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

1 Title Page 1 2 Filovirus antiviral activity of cationic amphiphilic drugs is associated with 3 lipophilicity and ability to induce phospholipidosis 4 5 Authors and affiliations: 6 Antonia P. Gunescha,b,c, Francisco J. Zapatero-Belinchona,b,c , Lukas Pinkertd, Eike 7 Steinmanne, Michael P. Mannsa,b, Gisbert Schneiderf, Thomas Pietschmannb,c, Mark 8 Brönstrupb,d, Thomas von Hahna,b,c,g# 9 10 a. Department of Gastroenterology, Hepatology and Endocrinology, Hannover 11 Medical School, Hannover, Germany 12 b. German Center for Infection Research (DZIF), Hannover-Braunschweig site, 13 Germany; 14 c. Institute of Experimental Virology, TWINCORE, Center for Experimental and 15 Clinical Infection Research Hannover, Hannover, Germany 16 d. Department of Chemical Biology, Helmholtz Centre for Infection Research 17 (HZI), Braunschweig, Germany 18 e. Department for Molecular & Medical Virology, Ruhr-Universität Bochum, 19 Bochum, Germany 20 f. Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical 21 Sciences, Eidgenössische Technische Hochschule (ETH) Zürich, Switzerland 22 g. Department of Gastroenterology and Interventional Endoscopy, Asklepios 23 Hospital Barmbek, Semmelweis University, Campus Hamburg, Germany 24 25 AAC Accepted Manuscript Posted Online 8 June 2020 Antimicrob. Agents Chemother. doi:10.1128/AAC.00143-20 Copyright © 2020 American Society for Microbiology. All Rights Reserved. on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 2 #Corresponding author: Prof. Dr. med. Thomas von Hahn, [email protected]. 26 Short running title: Antiviral structure-activity relationship of cationic amphiphilic drugs27 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 3 1. Abstract 28 Several cationic amphiphilic drugs (CADs) have been found to inhibit cell entry of 29 filoviruses and other enveloped viruses. Structurally unrelated CADs may have 30 antiviral activity, yet the underlying common mechanism and structure-activity 31 relationship are incompletely understood. 32 We aimed to understand how widespread antiviral activity is among CADs and which 33 structural and physico-chemical properties are linked to entry inhibition. 34 We measured inhibition of Marburg virus pseudoparticle (MARVpp) cell entry by 45 35 heterogeneous and mostly FDA-approved CADs and cytotoxicity in EA.hy926 cells. 36 We analysed correlation of antiviral activity with four chemical properties: pKa, ClogP, 37 molecular weight and distance between the basic group and hydrophobic ring 38 structures. Additionally, we quantified drug-induced phospholipidosis (DIPL) of a CAD 39 subset by flow cytometry. Structurally similar compounds (derivatives) and those with 40 similar chemical properties but unrelated structure (analogues) to strong inhibitors 41 were obtained by two in silico similarity search approaches and tested for antiviral 42 activity. Overall 11 out of 45 (24 %) CADs inhibited MARVpp by 40 % or more. The 43 strongest antiviral compounds were dronedarone, triparanol and quinacrine. 44 Structure-activity relationship studies revealed highly significant correlations between 45 antiviral activity, hydrophobicity (ClogP>4), and DIPL. Moreover, pKa and intra-46 molecular distance between hydrophobic and hydrophilic moieties correlated with 47 antiviral activity, but to a lesser extent. We also showed that in contrast to analogues, 48 derivatives had similar antiviral activity as the seed compound dronedarone. Overall, 49 one quarter of CADs inhibits MARVpp entry in vitro and antiviral activity of CADs 50 mostly relies on their hydrophobicity, yet is promoted by the individual structure. 