Simultaneous Screening for Selective SARS-CoV-2, Lassa, and Machupo Virus Entry Inhibitors

Abstract

Emerging highly pathogenic viruses can pose profound impacts on global health, the economy, and society. To meet that challenge, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) centers for early-stage identification and validation of novel antiviral drug candidates against viruses with pandemic potential. As part of this initiative, we established paired entry assays that simultaneously screen for inhibitors specifically targeting SARS-CoV-2 (SARS2), Lassa virus (LASV) and Machupo virus (MACV) entry. To do so we employed a dual pseudotyped virus (PV) infection system allowing us to screen ~650,000 compounds efficiently and cost-effectively. Adaptation of these paired assays into 1536 well-plate format for ultra-high throughput screening (uHTS) resulted in the largest screening ever conducted in our facility, with over 2.4 million wells completed. The paired infection system allowed us to detect two PV infections simultaneously: LASV + MACV, MACV + SARS2, and SARS2 + LASV. Each PV contains a different luciferase reporter gene which enabled us to measure the infection of each PV exclusively, albeit in the same well. Each PV was screened at least twice utilizing different reporters, which allowed us to select the inhibitors specific to a particular PV and to exclude those that hit off targets, including cellular components or the reporter proteins. All assays were robust with an average Z’ value ranging from 0.5 to 0.8. The primary screening of ~650,000 compounds resulted in 1,812, 1,506, and 2,586 unique hits for LASV, MACV, and SARS2, respectively. The confirmation screening narrowed this list further to 60, 40, and 90 compounds that are unique to LASV, MACV, and SARS2, respectively. Of these compounds, 8, 35, and 50 compounds showed IC₅₀ value < 10 μM, some of which have much greater potency and excellent antiviral activity profiles specific to LASV, MACV, and SARS2, and none are cytotoxic. These selected compounds are currently being studied for their mechanism of action and to improve their specificity and potency through chemical modification.

Introduction

In 2022, The National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers for Pathogens of Pandemic Concern which include paramyxoviruses, bunyaviruses, togaviruses, filoviruses (including Ebola viruses and Marburg virus), picornaviruses (including enteroviruses and other cold-causing viruses), arenaviruses, coronaviruses and flaviviruses (including yellow fever, dengue and Zika viruses). The main goal of the AViDD centers is to identify and validate viral targets and small molecules that inhibit viral infections. The team undertaking the research in this manuscript operates under the Midwest AViDD center (MWAC), one of the nine AViDD centers. The MWAC comprises 5 projects, each focusing on a different well-established antiviral target area such as entry, protease, nuclease, and helicase. To accomplish our goals, all projects were integrated into 3-pronged screening approaches, namely, ultra-High Throughput Screening (uHTS), DNA-Encoded Chemistry Technology (DEC-Tec), and Virtual Screening (VS), to screen large numbers of compounds effectively and obtain lead compounds to be studied more in depth. In this paper, we report the use of uHTS to identify small molecules that inhibit the entry steps of SARS2 and other RNA viruses of pandemic concern such as LASV and MACV.

SARS2 is the causative agent for COVID-19, which caused a worldwide pandemic in early 2020. Although Food and Drug Administration (FDA)-approved vaccines have been available since early 2021, COVD-19 is still affecting public health and negatively impacting our society and economics extensively. Currently, Remdesivir and Ritonavir-boosted Nirmatrelvir (Paxlovid) are FDA-approved drugs for COVID-19 treatment [1, 2]. These drugs target viral enzymes, such as RNA dependent RNA polymerase and main protease (M^pro), leaving a gap where no entry specific inhibitors are available yet. The entry of SARS2 is initiated by the interaction of the viral spike (S) protein with the host cellular receptor, angiotensin-converting enzyme 2 (ACE2) [3–5]. The S protein is pre-cleaved into two subunits, S1 and S2, by a protease, furin, in producer cells. When the S1 subunit on the released virus binds the receptor on the target cell, conformational changes occur in both the subunits which leads to another cleavage at the S2’ site internal to the S2 subunit. This cleavage is mediated either by serine proteases such as transmembrane serine protease 2 (TMPRSS2) on the target cell surface [6, 7] or by the lysosomal protease, cathepsin L in the target cells [8, 9]. The cleavage at the S2’ site exposes the fusion peptide in the S2 subunit, and this is essential for fusion between the viral and cellular membranes. Therefore, SARS2 entry into the target cell consists of several steps, and it would be ideal to identify small molecules that inhibit any of these steps including the interaction between S1 and the cellular receptor and the fusion mediated by the S2.

