Filovirus antiviral activity of cationic amphiphilic drugs is associated with lipophilicity and ability to induce phospholipidosis.

1 Ti le Page 1 2 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# 9 10 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 25 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. on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 2 #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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 3 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 4 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 5 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 6 102 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 7 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 8 (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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 9 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 16 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 17 (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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 18 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 19 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 20 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 21 476 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 1. WHO. 2019. WHO-Disease Ou b eak News. A ailable a : 490 h ps://www.who.in /cs /don/en/ (Accessed 2019-09-02) 491 2. Col a CEM, Lindsey B, Ghinai I, Johnson AM, Heymann DL. 2017. The Ebola 492 ou b eak , 2013 – 2016 : old lessons o new epidemics. Philos T ans R Soc 493 Lond B Biol Sci 372:1–24. 494 3. Sha e JG, G an DS, Schie elin JS, Boisen ML, Goba A, Ha ne JN, Le y 495 DC, Yenni RE, Moses LM, Fullah M, Momoh M, Fonnie M, Fonnie R, Kanneh 496 L, Ko oma VJ, Ka gbo K, O omassa hien D, Muncy IJ, Jones AB, Illick MM, 497 Kulakosky PC, Haislip AM, Bishop CM, Ellio DH, B own BL, Zhu H, Has ie KM, 498 Ande sen KG, Gi e SK, Tab izi S, Ta iyal R, S emlau M, Ma schine A, 499 Sampey DB, Spence JS, C oss RW, Geisbe JB, Fola in OA, Happi CT, Pi s 500 KR, Geske FJ, Geisbe TW, Saphi e EO, Robinson JE, Wilson RB, Sabe i PC, 501 Hende son LA, Khan SH, Bausch DG, B anco LM, Ga y RF. 2014. Lassa 502 Fe e in Pos -Con lic Sie a Leone. PLoS Negl T op Dis 8:1–13. 503 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 22 4. WHO. 2018. 2018 Annual e iew o diseases p io i ized unde he Resea ch 504 and De elopmen Bluep in . 505 5. Ma inez JP, Sasse F, B öns up M, Diez J, Meye hans A. 2015. An i i al d ug 506 disco e y: B oad-spec um d ugs om na u e. Na P od Rep 32:29–48. 507 6. Beke man E, Eina S. 2015. Comba ing eme ging i al h ea s. Science (80- ) 508 348:282–283. 509 7. Reaso MJ, Has ings KL, Ul ich RG. 2006. D ug-induced phospholipidosis: 510 issues and u u e di ec ions. Expe Opin D ug Sa 5:567–83. 511 8. Lüllmann H, Lüllmann-Rauch R, Wasse mann O. 1978. Lipidosis induced by 512 amphiphilic ca ionic d ugs. Biochem Pha macol 27:1103–1108. 513 9. Mo isse e G, Ammou y A, Rusu D, Ma gue y MC, Lodge R, Poubelle PE, 514 Ma ceau F. 2009. In acellula seques a ion o amioda one: Role o acuola 515 ATPase and mac oau ophagic ansi ion o he esul ing acuola 516 cy opa hology. B J Pha macol 157:1531–1540. 