions ha a e no oxic o non-in ec ed cells
(Figu e 2). We ob ained simila esul s wi h ZIKV, EV1, EV6, MERS-CoV, HBV, and HSV-2 i uses using
di e en cell lines (Figu e S5). Supplemen a y mo ies 1 and 2 u he ea i m ha he ea men o
panc ea ic cance cells in ec ed wi h SFV wi h ABT-263 a low moi a enua ed he eplica ion and sp ead
o he i us in hese cells. These esul s sugges ha Bcl-2i migh be used as b oad-spec um an i i als.
Vi uses 2017,9, 271 8 o 15
Vi uses 2017, 9, 271 8 o 15
Figu e 2. ABT-263 induces he p ema u e dea h o cells in ec ed wi h di e en i uses. (A) CTxG and
(B) CTG plo s showing he ABT-263 induces he p ema u e dea h o RPE cells in ec ed wi h FLUBV,
SFV4, o HSV-1 i uses (moi 1) bu no hose in ec ed wi h mock. Mean ± SD, n = 3; (C) able
summa izing he di e en ial e ec o Bcl-2i on he iabili y o i us- and mock-in ec ed cells,
exp essed as ΔAUCCxTG and ΔAUCCTG. I also shows he e ec o ABT-263 on i us p oduc ion in
d ug- (3 μM) and DMSO- ea ed RPE cells, which is exp essed in log10 old change (FC).
Impo an ly, ABT-263 induced he p ema u e dea h o RPE cells in ec ed wi h FLUBV, SFV, o
HSV-1 and lowe ed i us p oduc ion, all a concen a ions ha a e no oxic o non-in ec ed cells
(Figu e 2). We ob ained simila esul s wi h ZIKV, EV1, EV6, MERS-CoV, HBV, and HSV-2 i uses
using di e en cell lines (Figu e S5). Supplemen a y mo ies 1 and 2 u he ea i m ha he
ea men o panc ea ic cance cells in ec ed wi h SFV wi h ABT-263 a low moi a enua ed he
eplica ion and sp ead o he i us in hese cells. These esul s sugges ha Bcl-2i migh be used as
b oad-spec um an i i als.
We hypo hesized ha i al o any o he o eign RNA o DNA could igge ABT-263-sensi ized
apop osis. To es his, we ans ec ed RPE cells wi h IAV RNA ( RNA) o plasmid DNA (pDNA)
and ea ed ans ec ed cells wi h ABT-263. Vi al RNA- o pDNA, bu no mock- ans ec ed cells,
apidly died a e ABT-263 ea men (Figu e 3 and Figu e S6 A,B). I is impo an o no e ha cell
dea h was dependen on he amoun o ans ec ed RNA and pDNA, as well as on concen a ion o
ABT-263. Mo eo e , BH3 pep ides o Puma, Bad, and Bid induced MoMP in pDNA- ans ec ed ABT-
263- ea ed cells as ea ly as 3 h pos ans ec ion, in con as o cells ea ed wi h ABT-263 o cells
ans ec ed wi h pDNA (Figu e S6 F). As expec ed, ABT-263 did no accele a e he dea h o RPE o
A549 cells ea ed wi h bac e ial lipopolysaccha ides (LPS), which a e ecognized by di e en PRRs
o hose ha de ec in acellula o eign RNA o DNA (Figu e S6 C–E).
Figu e 2.
ABT-263 induces he p ema u e dea h o cells in ec ed wi h di e en i uses. (
A
) CTxG
and (
B
) CTG plo s showing he ABT-263 induces he p ema u e dea h o RPE cells in ec ed wi h
FLUBV, SFV4, o HSV-1 i uses (moi 1) bu no hose in ec ed wi h mock. Mean
±
SD, n= 3; (
C
) able
summa izing he di e en ial e ec o Bcl-2i on he iabili y o i us- and mock-in ec ed cells, exp essed
as
∆
AUC
CxTG
and
∆
AUC
CTG
. I also shows he e ec o ABT-263 on i us p oduc ion in d ug- (3
µ
M)
and DMSO- ea ed RPE cells, which is exp essed in log10 old change (FC).
We hypo hesized ha i al o any o he o eign RNA o DNA could igge ABT-263-sensi ized
apop osis. To es his, we ans ec ed RPE cells wi h IAV RNA ( RNA) o plasmid DNA (pDNA) and
ea ed ans ec ed cells wi h ABT-263. Vi al RNA- o pDNA, bu no mock- ans ec ed cells, apidly
died a e ABT-263 ea men (Figu e 3and Figu e S6 A,B). I is impo an o no e ha cell dea h was
dependen on he amoun o ans ec ed RNA and pDNA, as well as on concen a ion o ABT-263.
Mo eo e , BH3 pep ides o Puma, Bad, and Bid induced MoMP in pDNA- ans ec ed ABT-263- ea ed
cells as ea ly as 3 h pos ans ec ion, in con as o cells ea ed wi h ABT-263 o cells ans ec ed wi h
pDNA (Figu e S6 F). As expec ed, ABT-263 did no accele a e he dea h o RPE o A549 cells ea ed
wi h bac e ial lipopolysaccha ides (LPS), which a e ecognized by di e en PRRs o hose ha de ec
in acellula o eign RNA o DNA (Figu e S6C–E).
In he nex expe imen , we p e- ea ed RPE o A549 cells wi h compounds ha inhibi he
endocy ic up ake (oba oclax o SaliPhe) o he ansc ip ion and eplica ion o IAV (JNJ-872 o
gemci abin) [
46
,
51
] and in ec ed hem wi h IAV. ABT-263 did no show any e ec on hese
cells (Figu e S7). These esul s con i med ha in acellula o eign RNA/DNA o i s eplica ion
in e media es igge ed apop osis, and Bcl-2i accele a e his p ocess.