Moving onto LASV, it belongs to the Arenaviridae family and is the causative agent of Lassa hemorrhagic fever particularly prevalent in West Africa. Ribavirin is the only FDA-approved drug for treating Lassa fever [10]. Ribavirin is a nucleoside analog which binds the nucleotide-binding site of the viral polymerase. However, its toxicity, which is due to binding to the cellular polymerase [11], underscores an urgent need for developing anti-LASV drugs. There are two geographically separated serogroups of arenaviruses that infect mammals (mammarenaviruses): the Old World and the New World arenaviruses. LASV is an Old-World arenavirus and utilizes α-dystroglycan (α-DG) expressed on the cell surface as an entry receptor [12–14]. Further studies revealed that the glycoprotein (GP) of LASV also interacts with lysosome-associated membrane protein 1 (LAMP1), in addition to α-DG, for entry [12]. Recent work showed benzimidazole derivatives have broad-spectrum activity against different lineages of LASV [15, 16], targeting their GP [17, 18].

Finally, MACV also belongs to the Arenaviridae family but is a New World arenavirus and the causative agent for Bolivian hemorrhage fever (BHF) which was first discovered in 1959. Here again, Ribavirin is the only FDA-approved drug for treating BHF. All five pathogenic New World arenaviruses, including MACV, that cause various hemorrhagic fevers belong to clade B, and all clade B arenaviruses use transferrin receptor 1 (TFR1) as their entry receptor [19]. TFR1 is responsible for iron uptake for all living cells. The GP (in particular, GP1) of all five pathogenic New World arenaviruses bind the apical domain of human TFR1 [20, 21], making it an attractive molecular target for the development of broad-spectrum inhibitors of pathogenic arenavirus entry.

Our approach for discovering viral entry inhibitors utilizes pseudoviruses (PVs) that allow us to study pathogenic viruses safely at biosafety level 2 (BSL-2) while working with wild type SARS2 requires BSL-3, and LASV and MACV require BSL-4 facilities. Using PV enables us to work with a system that bears the same entry GP as the wild-type virus and also provides ease of modification, such as incorporating mutations into the GP when necessary [22]. This method has been effectively used for our target viruses: SARS2 [23–25], MACV [19, 26], LASV [18, 27, 28], and other well-known pathogenic viruses, such as HIV-1 and Influenza virus [22, 29–32]. In this study, we have developed a dual infection system that allows us to measure the infection of two different PVs simultaneously by using two different luciferases encoded by those PVs. We produced six PVs expressing the GP from SARS2, LASV, or MACV on the surface and containing a single reporter gene of either firefly or nano luciferase inside. The assay was adapted to a 1536-well plate format (1536 wpf) and used to screen approximately 650,000 compounds for each PV pair: MACV and LASV, MACV and SARS2, LASV and SARS2. By way of design, at the end of each phase of the HTS campaign, it allows us to collect duplicate data for each virus while collectively screening three different viruses which naturally excludes the compounds that are non-specifically active. The outcomes of the entire uHTS campaign including primary, confirmation. and counter screens as well as concentration-response profiling are all described herein.

Material and Methods

hACE2-H1299 cells

NCI-H1299 cell line was kindly provided by Joseph Kissil (Formerly at the Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL, USA), authenticated by ATCC Cell Line Authentication Service, and cells were maintained in RPMI 1640 (Life Technologies 61870–036) supplemented with 10% FBS at 37°C with 5% CO₂. To construct stable cell lines expressing hACE2, NCI-H1299 cells were first transduced with MLV-based PV, expressing full-length hACE2 and pseudotyped with VSV-G. Two days after transduction, the hACE2-expressing cells were selected under 3 μg / ml puromycin in the fresh culture media. To maintain the hACE2-H1299 cells, 1 μg / ml puromycin is always supplemented in the culture media.

hACE2-H1299 cells were expanded in culture media: RPMI-1640 (Gibco 61870) supplemented 10% FBS (Sigma F2442), 1 X Antibiotic-Antimycotic (Gibco 15240–062) and 1 μg / ml puromycin (InvivoGen ant-pr-1) in 5-layer cell culture flasks (CellPro TV55P). When the cell number reached the desired estimated total cell number (around 90% confluence of the cells in each flask), it was harvested and resuspended in recovery cell culture freezing media (Gibco 12648010) and stored in liquid nitrogen.