51 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 4 2. Introduction 52 Outbreaks of emerging viral diseases have repeatedly posed major challenges 53 to global health in the recent years (https://www.who.int/csr/don/en/). Important 54 examples include the Filoviridae Ebola virus (EBOV) and Marburg virus (MARV), 55 Arenaviridae Lassa virus (LASV), Coronaviridae like Middle East Respiratory 56 Syndrome coronavirus (MERS-CoV), or the Flaviviridae-member ZIKA. Some of 57 these infections are associated with high case-fatality rates, e.g. 50 % for EBOV and 58 MARV, 35 % for MERS-CoV (2), and up to 30 % for hospitalized LASV patients (3). 59 Thus according to the WHO-List of Blueprint priority diseases of 2018, the 60 aforementioned pathogens share priority status concerning the need for research 61 and development given that effective vaccines or direct acting antivirals are 62 unavailable in most cases (4). It would be of great value to have drugs that are active 63 against a broad range of viral pathogens (5, 6). Such agents would likely not target 64 specific components of individual viruses but rather cellular structures or processes 65 that are utilized by various unrelated viruses. An attractive target pathway concerns 66 cellular endosomal processes, since these are used by numerous pathogens 67 including members of the families Filoviridae, Arenaviridae, Rhabdoviridae, 68 Coronaviridae, Togaviridae, Flaviviridae and Bunyaviridae. 69 Cationic amphiphilic drugs (CADs) are a diverse group of compounds, many of which 70 are in medical use for various clinical indications. CADs are defined by a hydrophilic 71 group marked by a basic amine with high pKa (the negative logarithm of the acid 72 dissociation constant Ka) and hydrophobic group(s) characterised by several 73 aromatic or aliphatic structures (7). Due to their amphiphilic nature CADs 74 transmigrate membranes which can be enhanced by different substituents like 75 halogen residues. Upon reaching acidic compartments like late endosomes (LE) and 76 endolysosomes, the basic amine groups gets protonated, which leads to lysosomal 77 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 5 trapping of the compounds (8). Consequently, CADs primarily accumulate in acidified 78 compartments (9, 10). 79 Several studies, most of which analysed sets of approved drugs, have found that 80 various CADs have antiviral activity. The antiarrhythmics amiodarone, dronedarone 81 and verapamil (11) as well as the estrogen receptor antagonists clomifene and 82 toremifene (12) have been shown to inhibit filoviral entry in different cell lines. 83 Moreover, clomifene and toremifene were shown to elevate survival rates of mice 84 infected with mouse-adapted EBOV up to 90% (12). Of note, they could show that the 85 antiviral activity of these two CADs was unrelated to their clinical mechanism of 86 action as estrogen receptor modulators. Beyond filoviruses, CADs have also been 87 found to inhibit Arenaviridae LASV and Guanarito virus (GTOV) (13), Hepatitis C 88 virus (HCV) (14–18), Japanese encephalitis virus (JEV) (19), Severe acute 89 respiratory syndrome-related coronavirus (SARS-CoV) (20), and Eppstein-Barr virus 90 (EBV) (21). Moreover, it was found that the CADs amiodarone, bepridil, raloxifene 91 and amodiaquine besides several non-CAD compounds have broad antiviral effects 92 against all tested alphaand flaviviruses (with the exception of ZIKA virus), as well as 93 varying other viruses, but none inhibited Human immunodeficiency virus (HIV) (22). 94 Highest antiviral activity for all four CADs was found against EBOV virus like 95 particles. Despite these observations, it is not known so far how widespread antiviral 96 activity really is among CADs. Likewise, the mechanism of action behind the antiviral 97 activity is unclear. For this reason, we undertook a comprehensive study of antiviral 98 acitivity among 45 heterogeneous CAD compounds and correlated antiviral activity 99 with various physico-chemical properties. 