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Ti le Page 1
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Filo i us an i i al ac i i y o ca ionic amphiphilic d ugs is associa ed wi h 3
lipophilici y and abili y o induce phospholipidosis 4
5
Au ho s and a ilia ions: 6
An onia P. Gunescha,b,c, F ancisco J. Zapa e o-Belinchona,b,c , Lukas Pinke d, Eike 7
S einmanne, Michael P. Mannsa,b, Gisbe Schneide , Thomas Pie schmannb,c, Ma k 8
B öns upb,d, Thomas on Hahna,b,c,g#
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a. Depa men o Gas oen e ology, Hepa ology and Endoc inology, Hanno e 11
Medical School, Hanno e , Ge many 12
b. Ge man Cen e o In ec ion Resea ch (DZIF), Hanno e -B aunschweig si e, 13
Ge many; 14
c. Ins i u e o Expe imen al Vi ology, TWINCORE, Cen e o Expe imen al and 15
Clinical In ec ion Resea ch Hanno e , Hanno e , Ge many 16
d. Depa men o Chemical Biology, Helmhol z Cen e o In ec ion Resea ch 17
(HZI), B aunschweig, Ge many 18
e. Depa men o Molecula & Medical Vi ology, Ruh -Uni e si ä Bochum, 19
Bochum, Ge many 20
. Depa men o Chemis y and Applied Biosciences, Ins i u e o Pha maceu ical 21
Sciences, Eidgenössische Technische Hochschule (ETH) Zü ich, Swi ze land 22
g. Depa men o Gas oen e ology and In e en ional Endoscopy, Asklepios 23
Hospi al Ba mbek, Semmelweis Uni e si y, Campus Hambu g, Ge many 24
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AAC Accep ed Manusc ip Pos ed Online 8 June 2020
An imic ob. Agen s Chemo he . doi:10.1128/AAC.00143-20
Copy igh © 2020 Ame ican Socie y o Mic obiology. All Righ s Rese ed.
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#Co esponding au ho : P o . D . med. Thomas on Hahn, [email protected]. 26
Sho unning i le: An i i al s uc u e-ac i i y ela ionship o ca ionic amphiphilic d ugs27
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1. Abs ac 28
Se e al ca ionic amphiphilic d ugs (CADs) ha e been ound o inhibi cell en y o 29
ilo i uses and o he en eloped i uses. S uc u ally un ela ed CADs may ha e 30
an i i al ac i i y, ye he unde lying common mechanism and s uc u e-ac i i y 31
ela ionship a e incomple ely unde s ood. 32
We aimed o unde s and how widesp ead an i i al ac i i y is among CADs and which 33
s uc u al and physico-chemical p ope ies a e linked o en y inhibi ion. 34
We measu ed inhibi ion o Ma bu g i us pseudopa icle (MARVpp) cell en y by 45 35
he e ogeneous and mos ly FDA-app o ed CADs and cy o oxici y in EA.hy926 cells. 36
We analysed co ela ion o an i i al ac i i y wi h ou chemical p ope ies: pKa, ClogP, 37
molecula weigh and dis ance be ween he basic g oup and hyd ophobic ing 38
s uc u es. Addi ionally, we quan i ied d ug-induced phospholipidosis (DIPL) o a CAD 39
subse by low cy ome y. S uc u ally simila compounds (de i a i es) and hose wi h 40
simila chemical p ope ies bu un ela ed s uc u e (analogues) o s ong inhibi o s 41
we e ob ained by wo in silico simila i y sea ch app oaches and es ed o an i i al 42
ac i i y. O e all 11 ou o 45 (24 %) CADs inhibi ed MARVpp by 40 % o mo e. The 43
s onges an i i al compounds we e d oneda one, ipa anol and quinac ine. 44
S uc u e-ac i i y ela ionship s udies e ealed highly signi ican co ela ions be ween 45
an i i al ac i i y, hyd ophobici y (ClogP>4), and DIPL. Mo eo e , pKa and in a-46
molecula dis ance be ween hyd ophobic and hyd ophilic moie ies co ela ed wi h 47
an i i al ac i i y, bu o a lesse ex en . We also showed ha in con as o analogues, 48
de i a i es had simila an i i al ac i i y as he seed compound d oneda one. O e all, 49
one qua e o CADs inhibi s MARVpp en y in i o and an i i al ac i i y o CADs 50
mos ly elies on hei hyd ophobici y, ye is p omo ed by he indi idual s uc u e. 51