Vi uses 2017,9, 271 9 o 15
Vi uses 2017, 9, 271 9 o 15
Figu e 3. ABT-263 induces he p ema u e dea h o cells ans ec ed wi h IAV genomic RNA ( RNA)
o plasmid DNA (pDNA). (A) Fluo escen mic oscopy images showing ha ABT-263 kills RNA-
ans ec ed (160 ng) bu no mock- ans ec ed RPE cells a 8 h pos ans ec ion. Asymme ic cyanine
dye s ains he dsDNA o dead cells. Hoechs s ains DNA in li ing cells; (B) CTxG plo showing ha
ABT-263 (3 μM) induces ha p ema u e dea h o RPE cells ans ec ed wi h inc easing concen a ions
o RNA. Mean ± SD, n = 3; (C) Fluo escen and b igh ield mic oscopy o RPE cells showing ha
ABT-263 kills eGFP-exp essing plasmid ans ec ed (300 ng) bu no mock- ans ec ed RPE cells a 6
h pos ans ec ion; (D) CTG g aph showing ha he iabili y o ABT-263- ea ed (3 μM) cells
dec eases wi h inc easing concen a ions o ans ec ed plasmid DNA. Mean ± SD, n = 3.
In he nex expe imen , we p e- ea ed RPE o A549 cells wi h compounds ha inhibi he
endocy ic up ake (oba oclax o SaliPhe) o he ansc ip ion and eplica ion o IAV (JNJ-872 o
gemci abin) [46,51] and in ec ed hem wi h IAV. ABT-263 did no show any e ec on hese cells
(Figu e S7). These esul s con i med ha in acellula o eign RNA/DNA o i s eplica ion
in e media es igge ed apop osis, and Bcl-2i accele a e his p ocess.
In o de o p o ide u he clues on he mechanisms o Bcl-2i-sensi ized apop osis, we
in es iga ed he e ec o ABT-263 analogues (ABT-737, ABT-199, WEHI-539, A-1331852, and A-
1155463) on he iabili y and dea h o IAV-in ec ed and mock-in ec ed RPE cells a 24 hpi (Figu e 4).
In addi ion, we e alua ed he e ec o hese compounds on IAV eplica ion by i e ing he i uses
p oduced in cells ea ed wi h inc easing compound concen a ions. We calcula ed CC50, EC50, and SI
alues o each compound. We obse ed ha ABT-263 was mo e selec i e han s uc u ally simila
ABT-737 and ABT-199, whe eas A-1155463 was mo e selec i e han s uc u ally simila A-1331852
and WEHI-539 (Figu e 4 and Figu e 5A). Impo an ly, he ea men o non-in ec ed RPE cells wi h
hese compounds al e ed he le els o sec e ed adenine, adenosine, hypoxan hine, IMP, AMP,
inosine, and xan hosine, which belong o he ATP me abolic pa hway (Figu e S8). Al oge he , hese
esul s sugges ha A-1155463 may ep esen an an i i al lead candida e.
Figu e 3.
ABT-263 induces he p ema u e dea h o cells ans ec ed wi h IAV genomic RNA
( RNA) o plasmid DNA (pDNA). (
A
) Fluo escen mic oscopy images showing ha ABT-263 kills
RNA- ans ec ed (160 ng) bu no mock- ans ec ed RPE cells a 8 h pos ans ec ion. Asymme ic
cyanine dye s ains he dsDNA o dead cells. Hoechs s ains DNA in li ing cells; (
B
) CTxG plo
showing ha ABT-263 (3
µ
M) induces ha p ema u e dea h o RPE cells ans ec ed wi h inc easing
concen a ions o RNA. Mean
±
SD, n= 3; (
C
) Fluo escen and b igh ield mic oscopy o RPE cells
showing ha ABT-263 kills eGFP-exp essing plasmid ans ec ed (300 ng) bu no mock- ans ec ed
RPE cells a 6 h pos ans ec ion; (
D
) CTG g aph showing ha he iabili y o ABT-263- ea ed (3
µ
M)
cells dec eases wi h inc easing concen a ions o ans ec ed plasmid DNA. Mean ±SD, n= 3.
In o de o p o ide u he clues on he mechanisms o Bcl-2i-sensi ized apop osis, we in es iga ed
he e ec o ABT-263 analogues (ABT-737, ABT-199, WEHI-539, A-1331852, and A-1155463) on he
iabili y and dea h o IAV-in ec ed and mock-in ec ed RPE cells a 24 hpi (Figu e 4). In addi ion,
we e alua ed he e ec o hese compounds on IAV eplica ion by i e ing he i uses p oduced in
cells ea ed wi h inc easing compound concen a ions. We calcula ed CC
50
, EC
50
, and SI alues o
each compound. We obse ed ha ABT-263 was mo e selec i e han s uc u ally simila ABT-737 and
ABT-199, whe eas A-1155463 was mo e selec i e han s uc u ally simila A-1331852 and WEHI-539
(Figu es 4and 5A). Impo an ly, he ea men o non-in ec ed RPE cells wi h hese compounds al e ed
he le els o sec e ed adenine, adenosine, hypoxan hine, IMP, AMP, inosine, and xan hosine, which
belong o he ATP me abolic pa hway (Figu e S8). Al oge he , hese esul s sugges ha A-1155463
may ep esen an an i i al lead candida e.