100 101 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 6 102 3. Material and Methods 103 2.1 Pseudoparticle production and transduction 104 Pseudoparticles were produced as described before (23, 24). In brief, producer HEK-105 293T cells were seeded at 8*105 cells/well in a poly-L-lysine coated 6-well plate. After 106 24 h cells were co-transfected with 1 µg DNA using polyethyleneimine (PEI) to 107 produce HIV-based pseudoparticles bearing individual viral envelope gp. The 108 plasmids used were (1) a packaging plasmid containing HIV-based gag-pol genes 109 (25), (2) a plasmid encoding for the env gene of MARV (pCAGGS-MARVGP) (26) or 110 an empty vector pcDNA3.1 and (3) a gutted yet packaging competent HIV-based 111 transfer plasmid encoding for NLuc reporter gene (pWPI_NanoLuc_BLR) (in a ratio 112 1:1:4). After 6 h post-transfection, medium was exchanged for 3 % FCS DMEM 113 containing 5 % Penicillin/Streptomycin and pseudoparticles harvested, pooled and 114 filtered after 48 h and 72 h. For lentiviral transduction, target EA.hy926 cells were 115 seeded 1*104 cells / well in a 96-well plate. After incubation for 24 h, 50 µL of 116 MARVpp or 50 µL complete DMEM were mixed with polybrene (1:1000) and CADs to 117 an end-concentration of 5 µM and applied in triplicates. As a solvent control sterile 118 water, EtOH, or DMSO were diluted in DMEM complete in the same ratio as the 119 drugs. At 6 h post transduction (h.p.t), cells were washed once with phosphate 120 buffered saline (PBS) and 100 µL of complete DMEM were applied. Seventy-two 121 h.p.t, cells were washed with PBS and lysed. Next, 20 µL of cell lysate were 122 transferred to a 96-well luminometer plate, mixed with 80 µL of the luciferase 123 substrate coelenterazine, and incubated shortly in the dark before NLuc activity 124 quantification in a GloMax® plate luminometer (Promega, Madison, WI, USA), 125 representing gp-driven cell entry. 126 127 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 7 2.2 Cytotoxicity test 128 EA.hy926-NLuc were seeded at 1*104 cells/well in a 96-well plate. The next day, cells 129 were treated in triplicate with either complete DMEM, solvent control (complete 130 DMEM + solvent), or with diluted CADs at a final concentration of 5 µM. After 6 h, 131 cells were washed with PBS and incubated with 100 µL complete DMEM for another 132 72 h at 37 C. Then cells were lysed and luciferase activity was determined as an 133 aggregate measure of cell viability and proliferation. 134 135 Compounds and similarity search 136 The compounds amiodarone HCl (A8423), amorolfine HCl (SML0283), bepridil HCl 137 (B5016), clemastine fumarate salt (SML0445), clomiphene citrate salt (C6272), 138 dasatinib (CDS023389), dronedarone HCl (D9696), perhexiline maleate salt 139 (SML0120), promazine HCl (P6656), sertraline HCl (S6319), terconazole (32355), 140 toremifene citrate salt (T7204), triparanol (T5200), chlorcyclizine (SML1473), 141 clozapine (C6305), cyclizine HCl (C3090000), N-Desethylamiodarone HCl solution 142 (D-055), verapamil HCl (V4629), W-7 (N-(6-Aminohexyl)-5-chloro-1-143 naphthalenesulfonamide hydrochloride) (A3281), amantadine HCl (A1260), 144 Benzylamine (185701), chloroquine diphosphate salt (C6628), clomipramine HCl 145 (C7291), chlorpromazine HCl (C0982), enclomiphene HCl (SML0719), fenfluramine 146 HCl (F112), flupentixol dihydrochloride (Y0000064), fluphenazine dihydrochloride 147 (F4765), gentamicin sulfate (G1914), imipramine HCl (I0899), maprotiline HCl 148 (M9651), mianserin HCl (M2525), prochlorperazine dimaleate salt (P9178), 149 promethazine HCl (P4651), propranolol HCl (P0884), quinacrine dihydrochloride 150 (Q3251), teicoplanin (T0578), thioridazine HCl (T9025), trifluoperazine HCl (T6062), 151 tripelennamine HCl (T7511), U18666A (U3633), W-5 (N-(6-Aminohexyl)-1-152 naphthalenesulfonamide hydrochloride) (SML0657), zimelidine dihydrochloride 153 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 8 (Z101) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). 154 The compound Ro 48-8071 was purchased from Biomol GmbH (Hamburg, 155 Germany). 