517 10. Kazmi F, Hensley T, Pope C, Funk RS, Loewen GJ, Buckley DB, Pa kinson A. 518 2013. Lysosomal seques a ion ( apping) o lipophilic amine (ca ionic 519 amphiphilic) d ugs in immo alized human hepa ocy es (Fa2N-4 cells). D ug 520 Me ab Dispos 41:897–905. 521 11. Geh ing G, Roh mann K, A enchong N, Mi le E, Becke S, Dahlmann F, 522 Pöhlmann S, Vond an FWR, Da id S, Manns MP, Ciesek S, on Hahn T. 2014. 523 The clinically app o ed d ugs amioda one, d oneda one and e apamil inhibi 524 ilo i us cell en y. J An imic ob Chemo he 69:2123–2131. 525 12. Johansen LM, B annan JM, Delos SE, Shoemake CJ, S ossel A, Lea C, 526 Ho s om BG, DeWald LE, Scho nbe g KL, Scully C, Leha J, Hensley LE, 527 Whi e JM, Olinge GG. 2013. FDA-App o ed Selec i e Es ogen Recep o 528 Modula o s Inhibi Ebola Vi us In ec ion. Sci T ansl Med 5:190 a79-190 a79. 529 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 23 13. Klin wo h A, Nolden T, Wes haus S, Roh mann K, Da id S, Manns MP, Finke 530 S, Ciesek S, on Hahn T. 2015. Ca ionic amphiphilic d ugs enhance en y o 531 len i i al pa icles pseudo yped wi h abies i us glycop o ein in o non-neu onal 532 cells. An i i al Res 124:122–31. 533 14. Cheng Y-L, Lan K-H, Lee W-P, Tseng S-H, Hung L-R, Lin H-C, Lee F-Y, Lee 534 S-D, Lan K-H. 2013. Amioda one inhibi s he en y and assembly s eps o 535 hepa i is C i us li e cycle. Clin Sci 125:439–448. 536 15. Gas aminza P, Whi en-Baue C, Chisa i F V. 2010. Unbiased p obing o he 537 en i e hepa i is C i us li e cycle iden i ies clinical compounds ha a ge 538 mul iple aspec s o he in ec ion. P oc Na l Acad Sci 107:291–296. 539 16. Pe in PM, Haid S, B own RJP, Doe becke J, Schulze K, Zeilinge C, 540 Schaewen M Von, Helle B, Ve cau e en K, Bak ash YM, Vond an FWR, 541 Spee s a S, Awadh A, Mukh a o F, Schang LM, Ki schning A, Ploss A, 542 Pie schmann T. 2017. Fluna izine P e en s Hepa i is C Vi us Memb ane 543 Fusion in a Geno ype-dependen Manne by Ta ge ing he Po en ial Fusion 544 Pep ide wi hin E1. Hepa ology 63:49–62. 545 17. Owens CM, Mawhinney C, G enie JM, Al meye R, Lee MS, Bo isy AA, Leha 546 J, Johansen LM. 2010. Chemical combina ions elucida e pa hway in e ac ions 547 and egula ion ele an o Hepa i is C eplica ion. Mol Sys Biol 6:1–13. 548 18. He S, Lin B, Chu V, Hu Z, Hu X, Xiao J, Wang AQ, Schwei ze CJ, Li Q, 549 Imamu a M, Hi aga N, Sou hall N, Fe e M, Zheng W, Chayama K, Ma ugan 550 JJ, Liang TJ. 2015. Repu posing o he an ihis amine chlo cyclizine and ela ed 551 compounds o ea men o hepa i is C i us in ec ion. Sci T ansl Med 7:1–10. 552 19. Nawa M, Takasaki T, Yamada KI, Ku ane I, Aka suka T. 2003. In e e ence in 553 Japanese encephali is i us in ec ion o Ve o cells by a ca ionic amphiphilic 554 d ug, chlo p omazine. J Gen Vi ol 84:1737–1741. 555 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 24 20. S adle K, Ha HR, Ciminale V, Spi li C, Sale i G, Schia on M, B u omesso D, 556 Bigle L, Folla h F, Pe enazzo A, Ba i ussio A. 2008. Amioda one al e s la e 557 endosomes and inhibi s SARS co ona i us in ec ion a a pos -endosomal le el. 558 Am J Respi Cell Mol Biol 39:142–149. 559 21. Neme ow GR, Coope NR, Mulle -Ebe ha d HJ. 1984. In ec ion o B 560 lymphocy es by a human he pes i us, Eps ein-Ba i us, is blocked by 561 calmodulin an agonis s. Med Sci 81:4955–4959. 