Plasmids

The plasmid, named pQCXIP-hACE2, was constructed by cloning the full-length cDNA fragment encoding human ACE2 fused with a myc tag at the N-terminal into the retroviral vector pQCXIP (Clontech). Similarly, pQ-F-luc-deltaIRES, and pQ-N-luc-deltaIRES expressing firefly and nano luciferase, respectively, were also generated by replacing the IRES element of the pQCXIX (Clontech) vector with either F-luc or N-luc gene fragment. The previously constructed plasmid [33], named MLV-gag/pol, expressing the Gag-Pol protein of Moloney murine leukemia virus (MLV), was also used in this study. In addition, the following expression plasmids for different viral GPs were also used: pCAGGS-SARS2-S(BA.5)-dCT19 encodes the SARS2 S protein of the Omicron BA.5 lineage with 19 amino acids truncated at its C-terminus, pCAGGS-LASV-GPC expresses the full length LASV GP complex (GPC), pcDNA3.1-MACV-GPC expresses full length MACV GPC, and pCAGGS-VSV-G expresses full length vesicular stomatitis virus G protein, respectively. The truncated construct of SARS2 S protein was applied because this construct increases the S1-S2 association and the spike density on the virions which resulting in higher infectivity then full length [33].

Pseudovirus (PV) production

To produce the MLV-based PV for making stable cell lines expressing hACE2, 50–60% confluent HEK293T cells (ATCC) were co-transfected with the MLV-gag/pol, pQCXIP-hACE2, and pCAGGS-VSV-G at ratio 3:2:1 by calcium phosphate, and the supernatant was harvested at 48 hours post-transfection. SARS2 PVs with different reporters were produced by co-transfection of MLV-gag/pol, luciferase-expressing plasmid, and pCAGGS-SARS2(BA.5)-dCT19 into HEK293T cells at ratio 5:5:1, and the supernatant containing PVs were harvested at 43 hours post-transfection. For LASV and MACV PV, the ratio of MLV-gag/pol, luciferase-expressing plasmid, and GPC-expressing plasmid is 1:1:1 for the co-transfection, and the supernatant was harvested at 48 hours post-transfection. All PVs were clarified by 0.45 μm filter and aliquoted for storage at −80^oC.

PV infection on hACE2-expressing cell lines

To determine which cell line is the best in supporting all three PVs infection including LASV, MACV, and SARS2 PV, hACE2-Hela, hACE2-A549 and hACE2-H1299 cells (produced using the same method as hACE2-H1299 stated above) were first seeded in the white opaque 96-well plate (Costar 3917) at 4 × 10³ cells/well in 80 μl culture media. After overnight culture at 37°C with 5% CO₂ and 95% RH, the media was removed before the cells were infected with 80 μL of diluted PVs containing F-luc reporter (PV-F-luc) for 24 hours. After this incubation, the media was removed and 30 μL of 1×FLuc-Lysis Buffer (Luc-Pair^™ Firefly Luciferase HS Assay Kit GeneCopoeia Cat# LF-009) was added. Subsequently, 100 μL of 1× FLuc Assay Working Solution (Luc-Pair^™ Firefly Luciferase HS Assay Kit) was added to read the luminescence on the Molecular Devices SpectraMax Luminometer.

Assessment of paired luciferase reporters

For paired screens in which two different PVs carrying distinct luciferase reporters are mixed to infect the same cells, it is critical to generate separate signals specific for each PV infection. Since the signal of N-luc comes after F-luc reading by adding the Stop & Glo^® Reagent from the Nano-Glo^® Dual-Luciferase^® Reporter Assay System (Promega N1620) kit, to confirm whether the Stop & Glo^® Reagent will efficiently quench the F-luc signal, hACE2-H1229 cells were infected with PV-F-luc or PV-N-luc alone and detected by F-luc substrate followed by Stop & Glo^® Reagent containing N-luc substrate (Promega N1620). To rule out the possibility that the two paired PVs would interfere with each other during infection, the PV-F-luc was paired with PV-N-luc to infect hACE2-H1299 cells. At 48 hours post-infection (hpi), the luminescence was measured by Nano-Glo^® Dual-Luciferase^® Reporter Assay System (Promega N1620) kit and read with the BMG LABTECH PHERAstar.