156 Dronedarone-derivatives were received using SciFinder® software (Chemical 157 Abstracts Service, 2017). D-1 (N-Mesyldronedarone; TRC-M225785) and D-2 (S-158 Desmethyl S-Chloromethyl Dronedarone; TRC-D291470) were purchased from 159 BIOZOL Diagnostica Vertrieb GmbH (Eching, Germany). To obtain analogous CADs, 160 chemical advanced template search (CATS) software was used (Schneider et al., 161 1999). The compounds AC1 (1-(4-Ethyl-1-piperazinyl)-4-[1-(4-methylbenzyl)-1H-162 indol-3-yl]-1-butanone; E230-0651), CS3 (2-[(E)-2-{4-[Ethyl(4-163 methoxyphenyl)amino]phenyl}vinyl]-3-methyl-1.3-benzothiazol-3-ium; 7165-0012) 164 and QT1 (G119-1593; G119-1593) were obtained by ChemDiv (San Diego, CA, 165 USA). CS1 (2-{2-[(3.4-dichlorophenyl)thio]ethyl}-1-(2-oxo-2-phenylethyl)pyridinium 166 bromide; 5841320) and CS2 (1-{2-[4-(1-methyl-1-167 phenylethyl)phenoxy]ethyl}pyrrolidine; 7020606) were ordered from ChemBridge 168 Corporation (San Diego, CA, USA). DQ1 (2-(4-(sec-butyl)phenyl)-N’(1-169 phenethylpiperidin-4-ylidine)quinolone-4-carbohydrazide; IVK 9315735) was 170 purchased from SRC Alinda (Moscow, Russia) and CS4 (5-bromo-2-{[1-(3-171 fluorophenyl)ethyl]sulfanyl}pyridine; PB742999078) from Chemspace (Riga, Latvia). 172 CT1 ((E)-1-(5-chlorothiophen-2-yl)-3-(4-(diphenylamino)phenyl)prop-2-en-1-one; 173 Z46049784) was purchased from Enamine (Monmouth junction, NJ, USA). As 174 solvents water, ethanol (EtOH) (Carl Roth, Karlsruhe, Germany), or dimethylsulfoxid 175 (DMSO) (Carl Roth, Karlsruhe, Germany), were used. 176 177 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 9 Cell culture 178 Immortalized epithelial HEK-293T and EA.hy926, a fusion product of A549 and 179 human umbilical vein endothelial cells, were cultured in Dulbecco’s Modified Eagle 180 medium (DMEM) with 10 % fetal calf serum (FCS), 5 % Penicillin/Streptomycin, 5 % 181 non-essential aminoacids (NEAA) and 5 % L-glutamine (complete DMEM) at 37 °C 182 with 5 % CO2. Luciferase-expressing EA.hy926-NLuc cells were obtained by 183 transduction with VSV-Gpp harboring the nano luciferase (NLuc) gene and cultured 184 as previously described. 185 186 Phospholipidosis assay 187 EA.hy926 cells were seeded at 1*104 cells/well in a 96-well plate. The next day, 188 CADs and their solvents (DMSO and water) were diluted in culture medium. 189 Phospholipidosis was quantified using LipidTOX™ Green Phospholipidosis Detection 190 Reagent (H34350, Thermo Fisher Scientific, Waltham, MA, USA) according to a 191 modified version of a degradation assay (Mesens et al., 2009). In brief, LipidTOXTM 192 was diluted 1:500 in complete DMEM and sterile filtered. Then 50 µL of either CAD 193 mixture or controls were applied to the cells together with 50 µL of diluted LipidTox, 194 resulting in a final CAD concentration of 5 µM and a final LipidTox dilution of 1:1000. 195 After incubation for 6 h at 37 °C, cells were washed once with PBS. Medium was 196 exchanged for 100 µL culture medium and only the drugs (5µM) but no LipidTOXTM 197 were added a second time. After 24 h in total, cells were washed with PBS, 198 trypsinised, and transferred to FACS tubes. Cells were washed with 1 mL FACS 199 buffer (2 % FCS in PBS), centrifuged for 5 min at 1000 rpm and supernatant 200 discarded. In some replicates, cells were fixed with 100 µL 3-6 % PFA with no 201 influence on the results. Finally, CAD-induced lysosomal accumulation of LipidTOX™ 202 was quantified as intracellular green fluorescence by flow cytometry in BD 203 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 16 Previous studies showed that amiodarone, as well as its close relative dronedarone 358 act in low micromolar concentrations against pseudoparticles of different filovirus 359 species, GTOV and influenza H5N1. Furthermore, amiodarone was shown to inhibit 360 authentic EBOV, HCV, SARS and recently semliki forest virus, dengue virus, Sindbis 361 virus, Ross river virus, herpes simplex virus and the attenuated vaccine strain of 362 yellow fever virus. Conversely, ZIKV, HIV or vaccinia virus were not affected. (14, 15, 363 20, 22, 29–31) Triparanol was found to inhibit EBOV VLP as well as EBOV infection 364 and HCV replication (17, 32). Among a variety of other CADs, quinacrine, sertraline 365 and clomifene inhibited mainly EBOV VLPs, mouse-adapted EBOV and other 366 filovirus strains, including MARV (12, 33). To our knowledge, perhexiline has not 367 been investigated in the context of antivirals yet and was included in our study due to 368 reported association with DIPL (8). Furthermore, the amiodarone metabolite mono-N-369 desethylamiodarone (MDEA) was shown to have additive effects with amiodarone 370 (30). With our results, we partially confirmed and extended the previous studies. 