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2. In oduc ion 52
Ou b eaks o eme ging i al diseases ha e epea edly posed majo challenges 53
o global heal h in he ecen yea s (h ps://www.who.in /cs /don/en/). Impo an 54
examples include he Filo i idae Ebola i us (EBOV) and Ma bu g i us (MARV), 55
A ena i idae Lassa i us (LASV), Co ona i idae like Middle Eas Respi a o y 56
Synd ome co ona i us (MERS-CoV), o he Fla i i idae-membe ZIKA. Some o 57
hese in ec ions a e associa ed wi h high case- a ali y a es, e.g. 50 % o EBOV and 58
MARV, 35 % o MERS-CoV (2), and up o 30 % o hospi alized LASV pa ien s (3). 59
Thus acco ding o he WHO-Lis o Bluep in p io i y diseases o 2018, he 60
a o emen ioned pa hogens sha e p io i y s a us conce ning he need o esea ch 61
and de elopmen gi en ha e ec i e accines o di ec ac ing an i i als a e 62
una ailable in mos cases (4). I would be o g ea alue o ha e d ugs ha a e ac i e 63
agains a b oad ange o i al pa hogens (5, 6). Such agen s would likely no a ge 64
speci ic componen s o indi idual i uses bu a he cellula s uc u es o p ocesses 65
ha a e u ilized by a ious un ela ed i uses. An a ac i e a ge pa hway conce ns 66
cellula endosomal p ocesses, since hese a e used by nume ous pa hogens 67
including membe s o he amilies Filo i idae, A ena i idae, Rhabdo i idae, 68
Co ona i idae, Toga i idae, Fla i i idae and Bunya i idae. 69
Ca ionic amphiphilic d ugs (CADs) a e a di e se g oup o compounds, many o which 70
a e in medical use o a ious clinical indica ions. CADs a e de ined by a hyd ophilic 71
g oup ma ked by a basic amine wi h high pKa ( he nega i e loga i hm o he acid 72
dissocia ion cons an Ka) and hyd ophobic g oup(s) cha ac e ised by se e al 73
a oma ic o alipha ic s uc u es (7). Due o hei amphiphilic na u e CADs 74
ansmig a e memb anes which can be enhanced by di e en subs i uen s like 75
halogen esidues. Upon eaching acidic compa men s like la e endosomes (LE) and 76
endolysosomes, he basic amine g oups ge s p o ona ed, which leads o lysosomal 77
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apping o he compounds (8). Consequen ly, CADs p ima ily accumula e in acidi ied 78
compa men s (9, 10). 79
Se e al s udies, mos o which analysed se s o app o ed d ugs, ha e ound ha 80
a ious CADs ha e an i i al ac i i y. The an ia hy hmics amioda one, d oneda one 81
and e apamil (11) as well as he es ogen ecep o an agonis s clomi ene and 82
o emi ene (12) ha e been shown o inhibi ilo i al en y in di e en cell lines. 83
Mo eo e , clomi ene and o emi ene we e shown o ele a e su i al a es o mice 84
in ec ed wi h mouse-adap ed EBOV up o 90% (12). O no e, hey could show ha he 85
an i i al ac i i y o hese wo CADs was un ela ed o hei clinical mechanism o 86
ac ion as es ogen ecep o modula o s. Beyond ilo i uses, CADs ha e also been 87
ound o inhibi A ena i idae LASV and Guana i o i us (GTOV) (13), Hepa i is C 88
i us (HCV) (14–18), Japanese encephali is i us (JEV) (19), Se e e acu e 89
espi a o y synd ome- ela ed co ona i us (SARS-CoV) (20), and Epps ein-Ba i us 90
(EBV) (21). Mo eo e , i was ound ha he CADs amioda one, bep idil, aloxi ene 91
and amodiaquine besides se e al non-CAD compounds ha e b oad an i i al e ec s 92
agains all es ed alpha- and la i i uses (wi h he excep ion o ZIKA i us), as well as 93
a ying o he i uses, bu none inhibi ed Human immunode iciency i us (HIV) (22). 94
Highes an i i al ac i i y o all ou CADs was ound agains EBOV i us like 95
pa icles. Despi e hese obse a ions, i is no known so a how widesp ead an i i al 96
ac i i y eally is among CADs. Likewise, he mechanism o ac ion behind he an i i al 97