1536 well plate format uHTS protocol

hACE2-H1299 cells were seeded into a 1536 well assay plate (Aurora EWB0–42000A) at 250 cells / 2 μL / well. After addition of 10 nL of compounds, plates were incubated 24 hours at 37°C and 5% CO₂. Following the incubation, 2 μL / well of assay media (RPMI-1640 supplemented with 10 % FBS, 1X Antibiotic-Antimycotic and 1 μg / mL puromycin) was dispensed for column 1–3. For column 4–48, 2 μL / well of appropriate PV (contains either F-luc or N-luc) was added with following dilution ratio: LASV; 1:50, MACV; 1:50 and SARS2; 1:10 in assay media. The plates were further incubated for 48 hours at 37°C and 5% CO₂. The plates were then removed from that environment and incubated at room temperature for 10 minutes followed by addition of 2.5 μL / well of One Glo^® reagents (Promega N1650). After 10 minutes incubation at room temperature, firefly luciferase expression was measured using the PHERAstar. Subsequently, 2.5 μL / well of NanoDLR Stop & Glo^® reagents was added. After 10 minutes incubation at room temperature, nano luciferase activity was measured using the PHERAstar. The process is summarized in Table 1. High control wells contained hACE2-H1299 cells + assay media + vehicle (DMSO), and the low control and data wells had hACE2-H1299 cells + PV + test compound or vehicle.

Table 1:

PV Entry assay protocol in 96 wpf and 1536 wpf

Steps	96 wpf protocol	1,536 wpf protocol
1	Seed cells at 4000 cells / 80 μL / well	Thaw and dispense cells at 250 cells / 2 μL / well
2	Incubate plate at 37 °C, 5% CO2 for 16–18 hours	Add 10 nL of compound or vehicle
3	Remove media	Incubate plate at 37 °C, 5% CO2 for 24 hours
4	Add 80 μL / well of appropriate pair of PV or media	add 2 μL / well of appropriate pair of PV or media
5	Incubate plate at 37 °C, 5% CO2 for 48 hours	Incubate plate at 37 °C, 5% CO2 for 48 hours
6	Take plates out from incubator to adapt room temperature	Take plates out from incubator to adapt room temperature
7	Add 80 μL / well of ONE-Glo® Luciferase Assay Reagent	Add 2.5 μL / well of ONE-Glo® Luciferase Assay Reagent
8	Incubate plate at 37 °C, 5% CO2 for 10 minutes	Incubate plate at 37 °C, 5% CO2 for 10 minutes
9	Read firefly luminescence	Read firefly luminescence
10	Add 80 μL / well of NanoDLR™ Stop & Glo® Reagent	Add 2.5 μL / well of NanoDLR™ Stop & Glo® Reagent
11	Incubate plate at 37 °C, 5% CO2 for 10 minutes	Incubate plate at 37 °C, 5% CO2 for 10 minutes
12	Read nano luminescence	Read nano luminescence
	Virus dilution: 1:100 for LASV and MACV; 1:30 for SARS2	Virus dilution: 1:50 for LASV and MACV; 1:10 for SARS2

Cytotoxicity counterscreen

hACE2-H1299 cells were seeded at 250 cells / 2 μL / well for column 4–48 of the assay plates (Aurora EWB0–42000A). For column 1–3, we dispensed 2 μL/well of assay media. After addition of 10 nL of compounds, plates were incubated 24 hours at 37 °C and 5% CO2. Following that incubation, 2 μL / well of assay media to all wells. The plates were further incubated for 48 hours at 37°C and 5% CO2. The plates were then removed from that atmosphere and incubated at room temperature for 10 minutes followed by the addition of 4 μL / well of CellTiter-Glo reagent (Promega G7573). After 10 minutes incubation at room temperature, luminescence was measured using the PHERAstar.

UF Scripps Drug Discovery Library (UF-SDDL)

The UF Scripps Drug Discovery Library (UF-SDDL) is a collection of 646,275 small drug-like compounds that belong to The High-Throughput Molecular Screening Center at the Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology. The compounds are unique and individually pure, representing a wide diversity of drug-like small organic molecules used in traditional and non-traditional drug-discovery biology. This library has been curated from over 20 commercial sources, supplemented with academic sources, including compounds and sub-libraries prepared internally. The UF-SDDL compounds were selected based on scaffold novelty, physical properties (i.e., “drug-likeness”) and spatial connectivity. In its current state, the UF-SDDL has several focused sub-libraries for screening popular drug-discovery target classes (e.g., kinases/transferases, GPCRs, ion channels, nuclear receptors, hydrolases, transporters), with diverse chemistries (e.g., click-chemistry, PAINS-free collections, Fsp3 enriched, covalent inhibitors and natural product collections) and with desirable physical properties (“rule-of-five”, “rule-of-three”, polar surface area, etc.) [34–41].