562 22. Mazzon M, O ega-P ie o AM, Im ie D, Lu C, Hess L, Czieso S, G o e J, 563 Skel on JK, Fa leigh L, Buge JJ, W igh E, Tempe on N, Angell R, Oxen o d 564 S, Jacobs M, Ke ele R, Do ne M, Ma sh M. 2019. Iden i ica ion o B oad-565 Spec um An i i al Compounds by Ta ge ing Vi al En y. Vi uses 11:1–26. 566 23. Ku ne RH, Zhang XY, Reise J. 2009. P oduc ion, concen a ion and i a ion 567 o pseudo yped HIV-1-based len i i al ec o s. Na P o oc 4:495–505. 568 24. Ciesek S, Wes haus S, Wich M, Wapple I, Henschen S, Sa azin C, Hamdi N, 569 Abdelaziz AI, S assbu g CP, Wedemeye H, Manns MP, Pie schmann T, on 570 Hahn T. 2011. Impac o In a- and In e species Va ia ion o Occludin on I s 571 Func ion as Co ecep o o Au hen ic Hepa i is C Vi us Pa icles0. J Vi ol 572 85:7613–7621. 573 25. Flin M, on Hahn T, Zhang J, Fa quha M, Jones CT, Bal e P, Rice CM, 574 McKea ing JA. 2006. Di e se CD81 P o eins Suppo Hepa i is C Vi us 575 In ec ion. J Vi ol 80:11331–11342. 576 26. Mi le E, Kolesniko a L, S ecke T, Ga en W, Becke S. 2007. Role o he 577 T ansmemb ane Domain o Ma bu g Vi us Su ace P o ein GP in Assembly o 578 he Vi al En elope. J Vi ol 81:3942–3948. 579 27. Chemical Abs ac s Se ice. 2017. SciFinde . Columbus OH. 580 28. Schneide G, Neidha W, Gille T, Schmid G. 1999. “Sca old-Hopping” by 581 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 25 opological pha macopho e sea ch: A con ibu ion o i ual sc eening. Angew 582 Chemie - In Ed 38:2894–2896. 583 29. Mad id PB, Panchal RG, Wa en TK, Shu le AC, Endsley AN, G een CE, 584 Kolokol so A, Da ey ΔR, Mange ΠID, Gil L, Ba a i S, Tanga MJ. 2015. 585 E alua ion o Ebola Vi us Inhibi o s o D ug Repu posing. ACS In ec Dis 586 1:317–326. 587 30. Sala a C, Ba i ussio A, Munega o D, Calis i A, Ha HR, Bigle L, Fab is F, 588 Pa olin C, Palù G, Mi azimi A. 2015. Amioda one and me aboli e MDEA inhibi 589 Ebola i us in ec ion by in e e ing wi h he i al en y p ocess. Pa hog Dis 590 73:1–9. 591 31. Chockalingam K, Simeon RL, Rice CM, Chen Z. 2010. A cell p o ec ion sc een 592 e eals po en inhibi o s o mul iple s ages o he hepa i is C i us li e cycle. 593 P oc Na l Acad Sci 107:3764–3769. 594 32. Shoemake CJ, Scho nbe g KL, Delos SE, Scully C, Pajouhesh H, Olinge GG, 595 Johansen LM, Whi e JM. 2013. Mul iple Ca ionic Amphiphiles Induce a 596 Niemann-Pick C Pheno ype and Inhibi Ebola Vi us En y and In ec ion. PLoS 597 One 8:1–13. 598 33. Johansen LM, Dewald LE, Shoemake CJ, Ho s om BG, Lea - ooney CM, 599 S ossel A, Nelson E, Delos SE, Simmons JA, G enie JM, Pie ce LT, 600 Pajouhesh H, Lehá J, Hensley LE, Glass PJ, Whi e JM, Olinge GG. 2015. A 601 sc een o app o ed d ugs and molecula p obes iden i ies he apeu ics wi h an i 602 – Ebola i us ac i i y. Sci T ansl Med 7:1–14. 603 34. Zapa e o-Belinchón FJ, Die zel E, Dolnik O, Döhne K, Cos a R, He el B, 604 Veselko a B, Ki ui J, Klin wo h A, Manns MP, Pöhlmann S, Pie schmann T, 605 K ey T, Ciesek S, Ge old G, Sodeik B, Becke S, on Hahn T. 2019. 606 Cha ac e iza ion o he Filo i us-Resis an Cell Line SH-SY5Y Re eals 607 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 32 689 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 33 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 34 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 on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om on June 26, 2020 a Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om