ac i i y is unclea . Fo his eason, we unde ook a comp ehensi e s udy o an i i al 98
aci i i y among 45 he e ogeneous CAD compounds and co ela ed an i i al ac i i y 99
wi h a ious physico-chemical p ope ies. 100
101
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3. Ma e ial and Me hods 103
2.1 Pseudopa icle p oduc ion and ansduc ion 104
Pseudopa icles we e p oduced as desc ibed be o e (23, 24). In b ie , p oduce HEK-105
293T cells we e seeded a 8*105 cells/well in a poly-L-lysine coa ed 6-well pla e. A e 106
24 h cells we e co- ans ec ed wi h 1 µg DNA using polye hyleneimine (PEI) o 107
p oduce HIV-based pseudopa icles bea ing indi idual i al en elope gp. The 108
plasmids used we e (1) a packaging plasmid con aining HIV-based gag-pol genes 109
(25), (2) a plasmid encoding o he en gene o MARV (pCAGGS-MARVGP) (26) o 110
an emp y ec o pcDNA3.1 and (3) a gu ed ye packaging compe en HIV-based 111
ans e plasmid encoding o NLuc epo e gene (pWPI_NanoLuc_BLR) (in a a io 112
1:1:4). A e 6 h pos - ans ec ion, medium was exchanged o 3 % FCS DMEM 113
con aining 5 % Penicillin/S ep omycin and pseudopa icles ha es ed, pooled and 114
il e ed a e 48 h and 72 h. Fo len i i al ansduc ion, a ge EA.hy926 cells we e 115
seeded 1*104 cells / well in a 96-well pla e. A e incuba ion o 24 h, 50 µL o 116
MARVpp o 50 µL comple e DMEM we e mixed wi h polyb ene (1:1000) and CADs o 117
an end-concen a ion o 5 µM and applied in iplica es. As a sol en con ol s e ile 118
wa e , E OH, o DMSO we e dilu ed in DMEM comple e in he same a io as he 119
d ugs. A 6 h pos ansduc ion (h.p. ), cells we e washed once wi h phospha e 120
bu e ed saline (PBS) and 100 µL o comple e DMEM we e applied. Se en y- wo 121
h.p. , cells we e washed wi h PBS and lysed. Nex , 20 µL o cell lysa e we e 122
ans e ed o a 96-well luminome e pla e, mixed wi h 80 µL o he luci e ase 123
subs a e coelen e azine, and incuba ed sho ly in he da k be o e NLuc ac i i y 124
quan i ica ion in a GloMax® pla e luminome e (P omega, Madison, WI, USA), 125
ep esen ing gp-d i en cell en y. 126
127
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2.2 Cy o oxici y es 128
EA.hy926-NLuc we e seeded a 1*104 cells/well in a 96-well pla e. The nex day, cells 129
we e ea ed in iplica e wi h ei he comple e DMEM, sol en con ol (comple e 130
DMEM + sol en ), o wi h dilu ed CADs a a inal concen a ion o 5 µM. A e 6 h, 131
cells we e washed wi h PBS and incuba ed wi h 100 µL comple e DMEM o ano he 132
72 h a 37 C. Then cells we e lysed and luci e ase ac i i y was de e mined as an 133
agg ega e measu e o cell iabili y and p oli e a ion. 134
135
Compounds and simila i y sea ch 136
The compounds amioda one HCl (A8423), amo ol ine HCl (SML0283), bep idil HCl 137
(B5016), clemas ine uma a e sal (SML0445), clomiphene ci a e sal (C6272), 138
dasa inib (CDS023389), d oneda one HCl (D9696), pe hexiline malea e sal 139
(SML0120), p omazine HCl (P6656), se aline HCl (S6319), e conazole (32355), 140
o emi ene ci a e sal (T7204), ipa anol (T5200), chlo cyclizine (SML1473), 141
clozapine (C6305), cyclizine HCl (C3090000), N-Dese hylamioda one HCl solu ion 142
(D-055), e apamil HCl (V4629), W-7 (N-(6-Aminohexyl)-5-chlo o-1-143
naph halenesul onamide hyd ochlo ide) (A3281), aman adine HCl (A1260), 144
Benzylamine (185701), chlo oquine diphospha e sal (C6628), clomip amine HCl 145
(C7291), chlo p omazine HCl (C0982), enclomiphene HCl (SML0719), en lu amine 146
HCl (F112), lupen ixol dihyd ochlo ide (Y0000064), luphenazine dihyd ochlo ide 147
(F4765), gen amicin sul a e (G1914), imip amine HCl (I0899), map o iline HCl 148
(M9651), mianse in HCl (M2525), p ochlo pe azine dimalea e sal (P9178), 149