Screening data acquisition, normalization, representation, and analysis

All data files were uploaded into the UF Scripps institutional HTS database (Symyx Technologies, Santa Clara, CA) for plate QC and hit identification. Activity of each compound was calculated on a per-plate basis using the following equation:

$$\mathit{\text{Percent}}\mathit{\text{Response}}\mathit{\text{of}}\mathit{\text{compound}}=100\times \left(\frac{\mathit{\text{Test}}\mathit{\text{well}}-\mathit{\text{Median}}\mathit{\text{Data}}\mathit{\text{wells}}}{\mathit{\text{Median}}\mathit{\text{High}}\mathit{\text{Control}}-\mathit{\text{Median}}\mathit{\text{Data}}\mathit{\text{wells}}}\right)$$

Where the “High Control” represents wells containing hACE2-H1299 cells + DMSO, “Low Control” represents wells containing hACE2-H1299 + appropriate pair of PVs + DMSO and “Data Wells” contain the same including test compound. The Z’ and signal-to-background ratio (S:B) for this assay is calculated using the High Control and Low Control wells. Three PV pairs were screened separately: LASV firefly-luciferase (F-luc) + MACV nano-luciferase (N-luc); LASV F-luc + SARS2 N-luc; MACV F-luc + SARS2 N-luc.

The Z’ and signal-to-background ratio (S:B) were calculated using the High Control and Low Control wells using the following equations:

$$Z\prime =\frac{(3\times \mathit{\text{STDV}}\mathit{\text{High}}\mathit{\text{Control}}+3\times \mathit{\text{STDV}}\mathit{\text{Low}}\mathit{\text{Control}})}{\mathit{\text{ABS}}(\mathit{\text{AVR}}\mathit{\text{High}}\mathit{\text{Control}}-\mathit{\text{AVR}}\mathit{\text{Low}}\mathit{\text{Control}})}$$

$$S:B=\mathit{\text{AVG}}\mathit{\text{High}}\mathit{\text{Control}}÷\mathit{\text{AVG}}\mathit{\text{Low}}\mathit{\text{Control}}$$

In each case, a Z’ value greater than 0.5 was required for a plate to be considered acceptable [42]. If it were not acceptable it was rerun until it achieved a Z’ > 0.5 or evaluated further based on correlation between replicates.

Results

Development of Paired Entry Assays

In this study, we developed paired assays that enable us to monitor LASV, MACV, and SARS2 PV infection. Successful infection of these PVs results in firefly or nano luciferase expression and luminescence signal (Figure 1A), which is proportional to the individual PV infection level within a wide dynamic range and was measured as separate end-point readouts. Using two PVs, each of which encodes a different luciferase, enabled us to test them simultaneously. Frist, we focused on making three cell lines stably expressing hACE2 (namely hACE2-A549, hACE2-Hela, and hACE2-H1299, respectively) to select one that supports efficient SARS2 infection. This is because while most or all cell lines naturally express the receptor for LASV or MACV, respectively, ACE2 is rarely expressed in many cell lines. Then we evaluated their permissibility to the infection by SARS2, LASV, and MACV PV expressing F-luc reporter. As shown in Figure. 1B, hACE2-H1299 cells demonstrated the highest luminescence signal at 24 hours post-infection (hpi) for all PVs, which suggests hACE2-H1299 cell line is the best among these three tested cell lines for the SARS2, LASV, and MACV PV infection. In addition, we further optimized the luciferase reporter pairing for the uHTS by comparing the signal intensity and specificity for each reporter upon PV infection. To do so, the hACE2-H1299 cells were simultaneously infected with two PVs, one expressing F-luc and the other expressing N-luc (Figure. 2A). The F-luc signal was first detected using the F-luc substrate followed by the Stop & Glo^® reagent that quenches the F-luc signal and allows for the N-luc signal detection. As shown in the left panel of Figure. 2B, the F-luc substrate specifically responded to F-luc but not to N-luc. The Stop & Glo^® reagent quenched F-luc signal completely as shown in the right panel of Figure. 2B, meaning there is no spillover signal from F-luc to N-luc. In other words, N-luc signal specifically corresponds to the indicated PV infection. Before running the large-scale uHTS, we also tested whether the co-infection of two different PVs might interfere with each other, and the result showed that there was no such interference regardless of the PV combinations (Figure. 2C). The final protocol is summarized in Table 1.

Figure 1: Selection of the susceptible cell line for robust infection of three PVs.