371 Notably, we could show that the drugs dronedarone and triparanol followed by 372 quinacrine, sertraline, amiodarone, perhexiline, clomifene and N-desethylamiodarone 373 have higher antiviral potential within the broad field of described CADs. Furthermore, 374 we show that candidates previously not associated with antiviral effects but only with 375 DIPL (e.g. perhexiline) may also be potent antivirals or templates for further drug 376 development. Experiments were performed in the endothelium/lung-hybrid cell line 377 EA.hy926 that is easy to cultivate and gives low background. Besides monocytes, 378 macrophages, endothelial cells, hepatocytes and fibroblasts, this cell line was shown 379 to be susceptible to several enveloped viruses including filoviruses (11, 13, 34, 35). 380 Concentration-response analyses showed that the six strongest CADs act at low 381 micromolar concentrations. The IC50 values for amiodarone and sertraline are 382 comparable to the reported plasma concentrations (Cmax(amiodarone) = 1.5 – 2.5 mg/L 383 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 17 (2.2 – 3.7 µmol/L) and Cmax(sertraline) = 0.352 µmol/L, which can accumulate up to 20-384 fold in the liver (36–38). In case of dronedarone, the determined IC50 value is 10-fold 385 higher than the plasma concentration achieved in treatment of arrhythmia 386 (Cmax(dronedarone) = 84 – 147 µg/L (0.14 – 0.25 µmol/L)) (39). The selectivity index of 387 these CADs is relatively narrow in vitro. However, it may be worth to probe whether a 388 therapeutic window exists in vivo, which might be acceptable when dealing with a 389 severe or even life-threating disease or in an outbreak setting triggered by emerging 390 enveloped viruses, given that they act against a range of viruses. 391 In a previous study, Shoemaker et al. identified six highly antiviral CADs among 392 various sterol pathway inhibitors. These CADs were characterized with a positively 393 charged amine group, alkaline pKa (> 8.8), small MW, high hydrophobicity, and they 394 caused cholesterol accumulation (32). However, it is not known whether the previous 395 observations apply necessarily to all antivirally active CADs and which set of 396 structural features define antiviral activity. To understand the relationship between 397 CAD structure and antiviral activity we initially focused on a limited number of 398 properties including pKa, ClogP, MW, and linker length. Hydrophobicity is an 399 important determinant of membrane permeability and bioavailability of a molecule, 400 and has been described as one of the key factors for ligand binding together with 401 molar refractivity and formal charge density (8, 40, 41). We could show that strongly 402 hydrophobic compounds have significantly stronger capacity to inhibit viral entry. The 403 reason for this observation is so far unclear, but it may suggest that CADs need to be 404 able to traverse membranes in order to exert their antiviral effect. Apart from the 405 CAD-determining amine and hydrophobic groups, we were not able to identify a set 406 of functional groups that is required for antiviral activity. 