p ome hazine HCl (P4651), p op anolol HCl (P0884), quinac ine dihyd ochlo ide 150
(Q3251), eicoplanin (T0578), hio idazine HCl (T9025), i luope azine HCl (T6062), 151
ipelennamine HCl (T7511), U18666A (U3633), W-5 (N-(6-Aminohexyl)-1-152
naph halenesul onamide hyd ochlo ide) (SML0657), zimelidine dihyd ochlo ide 153
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(Z101) we e pu chased om Sigma-Ald ich Chemie GmbH (Tau ki chen, Ge many). 154
The compound Ro 48-8071 was pu chased om Biomol GmbH (Hambu g, 155
Ge many). 156
D oneda one-de i a i es we e ecei ed using SciFinde ® so wa e (Chemical 157
Abs ac s Se ice, 2017). D-1 (N-Mesyld oneda one; TRC-M225785) and D-2 (S-158
Desme hyl S-Chlo ome hyl D oneda one; TRC-D291470) we e pu chased om 159
BIOZOL Diagnos ica Ve ieb GmbH (Eching, Ge many). To ob ain analogous CADs, 160
chemical ad anced empla e sea ch (CATS) so wa e was used (Schneide e al., 161
1999). The compounds AC1 (1-(4-E hyl-1-pipe azinyl)-4-[1-(4-me hylbenzyl)-1H-162
indol-3-yl]-1-bu anone; E230-0651), CS3 (2-[(E)-2-{4-[E hyl(4-163
me hoxyphenyl)amino]phenyl} inyl]-3-me hyl-1.3-benzo hiazol-3-ium; 7165-0012) 164
and QT1 (G119-1593; G119-1593) we e ob ained by ChemDi (San Diego, CA, 165
USA). CS1 (2-{2-[(3.4-dichlo ophenyl) hio]e hyl}-1-(2-oxo-2-phenyle hyl)py idinium 166
b omide; 5841320) and CS2 (1-{2-[4-(1-me hyl-1-167
phenyle hyl)phenoxy]e hyl}py olidine; 7020606) we e o de ed om ChemB idge
168
Co po a ion (San Diego, CA, USA). DQ1 (2-(4-(sec-bu yl)phenyl)-N’(1-169
phene hylpipe idin-4-ylidine)quinolone-4-ca bohyd azide; IVK 9315735) was 170
pu chased om SRC Alinda (Moscow, Russia) and CS4 (5-b omo-2-{[1-(3-171
luo ophenyl)e hyl]sul anyl}py idine; PB742999078) om Chemspace (Riga, La ia). 172
CT1 ((E)-1-(5-chlo o hiophen-2-yl)-3-(4-(diphenylamino)phenyl)p op-2-en-1-one; 173
Z46049784) was pu chased om Enamine (Monmou h junc ion, NJ, USA). As 174
sol en s wa e , e hanol (E OH) (Ca l Ro h, Ka ls uhe, Ge many), o dime hylsul oxid 175
(DMSO) (Ca l Ro h, Ka ls uhe, Ge many), we e used. 176
177
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Cell cul u e 178
Immo alized epi helial HEK-293T and EA.hy926, a usion p oduc o A549 and 179
human umbilical ein endo helial cells, we e cul u ed in Dulbecco’s Modi ied Eagle 180
medium (DMEM) wi h 10 % e al cal se um (FCS), 5 % Penicillin/S ep omycin, 5 % 181
non-essen ial aminoacids (NEAA) and 5 % L-glu amine (comple e DMEM) a 37 °C 182
wi h 5 % CO2. Luci e ase-exp essing EA.hy926-NLuc cells we e ob ained by 183
ansduc ion wi h VSV-Gpp ha bo ing he nano luci e ase (NLuc) gene and cul u ed 184
as p e iously desc ibed. 185
186
Phospholipidosis assay 187
EA.hy926 cells we e seeded a 1*104 cells/well in a 96-well pla e. The nex day, 188
CADs and hei sol en s (DMSO and wa e ) we e dilu ed in cul u e medium. 189
Phospholipidosis was quan i ied using LipidTOX™ G een Phospholipidosis De ec ion 190
Reagen (H34350, The mo Fishe Scien i ic, Wal ham, MA, USA) acco ding o a 191
modi ied e sion o a deg ada ion assay (Mesens e al., 2009). In b ie , LipidTOXTM 192
was dilu ed 1:500 in comple e DMEM and s e ile il e ed. Then 50 µL o ei he CAD 193
mix u e o con ols we e applied o he cells oge he wi h 50 µL o dilu ed LipidTox, 194
esul ing in a inal CAD concen a ion o 5 µM and a inal LipidTox dilu ion o 1:1000. 195
A e incuba ion o 6 h a 37 °C, cells we e washed once wi h PBS. Medium was 196
exchanged o 100 µL cul u e medium and only he d ugs (5µM) bu no LipidTOXTM 197
we e added a second ime. A e 24 h in o al, cells we e washed wi h PBS, 198
ypsinised, and ans e ed o FACS ubes. Cells we e washed wi h 1 mL FACS 199
bu e (2 % FCS in PBS), cen i uged o 5 min a 1000 pm and supe na an 200
