key: cord-0331481-eagq0cjl
authors: Cao, Junyuan; Liu, Yang; Dong, Siqi; Zhou, Minmin; Guo, Jiao; Jia, Xiaoying; Zhang, Yueli; Hou, Yuxiao; Xiao, Gengfu; Wang, Wei
title: Screening and Identification of Lujo Virus Entry Inhibitors from an FDA-Approved Drugs Library
date: 2021-09-02
journal: bioRxiv
DOI: 10.1101/2021.09.01.458657
sha: 8c7de5c83a264dd175386be691bcf389b6108c62
doc_id: 331481
cord_uid: eagq0cjl

The Lujo virus (LUJV) belongs to the Old World (OW) genus Mammarenavirus (family Arenaviridae); it is categorized as a biosafety level (BSL) 4 agent. Currently, there are no U.S. Food and Drug Administration (FDA)-approved drugs or vaccines specifically for LUJV or other pathogenic OW mammarenaviruses. Here, a high-throughput screening of an FDA-approved drug library was conducted using pseudotype viruses bearing LUJV envelope glycoprotein (GPC) to identify inhibitors of LUJV entry. Three hit compounds, trametinib, manidipine, and lercanidipine, were identified as LUJV entry inhibitors in the micromolar range. Mechanistic studies revealed that trametinib inhibited LUJV GPC-mediated membrane fusion by targeting C410 (located in the transmembrane (TM) domain), while manidipine and lercanidipine inhibited LUJV entry by acting as calcium channel blockers. Meanwhile, all three hits extended their antiviral spectra to the entry of other pathogenic mammarenaviruses. Furthermore, all three could inhibit the authentic prototype mammarenavirus, lymphocytic choriomeningitis virus (LCMV), and could prevent infection at the micromolar level. This study shows that trametinib, manidipine, and lercanidipine are candidates for LUJV therapy, and highlights the critical role of calcium in LUJV infection. The presented findings reinforce the notion that the key residue(s) located in the TM domain of GPC provide an entry-targeted platform for designing mammarenavirus inhibitors. IMPORTANCE To date, only one LUJV outbreak has been recorded; it occurred in 2008 and resulted in a fatality rate of 80% (4/5 cases). Pathogenesis studies and therapeutic strategies are therefore urgently needed. Repurposing approved drugs can accelerate the development of drug design and facilitate the understanding of infectious mechanisms. Here, three compounds, trametinib, manidipine, and lercanidipine, were identified as entry inhibitors against LUJV. Studying the underling mechanisms revealed that a key residue (C410) in LUJV GPC modulates its sensitivity/resistance to trametinib and demonstrated the critical role of calcium in LUJV infection.

The mammarenavirus RNA genome encodes viral polymerase, nucleoproteins, matrix 64 protein (Z), and glycoprotein complex (GPC). GPC is synthesized as a polypeptide 65 precursor that is sequentially cleaved by signal peptidase and the cellular protease subtilisin kexin isozyme-1/site-1 protease to generate the three subunits of the mature 67 complex: the retained stable signal peptide (SSP), the receptor-binding subunit GP1, 68 and the membrane fusion subunit GP2 (8-11). These three non-covalently bound 69 subunits form a (SSP/GP1/GP2) 3 trimeric complex that is present at the surface of the 70 mature virion; it plays an essential role in virus entry. The relatively conserved 71 mammarenavirus SSP and GP2 form an interface that not only contributes to the 72 stabilization of the prefusion conformation of GPC, but also provides an "Achilles' 73 heel" that can be targeted by the entry inhibitors.

To date, no vaccines or specific antiviral agents against LUJV have been developed.

To address this issue, here we screened a U.S. Food and Drug Administration 76 (FDA)-approved drug library of 1,775 compounds. Drug repurposing is a strategy that 77 is used to accelerate the discovery and development of new and emerging pathogens.

Meanwhile, understanding the antiviral mechanisms of potential drugs that could 79 combat LUJV could provide novel insights into its pathogenesis. The safety, 80 pharmacokinetics, and mechanisms of approved drugs have been intensively 81 investigated. Here, therefore, we screened drugs that targeted the entry step of LUJV 82 infection, as this could block viral replication and spreading at an early stage. After 83 three rounds of screening, trametinib, manidipine, and lercanidipine were found to be 84 highly effective against LJUV entry; thus, they could offer potential new therapies to 85 treat LUHF. 89 As studies of LUJV require BSL-4 equipment, we utilized a replication-competent concentration-dependent inhibitory effects. Among these four compounds, the top 107 three hits (trametinib, manidipine, and lercanidipine) were selected for further 108 investigation; ospemifene was eliminated because of its cytotoxicity (Fig. 1B) .

Trametinib is an inhibitor of mitogen-activated protein kinase (MAPK), while manidipine and lercanidipine are dihydropyridine (DHP) voltage-gated Ca 2+ channel 111 antagonists. We evaluated the 50% inhibitory concentration (IC 50 ) of the hit 112 compounds using LUJVpv (Fig. 1B) . Furthermore, the inhibitory effects were 113 confirmed using LUJVrv on both Vero and A549 human epithelial cell lines (Fig. 1C) .