407 We further analysed CADs with either structural (derivatives) or functional 408 (analogues) similarity to strong antiviral CADs of our screen (28, 42). The fact that 409 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 18 only the derivatives, but none of the analogues reached the level of inhibition seen 410 with their respective seed compounds suggests that structural architecture seemed to 411 have more impact on the CADs activity than chemical properties only. Nevertheless, 412 the analogues’ activity rate is far higher than the one obtained by screening random 413 collections. However, these data show that hydrophobicity alone is not a certain 414 predictor for antiviral activity, but rather a combination of chemical properties, which 415 is also suggested for DIPL-induction (43, 44). An extended multiparametric analysis 416 would be useful to identify a larger set of (interdependent) parameters that enable 417 and enhance activity (28). 418 In addition, our studies showed a strong association between CAD antiviral activity 419 and the ability to induce DIPL in target cells, i.e. lysosomal accumulation of charged 420 phospholipids, an effect that has been reported for many CADs (8, 45). DIPL shows 421 morphological resemblance of Niemann-Pick type C disease (NPC-1), an inherited 422 and generally fatal lipid-storage disorder, and similar diseases (8, 46). Amiodarone 423 preferentially accumulates in the lung, causes DIPL and is associated with lung 424 dysfunction (47). However, the CADs in clinical use have not been linked to 425 Niemann-Pick like symptoms and a clear general link between cellular DIPL and 426 organ toxicity has not been demonstrated. Moreover, tissue accumulation seems to 427 be reversible within a few days (48). Nevertheless, DIPL is considered a concern in 428 drug development. In our study, we found that DIPL strongly correlates with both 429 antiviral activity and with hydrophobicity. However, visible DIPL itself is not required 430 for antiviral activity of CADs since effects like EBOV VLP inhibition (32) or lysosomal 431 calcium flux changes (49) precede DIPL induction for hours. In conclusion, DIPL 432 induction seems to be more prominent among CADs with antiviral activity. In fact, in 433 our series all CADs with antiviral activity also induce DIPL to some extent. Yet, the 434 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 19 ability to induce DIPL is not the only determinant of antiviral activity, as some CADs 435 with similar DIPL values have quite different degrees of antiviral activity. 436 Filoviruses are late-penetrating viruses that are taken up into endosomes following 437 low-specificity interactions with one or more of various cell surface attachment factors 438 (34). Within the endosome, they undergo gp priming by low-pH dependent cathepsin 439 and interact with their endolysosomal receptor NPC1, a cholesterol transporter (50). 440 This enables membrane fusion, thereby releasing the nucleocapsid into the cytosol 441 (51, 52). It is conceivable that CADs modulate endosomal or endolysosomal 442 membranes or membrane-attached proteins in a manner that perturbs viral 443 penetration. Direct evidence or a clear concept of how this might occur is as yet 444 lacking. So far, it has not even been fully established whether CADs exert their 445 antiviral effects on the host cell or the viral particle. It has been reported for a group 446 of structurally diverse EBOV-inhibiting CADs – of which toremifen, bepridil, and 447 sertraline were tested in our study – that these compounds decrease the overall 448 stability of the GP1-GP2 dimers, thus inhibiting fusion (53, 54). Another recent study 449 described that the CAD flunarizine as well as the structurally similar fluphenazine, 450 trifluoperazine, chlorcyclizine and chlorpromazine seem to inhibit membrane fusion of 451 HCV particles by targeting a specific hydrophobic region in the E1 protein (16, 55). 452 Moreover, the antiviral spectrum of amiodarone, bepridil, amodiaquine and raloxifene 453 together with lipid mixing assays suggest that mostly late penetrating viruses and a 454 late entry step, most probably membrane fusion, are affected by these CADs (22). 