disca ded. In some eplica es, cells we e ixed wi h 100 µL 3-6 % PFA wi h no 201
in luence on he esul s. Finally, CAD-induced lysosomal accumula ion o LipidTOX™ 202
was quan i ied as in acellula g een luo escence by low cy ome y in BD 203
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P e ious s udies showed ha amioda one, as well as i s close ela i e d oneda one 358
ac in low mic omola concen a ions agains pseudopa icles o di e en ilo i us 359
species, GTOV and in luenza H5N1. Fu he mo e, amioda one was shown o inhibi 360
au hen ic EBOV, HCV, SARS and ecen ly semliki o es i us, dengue i us, Sindbis 361
i us, Ross i e i us, he pes simplex i us and he a enua ed accine s ain o 362
yellow e e i us. Con e sely, ZIKV, HIV o accinia i us we e no a ec ed. (14, 15, 363
20, 22, 29–31) T ipa anol was ound o inhibi EBOV VLP as well as EBOV in ec ion 364
and HCV eplica ion (17, 32). Among a a ie y o o he CADs, quinac ine, se aline 365
and clomi ene inhibi ed mainly EBOV VLPs, mouse-adap ed EBOV and o he 366
ilo i us s ains, including MARV (12, 33). To ou knowledge, pe hexiline has no 367
been in es iga ed in he con ex o an i i als ye and was included in ou s udy due o 368
epo ed associa ion wi h DIPL (8). Fu he mo e, he amioda one me aboli e mono-N-369
dese hylamioda one (MDEA) was shown o ha e addi i e e ec s wi h amioda one 370
(30). Wi h ou esul s, we pa ially con i med and ex ended he p e ious s udies. 371
No ably, we could show ha he d ugs d oneda one and ipa anol ollowed by 372
quinac ine, se aline, amioda one, pe hexiline, clomi ene and N-dese hylamioda one 373
ha e highe an i i al po en ial wi hin he b oad ield o desc ibed CADs. Fu he mo e, 374
we show ha candida es p e iously no associa ed wi h an i i al e ec s bu only wi h 375
DIPL (e.g. pe hexiline) may also be po en an i i als o empla es o u he d ug 376
de elopmen . Expe imen s we e pe o med in he endo helium/lung-hyb id cell line 377
EA.hy926 ha is easy o cul i a e and gi es low backg ound. Besides monocy es, 378
mac ophages, endo helial cells, hepa ocy es and ib oblas s, his cell line was shown 379
o be suscep ible o se e al en eloped i uses including ilo i uses (11, 13, 34, 35). 380
Concen a ion- esponse analyses showed ha he six s onges CADs ac a low 381
mic omola concen a ions. The IC50 alues o amioda one and se aline a e 382
compa able o he epo ed plasma concen a ions (Cmax(amioda one) = 1.5 – 2.5 mg/L 383
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(2.2 – 3.7 µmol/L) and Cmax(se aline) = 0.352 µmol/L, which can accumula e up o 20-384
old in he li e (36–38). In case o d oneda one, he de e mined IC50 alue is 10- old 385
highe han he plasma concen a ion achie ed in ea men o a hy hmia 386
(Cmax(d oneda one) = 84 – 147 µg/L (0.14 – 0.25 µmol/L)) (39). The selec i i y index o 387
hese CADs is ela i ely na ow in i o. Howe e , i may be wo h o p obe whe he a 388
he apeu ic window exis s in i o, which migh be accep able when dealing wi h a 389
se e e o e en li e- h ea ing disease o in an ou b eak se ing igge ed by eme ging 390
en eloped i uses, gi en ha hey ac agains a ange o i uses. 391
In a p e ious s udy, Shoemake e al. iden i ied six highly an i i al CADs among 392
a ious s e ol pa hway inhibi o s. These CADs we e cha ac e ized wi h a posi i ely 393
cha ged amine g oup, alkaline pKa (> 8.8), small MW, high hyd ophobici y, and hey 394
caused choles e ol accumula ion (32). Howe e , i is no known whe he he p e ious 395
obse a ions apply necessa ily o all an i i ally ac i e CADs and which se o 396
s uc u al ea u es de ine an i i al ac i i y. To unde s and he ela ionship be ween 397