(A) Diagram of PV infection. The PV particle enters the susceptible cells through the interaction between the surface viral glycoprotein and its cellular receptor hACE2, TFR1, and a-DG for SARS2, MACV, and LASV, respectively. Since most cell lines express low ACE2, H1299, A549, and Hela cells with naturally expressing TFR1 and a-DG are stably transduced to over express hACE2. (B) The hACE2-expressing H1299, A549 and Hela were infected with 100-fold diluted LASV, MACV, or 30-fold diluted SARS2 PV containing F-luc reporter. The cells were harvested at 24 hpi for luminescence detection. The data is presented as mean values ± SEM from three independent experiments with two technical repeats. The statistical significance was calculated by one-way ANOVA (****, P < 0.0001).

Figure 2: Development of paired PV entry assay.

(A) Paired PV entry assay principle. Susceptible cells were infected with an appropriate pair of PVs which contained either nano or firefly luciferase expressing genome. The successful infection is monitored by luminescent signal which is proportional to the infection level. When PV entry is inhibited, the low signal is observed. (B) hACE2-H1299 cells were infected with 100-fold diluted LASV, MACV, or 30-fold diluted SARS2 PV containing either F-luc or N-luc reporter. (C) hACE2-H1299 cells were infected with 100-fold diluted paired PVs containing F-luc and N-luc as indicated. At 48 hpi, the cells were harvested to read F-luc signal first with the F-luc substrate and then added Stop & Glo^® reagent to read N-luc signal. Three independent experiments with four technical repeats were conducted. The statistical analysis was not applied to this experiment.

Optimization of PV Entry assay for uHTS

To adapt the assay in 1,536 wpf, we first assessed optimal cell number per well. 125, 250, 500 and 1000 cells / well were used and single infection was performed with each PV diluted at 1:100. 250 cells / well showed the best balance between signal and statistics (Figure S1A). The Z’ of SARS2 N-luc or F-luc was less than 0.5 for all cell numbers, and thus we reasoned that a higher concentration would improve the SARS2 assay outcomes. Hence, we tested lower dilutions of SARS2 PV using 250 cells /well. Infection of SARS2 N-luc diluted at 1:12.5 showed Z’ = 0.52 whereas SARS2 F-luc at the same dilution showed Z’ = 0.39 (Figure S1B). Based on these results, we decided to use only SARS2 N-luc for the primary screen at a 1:10 dilution which resulted in a better Z’ than the 1:12.5 dilution outcomes. LASV and MACV F-luc likewise showed linear reduction of luminescent signal which was proportional to PV dilution. Notably, the outcomes from 1:50 PV infection showed Z’ ≥ 0.5 (Figure S1 C and D). The LASV and MACV N-luc demonstrated a similar effect as the F-luc virus except at the higher concentrations of PV (Figure S1 C and D), presumably due to cytotoxicity. LASV and MACV N-luc diluted at 1:50 showed the best Z’ at 0.73 and 0.75, respectively, and therefore we decided to use this dilution for LASV and MACV in the primary screen. The final protocol for uHTS is summarized in Table 1.

Primary uHTS

The first step of the uHTS campaign was to screen the 650,000 UF-SDDL libraries for PV entry inhibition. In this primary screen, compounds were tested in singlicate at a single nominal concentration of 5 μM. A summary of the results of the primary screening assays is shown in Figure 3A. To determine an appropriate hit cut-off for each assay we applied what we call a DMSO-based cut-off which incorporates a mathematical algorithm where three values are calculated: (1) the average (AVG) activity value for all wells treated with DMSO, (2) 3 times the standard deviation (SD) value for the same set of wells, and (3) the sum of these two values. The value from (3) was used as a cutoff parameter, i.e. any compound that exhibited greater percent inhibition than the cutoff parameter was declared active; this was applied to each individual plate. This analysis was applied to all primary HTS assays except the MACV N-Luc assay which utilized the average activity of all samples tested and 3 times the SD as the cut-off. The reason being is so that we were able to set the cut-off for hit compounds very close to the noise of the sample field for each of the assays, thus retaining as many hits as possible for the next stage. All screen assays were robust, and we observed the following: The LASV F-luc (paired with MACV N-luc) assay performance was consistent with an average Z’ of 0.69 ± 0.05 and an average S:B of 2,508 ± 256 (n=522 plates; Figure 3A orange star in the left panel). Using the “AVG + 3*SD of DMSO plates (n=15) based Cut-off” criteria yielded 27,166 active compounds (“hits”). The MACV N-luc (paired with LASV F-luc) assay showed an excellent average Z’, 0.83 ± 0.04, and an average S:B of 43.11 ± 1.17 (Figure, 3A blue circle in the left panel). Using standard AVG + 3*SD of sample field cut-off yielded 14,899 hits. Subsequently, The MACV F-luc assay, which was paired with SARS2 N-luc performance, was consistent with an average Z’ of 0.63 ± 0.06 and an average S:B of 2,321 ± 263 (Figure 3A, blue star in the middle panel) For this assay, using the AVG + 3*SD of DMSO plates (n=22) based cut-off criteria yielded 31,948 hits. The dual infection pair, SARS2 N-luc (paired with MACV F-luc), similarly showed great assay performance with an average Z’ of 0.61 ± 0.06 and an average S:B of 42.99 ± 3.03 (Figure 3A, the green circle in the middle panel). Using standard AVG + 3*SD of DMSO plates (n=22) based cut-off criteria identified 25,281 hits. Furthermore, the LASV F-luc assay, which was paired with the SARS2 N-luc assay, performed consistent with an average Z’ of 0.68 ± 0.06 and an average S:B of 2486 ± 243 (Figure 3A, the orange star in the right panel). Using the AVG + 3*SD of DMSO plates (n=9) based cut-off criteria yielded 28,391 hits. The paired SARS2 N-luc assay (paired with LASV F-luc) performance showed an average Z’ of 0.47 ± 0.12 and an average S:B of 43.63 ± 4.29 (Figure 3A, the green circle in the right panel). The cut-off criteria based on the standard AVG + 3*SD of DMSO wells (n=9) identified 14,651 hits. Figure 3B represents the Venn analysis of hits common to the same PV, and there were 21,218, 13,260 and 12,166 compounds identified for LASV, SARS2 and MACV, respectively. Of those, 1,812, 2,586 and 1,506 compounds were identified as unique hits to LASV, SARS2 and MACV, respectively (Figure 3B) and subjected for cherry-picking for the confirmation screen. All but 24 compounds were available for confirmation screen.