455 However, given that we and others have described a range of structurally diverse 456 CADs as inhibitors of cell entry by different unrelated virus species it seems likely that 457 besides these virus-specific effects there is a shared mechanism common to diverse 458 CADs affecting a range of enveloped viruses. The correlation of antiviral effects and 459 early events of DIPL points towards a cell-based effect of CADs that takes place in 460 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 20 the endolysosomal compartment. Thus, we consider that the perturbation of late 461 endosomal homeostasis might be the underlying antiviral mechanism of CADs. The 462 clearer elucidation of the antiviral mechanism of some CADs is part of an ongoing 463 follow-up study. 464 In summary, we describe the most comprehensive evaluation to date of the antiviral 465 properties among CADs and show how antiviral activity is linked to structural 466 features. Most notably, we show that antiviral activity is strongly associated with 467 hydrophobicity and DIPL induction in target cells, corroborating previous studies (32). 468 Overall, our findings help clarify the structure-activity relationship of antiviral CADs. 469 Furthermore, using an in silico screening approach for identification of structurally 470 and functionally related compounds, we identified novel CADs with antiviral 471 properties. By showing that CAD-antiviral activity is partially fingerprinted on their 472 molecular architecture, we set a basis for the structure-based design of potent 473 antiviral CADs. 474 475 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 21 476 5. Acknowledgements 477 We thank Petra Schneider (ETH, Pharmaceutical Sciences, Zürich, Switzerland) for 478 help with CATS software, Barbara Hertel (University of Potsdam, Department for food 479 chemistry, Potsdam, Germany) for excellent experimental support and Beate Sodeik 480 (MHH, Institute of Virology, Hannover, Germany) for helpful discussions. 481 6. Funding 482 This work was supported by Deutsches Zentrum für Infektionsforschung (DZIF, 483 German Center for Infection Research; grant number 05807) and German Research 484 Foundation (DFG) via SFB738 (Research Project B2). AG is PhD student of ZIB and 485 HBRS. 486 7. Declaration of interests 487 None. 488 8. References 489 1. WHO. 2019. WHO-Disease Outbreak News. Available at: 490 https://www.who.int/csr/don/en/ (Accessed 2019-09-02) 491 2. Coltart CEM, Lindsey B, Ghinai I, Johnson AM, Heymann DL. 2017. The Ebola 492 outbreak , 2013 – 2016 : old lessons for new epidemics. Philos Trans R Soc 493 Lond B Biol Sci 372:1–24. 494 3. 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A cell protection screen 592 reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle. 593 Proc Natl Acad Sci 107:3764–3769. 594 32. Shoemaker CJ, Schornberg KL, Delos SE, Scully C, Pajouhesh H, Olinger GG, 595 Johansen LM, White JM. 2013. Multiple Cationic Amphiphiles Induce a 596 Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection. PLoS 597 One 8:1–13. 598 33. Johansen LM, Dewald LE, Shoemaker CJ, Hoffstrom BG, Lear-rooney CM, 599 Stossel A, Nelson E, Delos SE, Simmons JA, Grenier JM, Pierce LT, 600 Pajouhesh H, Lehár J, Hensley LE, Glass PJ, White JM, Olinger GG. 2015. A 601 screen of approved drugs and molecular probes identifies therapeutics with anti 602 – Ebola virus activity. Sci Transl Med 7:1–14. 603 34. Zapatero-Belinchón FJ, Dietzel E, Dolnik O, Döhner K, Costa R, Hertel B, 604 Veselkova B, Kirui J, Klintworth A, Manns MP, Pöhlmann S, Pietschmann T, 605 Krey T, Ciesek S, Gerold G, Sodeik B, Becker S, von Hahn T. 2019. 