CAD s uc u e and an i i al ac i i y we ini ially ocused on a limi ed numbe o 398
p ope ies including pKa, ClogP, MW, and linke leng h. Hyd ophobici y is an 399
impo an de e minan o memb ane pe meabili y and bioa ailabili y o a molecule, 400
and has been desc ibed as one o he key ac o s o ligand binding oge he wi h 401
mola e ac i i y and o mal cha ge densi y (8, 40, 41). We could show ha s ongly 402
hyd ophobic compounds ha e signi ican ly s onge capaci y o inhibi i al en y. The 403
eason o his obse a ion is so a unclea , bu i may sugges ha CADs need o be 404
able o a e se memb anes in o de o exe hei an i i al e ec . Apa om he 405
CAD-de e mining amine and hyd ophobic g oups, we we e no able o iden i y a se 406
o unc ional g oups ha is equi ed o an i i al ac i i y. 407
We u he analysed CADs wi h ei he s uc u al (de i a i es) o unc ional 408
(analogues) simila i y o s ong an i i al CADs o ou sc een (28, 42). The ac ha 409
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only he de i a i es, bu none o he analogues eached he le el o inhibi ion seen 410
wi h hei espec i e seed compounds sugges s ha s uc u al a chi ec u e seemed o 411
ha e mo e impac on he CADs ac i i y han chemical p ope ies only. Ne e heless, 412
he analogues’ ac i i y a e is a highe han he one ob ained by sc eening andom 413
collec ions. Howe e , hese da a show ha hyd ophobici y alone is no a ce ain 414
p edic o o an i i al ac i i y, bu a he a combina ion o chemical p ope ies, which 415
is also sugges ed o DIPL-induc ion (43, 44). An ex ended mul ipa ame ic analysis 416
would be use ul o iden i y a la ge se o (in e dependen ) pa ame e s ha enable 417
and enhance ac i i y (28). 418
In addi ion, ou s udies showed a s ong associa ion be ween CAD an i i al ac i i y 419
and he abili y o induce DIPL in a ge cells, i.e. lysosomal accumula ion o cha ged 420
phospholipids, an e ec ha has been epo ed o many CADs (8, 45). DIPL shows 421
mo phological esemblance o Niemann-Pick ype C disease (NPC-1), an inhe i ed 422
and gene ally a al lipid-s o age diso de , and simila diseases (8, 46). Amioda one 423
p e e en ially accumula es in he lung, causes DIPL and is associa ed wi h lung 424
dys unc ion (47). Howe e , he CADs in clinical use ha e no been linked o 425
Niemann-Pick like symp oms and a clea gene al link be ween cellula DIPL and 426
o gan oxici y has no been demons a ed. Mo eo e , issue accumula ion seems o 427
be e e sible wi hin a ew days (48). Ne e heless, DIPL is conside ed a conce n in 428
d ug de elopmen . In ou s udy, we ound ha DIPL s ongly co ela es wi h bo h 429
an i i al ac i i y and wi h hyd ophobici y. Howe e , isible DIPL i sel is no equi ed 430
o an i i al ac i i y o CADs since e ec s like EBOV VLP inhibi ion (32) o lysosomal 431
calcium lux changes (49) p ecede DIPL induc ion o hou s. In conclusion, DIPL 432
induc ion seems o be mo e p ominen among CADs wi h an i i al ac i i y. In ac , in 433
ou se ies all CADs wi h an i i al ac i i y also induce DIPL o some ex en . Ye , he 434
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abili y o induce DIPL is no he only de e minan o an i i al ac i i y, as some CADs 435
wi h simila DIPL alues ha e qui e di e en deg ees o an i i al ac i i y. 436
Filo i uses a e la e-pene a ing i uses ha a e aken up in o endosomes ollowing 437
low-speci ici y in e ac ions wi h one o mo e o a ious cell su ace a achmen ac o s 438
(34). Wi hin he endosome, hey unde go gp p iming by low-pH dependen ca hepsin 439
and in e ac wi h hei endolysosomal ecep o NPC1, a choles e ol anspo e (50). 440
This enables memb ane usion, he eby eleasing he nucleocapsid in o he cy osol 441
(51, 52). I is concei able ha CADs modula e endosomal o endolysosomal 442