To validate the antiviral effects, trametinib, manidipine, and lercanidipine were 115 purchased from other commercial sources and tested; the cytotoxic and antiviral 116 effects were similar to the results shown in Fig. 1 . The unique retained SSP, together with GP2, provides an "Achilles's heel" that can be 120 targeted by many arenavirus entry inhibitors. We carried out a membrane fusion assay 121 to test whether the three hit compounds act by targeting the SSP-GP2 interface. LUJVrv was also conducted in dimethyl sulfoxide (DMSO) as a control. In the 157 trametinib-treated group, resistance was detected after three rounds of passaging; it 158 increased in passage P4 and then remained stable through passages P5 to P6 (Fig. 3A) .

To identify the viral genetic determinant(s) responsible for the resistance, we with acid triggering, similar to that of C410G (Fig. 3D ). When treated with trametinib, 180 fusogenicity was inhibited in the C412G group, but not in the C410G/C412G group, 181 suggesting that only C410 served as the viral target of trametinib. Furthermore, 182 LUJV C412G pv was still as sensitive to trametinib as that of WT, whereas 183 LUJV C410G/C412G pv was resistant to trametinib, similar to LUJV C410G pv (Fig. 4B ). This 184 confirms that only C410G contributed to the resistance to trametinib. 185 Notably, we attempted to identify the resistance of LUJVrv to manidipine and Sequencing of trametinib-resistant LASVrv revealed that the viral target was F446L, 212 which is located in the TM of GP2 (Fig. 6B ). F446 is highly conserved in mammals, 213 and has been reported to confer resistance to other structurally distinct membrane 214 fusion inhibitors (15). The sensitivity/resistance was further tested in the presence of 215 trametinib by introducing the F446L mutant into LASVpv, MOPVpv, and LCMVpv.

As shown in Fig. 6C to E, LASV F446L pv, MOPVpv, and LCMVpv (containing the 217 corresponding F446L mutant) conferred resistance to trametinib, confirming that 218 F446 served as the viral target of trametinib in LASV, MOPV, and LCMV. 219 We also investigated the broad-spectrum antiviral effects of manidipine and 220 lercanidipine. Both showed robust inhibition against NW mammarenavirus entry, with 221 IC 50 values ranging from ~ 0.1-1 µM. At the tested concentration, the dose-response 222 curves of both manidipine and lercanidipine for NW viruses showed the typical "s" 223 model, with 2-log spans (Fig. 7A-E, Fig. 7J-N) . These results are in line with In mammarenavirus GPC, the retained SSP interacts with the membrane-proximal external region, as well as the TM domain of GP2. Thus, it stabilizes the pre-fusion 265 conformation of GPC and provides an "Achilles' heel" that can be targeted by distinct 266 entry inhibitors (13-15, 24-27) . To our knowledge, this is the first study to report entry 267 inhibitors targeting the LUJV SSP-GP2 interface, and to identify their viral target(s). VSVpv (MOI: 0.1). After 6 h, the duplicate cell lysates were subjected to qPCR (C) 558 and Rluc assays (D), respectively. Data are presented as presented as means ± SD 559 from more than five independent experiments (****, P <0.0001; ***, P <0.001; **, P 560 <0.01; *, P <0.05).

Genetic detection and characterization 392

Arenaviruses. I. The epidemiology molecular and cell biology of 395 arenaviruses. Introduction

The curious case of arenavirus entry, and its inhibition

Structural basis for receptor recognition by Lujo 399 virus

NRP2 and CD63 Are Host Factors for Lujo Virus Cell 402

Fields Virology

Animal models, prophylaxis, and therapeutics for arenavirus infections

Signal peptide of Lassa virus glycoprotein GP-C 408 exhibits an unusual length

Structure-function relationship of the 410 mammarenavirus envelope glycoprotein

X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation

The Lassa virus glycoprotein 415 precursor GP-C is proteolytically processed by subtilase SKI-1/S1P

Properties of replication-competent vesicular stomatitis virus 419 vectors expressing glycoproteins of filoviruses and arenaviruses

Screening 421 and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drugs Library

Structure-activity relationship optimization for lassa virus fusion inhibitors targeting the 425 transmembrane domain of GP2

Screening of Botanical 427 Drugs against Lassa Virus Entry

Characterizing the Lassa Virus Envelope 429

Glycoprotein Membrane Proximal External Region for Its Role in Fusogenicity

Palmitoylation of virus proteins

Ebola virus glycoprotein: proteolytic 433 processing, acylation, cell tropism, and detection of neutralizing antibodies

BRAF mutations are associated with increased iron regulatory protein-2 expression 437 in colorectal tumorigenesis

CACNA1S haploinsufficiency confers resistance to New World 439 arenavirus infection

siRNA screen for genes that affect 441

Junin virus entry uncovers voltage-gated calcium channels as a therapeutic target

Antiviral potential of ERK/MAPK and

PI3K/AKT/mTOR signaling modulation for Middle East respiratory syndrome coronavirus 446 infection as identified by temporal kinome analysis

MEK inhibitors reduce cellular expression of ACE2, pERK

NK-mediated cytotoxicity and attenuating inflammatory cytokines relevant to SARS-CoV-2 452 infection

Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the

Lassa Virus Envelope Glycoprotein

Identification of the dietary supplement capsaicin as an 457 inhibitor of Lassa virus entry

Unique small molecule entry inhibitors of hemorrhagic 460 fever arenaviruses

Identification of a 462 broad-spectrum arenavirus entry inhibitor

Host Calcium Channels and Pumps in Viral Infections. Cells 9

Screening and Identification of Lujo Virus Inhibitors 466

Using a Recombinant Reporter Virus Platform

Selective estrogen-receptor modulators --mechanisms of 468 action and application to clinical practice