606 Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals 607 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 32 689 Figure 1: Antiviral activity against MARVpp gp-drven cell entry and cytotoxicity 690 of 45 CADs. (a) EA.hy926 target cells were transduced with MARVpp encoding an 691 NLuc reporter gene in a single-point approach in the presence of various CADs at 5 692 µM. Results were normalised to solvent control. (b) Cytotoxic and antiproliferative 693 effects were evaluated for CADs at 5 µM in EA.hy926 cells stably expressing Nluc. 694 Box-whiskers plots show the median, interquartile range and standard deviation of 695 n=3 independent experiments. Asterisks indicate statistical significance of differences 696 between compounds and solvent control that was calculated by One-way ANOVA 697 with correction for multiple comparisons. (*p<0.01; **p<0.001;***p<0.0001) 698 699 Figure 2: Correlation of MARVpp antiviral activity of 45 CADs and their 700 physico-chemical properties. 701 Residual transductoin of 45 CADs against MARVpp gp-driven cell entry of single-702 point assays were normalised and the average of n=3 independent experiments was 703 correlated with (a) pKa, (b) ClogP, (c) molecular weight and (d) linker length of all 704 tested CADs. The prototypical CAD amiodarone is labelled. Non-parametric 705 Spearman-correlation was computed and the correlation coefficient R and p-value 706 represent statistical significance (ns: p > 0.12; *p < 0.03; **p < 0.002; ***p < 0.0002; 707 ****p < 0.0001). 708 709 Figure 3: Correlation of MARVpp antiviral activity, DIPL induction and 710 hydrophobicity. 711 (a) EA.hy926 cells were treated in triplicate with 16 different CADs at a 5 µM 712 concentration and the fluorescent phospholipid LipidTox™ Green (1:1). After 6 h 713 incubation, medium was exchanged and compounds (without LipidTox™) were 714 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 33 added again for extra 18 h. Fluorescence measured by FACS represents DIPL 715 induction by CADs. Delta mean fluorescence intensity (∆MFI) was calculated by first 716 substracting the average solvent control from single values. Averages of n=3 717 independent stainings were calculated and plotted against average antiviral activity. 718 (b) DIPL induction was correlated with ClogP of the investigated 16 CADs. 719 Correlation coefficients R were determined via Spearman-correlation (ns: p > 0.12; 720 *p < 0.03; **p < 0.002; ***p < 0.0002; ****p < 0.0001). 721 722 Figure 4: Antiviral activity and cytotoxicity of dronedarone structural 723 derivatives. 724 Dronedarone-derivatives D-1 and D-2 were found by SciFinder® similarity search 725 and applied in a 5 µM concentration (a) together with MARVpp on EA.hy926 cells or 726 (b) on EA.hy-NLuc cells for 6 h. Luciferase activity was determined after 72 h.p.t. and 727 triplicate values of n=3 independent experiments were normalised to solvent control. 728 Statistical difference was calculated by One-way ANOVA corrected for multiple 729 comparisons and is represented by asterisks (*p<0.01; **p<0.001;***p<0.0001). 730 731 Figure 5: Inhibition of MARVpp gp-mediated cell entry and cytotoxicity of 732 additional CADs identified by CATS analysis. 733 New CADs were identified in a CATS-screen for compounds with similar chemical 734 properties to seed compounds. Eight double hits were chosen which fitted the 735 requirements of being a CAD, having diverse functional groups, and having ClogP 736 > 4. In order to simplify compound nomenclature, new CADs were abbreviated 737 according to the initials of their two seed compounds. (a) For analysis of viral entry 738 inhibition, 5 µM CADs were applied together with MARVpp on EA.hy926 cells and 739 luciferase activity was measured in transduced cells 72 h.p.t. (b) Cytotoxicity of new 740 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 34 CADs, seed compounds and solvent controls was measured in EA.hy-NLuc cells 741 treated with 5 µM CADs for 6 h. Normalised results of n=3 experiments with three 742 technical replicates are shown and asterisks indicate statistical significance to solvent 743 control calculated by One-way ANOVA including multiple comparisons test. (*p<0.01; 744 **p<0.001;***p<0.0001) 745 on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from on June 26, 2020 at Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from