memb anes o memb ane-a ached p o eins in a manne ha pe u bs i al 443
pene a ion. Di ec e idence o a clea concep o how his migh occu is as ye 444
lacking. So a , i has no e en been ully es ablished whe he CADs exe hei 445
an i i al e ec s on he hos cell o he i al pa icle. I has been epo ed o a g oup 446
o s uc u ally di e se EBOV-inhibi ing CADs – o which o emi en, bep idil, and 447
se aline we e es ed in ou s udy – ha hese compounds dec ease he o e all 448
s abili y o he GP1-GP2 dime s, hus inhibi ing usion (53, 54). Ano he ecen s udy 449
desc ibed ha he CAD luna izine as well as he s uc u ally simila luphenazine, 450
i luope azine, chlo cyclizine and chlo p omazine seem o inhibi memb ane usion o 451
HCV pa icles by a ge ing a speci ic hyd ophobic egion in he E1 p o ein (16, 55). 452
Mo eo e , he an i i al spec um o amioda one, bep idil, amodiaquine and aloxi ene 453
oge he wi h lipid mixing assays sugges ha mos ly la e pene a ing i uses and a 454
la e en y s ep, mos p obably memb ane usion, a e a ec ed by hese CADs (22). 455
Howe e , gi en ha we and o he s ha e desc ibed a ange o s uc u ally di e se 456
CADs as inhibi o s o cell en y by di e en un ela ed i us species i seems likely ha 457
besides hese i us-speci ic e ec s he e is a sha ed mechanism common o di e se 458
CADs a ec ing a ange o en eloped i uses. The co ela ion o an i i al e ec s and 459
ea ly e en s o DIPL poin s owa ds a cell-based e ec o CADs ha akes place in 460
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he endolysosomal compa men . Thus, we conside ha he pe u ba ion o la e 461
endosomal homeos asis migh be he unde lying an i i al mechanism o CADs. The 462
clea e elucida ion o he an i i al mechanism o some CADs is pa o an ongoing 463
ollow-up s udy. 464
In summa y, we desc ibe he mos comp ehensi e e alua ion o da e o he an i i al 465
p ope ies among CADs and show how an i i al ac i i y is linked o s uc u al 466
ea u es. Mos no ably, we show ha an i i al ac i i y is s ongly associa ed wi h 467
hyd ophobici y and DIPL induc ion in a ge cells, co obo a ing p e ious s udies (32). 468
O e all, ou indings help cla i y he s uc u e-ac i i y ela ionship o an i i al CADs. 469
Fu he mo e, using an in silico sc eening app oach o iden i ica ion o s uc u ally 470
and unc ionally ela ed compounds, we iden i ied no el CADs wi h an i i al 471
p ope ies. By showing ha CAD-an i i al ac i i y is pa ially inge p in ed on hei 472
molecula a chi ec u e, we se a basis o he s uc u e-based design o po en 473
an i i al CADs. 474
475
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476
5. Acknowledgemen s 477
We hank Pe a Schneide (ETH, Pha maceu ical Sciences, Zü ich, Swi ze land) o 478
help wi h CATS so wa e, Ba ba a He el (Uni e si y o Po sdam, Depa men o ood 479
chemis y, Po sdam, Ge many) o excellen expe imen al suppo and Bea e Sodeik 480
(MHH, Ins i u e o Vi ology, Hanno e , Ge many) o help ul discussions. 481
6. Funding 482
This wo k was suppo ed by Deu sches Zen um ü In ek ions o schung (DZIF, 483
Ge man Cen e o In ec ion Resea ch; g an numbe 05807) and Ge man Resea ch 484
Founda ion (DFG) ia SFB738 (Resea ch P ojec B2). AG is PhD s uden o ZIB and 485
HBRS. 486
7. Decla a ion o in e es s 487
None. 488
8. Re e ences 489
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689
Figu e 1: An i i al ac i i y agains MARVpp gp-d en cell en y and cy o oxici y 690
o 45 CADs. (a) EA.hy926 a ge cells we e ansduced wi h MARVpp encoding an 691
NLuc epo e gene in a single-poin app oach in he p esence o a ious CADs a 5 692
µM. Resul s we e no malised o sol en con ol. (b) Cy o oxic and an ip oli e a i e 693