Figure 3: Illustration of uHTS screening.

(A) Results of primary screening. In the primary screening, 649,568 compounds were screened at 5 μM in singlicate. (B) Venn analysis of hit compounds from primary screening. Each PV was tested at least twice and 21,218, 12,166 and 13,260 compounds were identified as common hits for LASV, MACV, and SARS2, respectively. Of those, 1,812, 1,506 and 2,586 compounds were identified as unique hits to LASV, MACV and SARS2, respectively. (C) Results of confirmation screening. In this stage, 5,880 compounds were tested at 5 μM in triplicate. At this stage, an additional SARS2 F-luc + LASV N-luc pair was included. (D) Venn analysis of hit compounds from confirmation screening. Each PV was tested at least twice or three times and 135, 110 and 153 compounds were identified as common hits for LASV, MACV and SARS2, respectively. Of those, 60, 40, and 90 compounds were identified as unique hits to LASV, MACV, and SARS2, respectively. (E) Results of titration screening. In this stage, 202 compounds were tested with 10 points, 3-fold serial diluted starting at 13.1 μM in triplicate. At this stage, as well as SARS2 F-luc + LASV N-luc pair, cytotoxicity assay was included.

Confirmation Screen

The confirmation screen used the same reagents and detection system as the primary screening assays but tested each of the available compounds in triplicate, albeit at a single concentration (nominally 5 μM). A total of 5,880 compounds underwent this procedure. Again, each screen was run separately with an appropriate pair of PVs, which was the same as the primary screen and is summarized in Figure 3C. Both LASV F-luc and its pair, MACV N-luc, showed excellent performance with average Z’, 0.60 ± 0.06 and 0.85 ± 0.03, respectively (Figure 3C orange star and blue circle). The primary screen hit cut-off (50.12%) was applied for the LASV F-luc confirmation assay, and 146 hits were confirmed their activity. Similarly, 111 hits (hit rate: 7.4%) were identified from the MACV N-luc (paired with LASV F-luc) confirmation assay, in this case using an interval-based AVG + 3*SD of sample field hit cut-off equal to 38.12 % inhibition. This identified 295 compounds (hit rate: 19.6%), for the MACV F-luc confirmation assay, which was paired with SARS2 N-luc (Figure 3C blue star and green circle). This was expected as we saw a similar pattern of approximately twice as many hits found in the F-luc screen than the N-luc screen during the primary screen. This hit number was obtained by using the same hit cut-off as primary screening, 45.72% inhibition. Simultaneously, the paired SARS2 N-luc screened identified 442 hits (hit rate: 17.1%) using the same hit cut-off as primary screening, 40.55% inhibition. The third pair, LASV F-luc and SARS2 N-luc (Figure 3C orange star and green circle), confirmed 301 (hit rate: 16.6%) and 163 (hit rate: 6.3%;) compounds applying same hit cut-off as primary screening, 44.66% and 57.35% inhibition, respectively. An additional assay, a pair of SARS2 F-luc and LASV N-luc was conducted for a better selection of compounds for the next level. The SARS2 F-luc confirmation assay yielded an average Z’ of 0.51 ± 0.04 and a S:B of 1928 ± 249 (Figure 3C green star). Using a hit cut-off of 63.28 % inhibition (interval AVG + 3*SD of DMSO plates-based hit cut-off), 219 hits confirmed their activity (hit rate: 8.5%). The LASV N-luc confirmation assay yielded an average Z’ of 0.87 ± 0.02 and a S:B of 47.65 ± 0.77 (Figure 3C orange circle). Using a hit cut-off of 18.58 % inhibition (interval AVG + 3*SD of DMSO plates-based hit cut-off), 379 hits confirmed their activity (hit rate: 20.9%). We then performed a Venn analysis of hits from each PV, to eliminate non-specific compounds. There were 135, 153 and 110 compounds identified as common hits between F-luc and N-luc PV for LASV, SARS2 and MACV, respectively (Figure 3D). Of these 60, 90 and 40 compounds were identified as unique hits for LASV, SARS2, and MACV, respectively, and subjected to cherry-picking for the titration assay (Figure 3D).