e ec s we e e alua ed o CADs a 5 µM in EA.hy926 cells s ably exp essing Nluc. 694
Box-whiske s plo s show he median, in e qua ile ange and s anda d de ia ion o 695
n=3 independen expe imen s. As e isks indica e s a is ical signi icance o di e ences 696
be ween compounds and sol en con ol ha was calcula ed by One-way ANOVA 697
wi h co ec ion o mul iple compa isons. (*p<0.01; **p<0.001;***p<0.0001) 698
699
Figu e 2: Co ela ion o MARVpp an i i al ac i i y o 45 CADs and hei 700
physico-chemical p ope ies. 701
Residual ansduc oin o 45 CADs agains MARVpp gp-d i en cell en y o single-702
poin assays we e no malised and he a e age o n=3 independen expe imen s was 703
co ela ed wi h (a) pKa, (b) ClogP, (c) molecula weigh and (d) linke leng h o all 704
es ed CADs. The p o o ypical CAD amioda one is labelled. Non-pa ame ic 705
Spea man-co ela ion was compu ed and he co ela ion coe icien R and p- alue 706
ep esen s a is ical signi icance (ns: p > 0.12; *p < 0.03; **p < 0.002; ***p < 0.0002; 707
****p < 0.0001). 708
709
Figu e 3: Co ela ion o MARVpp an i i al ac i i y, DIPL induc ion and 710
hyd ophobici y. 711
(a) EA.hy926 cells we e ea ed in iplica e wi h 16 di e en CADs a a 5 µM 712
concen a ion and he luo escen phospholipid LipidTox™ G een (1:1). A e 6 h 713
incuba ion, medium was exchanged and compounds (wi hou LipidTox™) we e 714
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added again o ex a 18 h. Fluo escence measu ed by FACS ep esen s DIPL 715
induc ion by CADs. Del a mean luo escence in ensi y (∆MFI) was calcula ed by i s 716
subs ac ing he a e age sol en con ol om single alues. A e ages o n=3 717
independen s ainings we e calcula ed and plo ed agains a e age an i i al ac i i y. 718
(b) DIPL induc ion was co ela ed wi h ClogP o he in es iga ed 16 CADs. 719
Co ela ion coe icien s R we e de e mined ia Spea man-co ela ion (ns: p > 0.12; 720
*p < 0.03; **p < 0.002; ***p < 0.0002; ****p < 0.0001). 721
722
Figu e 4: An i i al ac i i y and cy o oxici y o d oneda one s uc u al 723
de i a i es. 724
D oneda one-de i a i es D-1 and D-2 we e ound by SciFinde ® simila i y sea ch 725
and applied in a 5 µM concen a ion (a) oge he wi h MARVpp on EA.hy926 cells o 726
(b) on EA.hy-NLuc cells o 6 h. Luci e ase ac i i y was de e mined a e 72 h.p. . and 727
iplica e alues o n=3 independen expe imen s we e no malised o sol en con ol. 728
S a is ical di e ence was calcula ed by One-way ANOVA co ec ed o mul iple 729
compa isons and is ep esen ed by as e isks (*p<0.01; **p<0.001;***p<0.0001). 730
731
Figu e 5: Inhibi ion o MARVpp gp-media ed cell en y and cy o oxici y o 732
addi ional CADs iden i ied by CATS analysis. 733
New CADs we e iden i ied in a CATS-sc een o compounds wi h simila chemical 734
p ope ies o seed compounds. Eigh double hi s we e chosen which i ed he 735
equi emen s o being a CAD, ha ing di e se unc ional g oups, and ha ing ClogP 736
> 4. In o de o simpli y compound nomencla u e, new CADs we e abb e ia ed 737
acco ding o he ini ials o hei wo seed compounds. (a) Fo analysis o i al en y 738
inhibi ion, 5 µM CADs we e applied oge he wi h MARVpp on EA.hy926 cells and 739
luci e ase ac i i y was measu ed in ansduced cells 72 h.p. . (b) Cy o oxici y o new 740
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CADs, seed compounds and sol en con ols was measu ed in EA.hy-NLuc cells 741
ea ed wi h 5 µM CADs o 6 h. No malised esul s o n=3 expe imen s wi h h ee 742
echnical eplica es a e shown and as e isks indica e s a is ical signi icance o sol en 743
con ol calcula ed by One-way ANOVA including mul iple compa isons es . (*p<0.01; 744
**p<0.001;***p<0.0001) 745
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