Titration Assay

Based on the results from the confirmation assays, 202 compounds were selected, including 12 that overlapped between the MACV and LASV outcomes, for the titration assays (Figure 3D). The titration assay employed the same reagents, protocols, pairs of PVs and detection systems as the confirmation assays but tested each of the selected compounds for a 10-point dose-response titration (3-fold dilutions) in triplicate (Figure 3E). At this stage, in addition to the infection assays using F-luc and N-luc PV pairs, a cytotoxicity assay was separately conducted. In the cytotoxicity assay, we used the same protocol and reagents as PV entry assay except that 2 μL media, instead of PV, was added per well and that CellTiter Glo (4 μL / well) was used for the detection of viable cells. A four-parameter equation describing a sigmoidal dose-response curve was then fitted with an adjustable baseline using Assay Explorer software (Symyx Technologies Inc.). The IC₅₀ values were determined by extrapolating the X values (concentration of compounds) from the fitted curves that correspond to the 50% inhibition of infection level on the Y axis. Only the compounds with an IC₅₀ less than 10 μM were considered active. Based on this rule, 34, 82 and 1 compounds were active specifically for LASV, SARS2 and MACV, respectively, with no cytotoxicity.

Verification Assay

To verify the potency and specificity of our high-throughput screening (HTS), the MACV entry inhibitor candidate (ID# SR-01000464326–1, with its chemical structure shown in Figure 4A) was selected for the further verification assay. In this assay, hACE2-H1299 cells in a 96-well plate were infected with MACV-, LASV-, and SARS2-Fluc PV in the presence of various concentrations of this compound. As shown in Figure 4B, the compound specifically and potently inhibited MACV entry in a dose-dependent manner compared to the other two viruses. Similarly, other potent unique compounds against LASV and SARS2 will be studied further and are the topic of a future publication.

Figure 4: Verification of the MACV entry inhibitor candidate.

(A) The identification number and chemical structure of the MACV entry inhibitor candidate. The chemical structure and its related information can be retrieved from PubChem using the identification number listed above. (B) The relative entry inhibition of the compound against MACV (blue), LASV (purple), and SARS2-BA.5 (green) PV on hACE2-H1299 cells. The cytotoxicity (grey) of this compound was also measured with the CellTiter kit (Promega). The Y-axis shows the relative inhibition by normalizing the luminescence values of compound-treated groups to that of the DMSO-treated group. The dots on the X-axis represent 0.1, 0.3, 1, 10, and 30 μM in the Log2 scale. The IC₅₀ was calculated by fitting the curve with [Inhibitor] vs. normalized response and Variable slope in GraphPad Prism. The IC₅₀ values of the compound against MACV (blue), LASV (purple) PV are listed on the right, N/A means the IC₅₀ value is not applicable. This graph is one representative of three independent experiments. Error bars are shown in SEM, N=2 replicates per concentration per experiment.

Discussion

Viral entry into host cells initiates virus infection, which includes receptor engagement and membrane fusion. Thus, intervention strategies that interfere with these steps are expected to inhibit viral infection. Based on this scenario, we sought to identify entry inhibitors targeting the entry steps of three highly pathogenic viruses, including SARS2, LASV, and MACV. In this study, we produced PVs that are coated with the SARS2, LASV or MACV GP and contain a reporter gene of either firefly or nano luciferase. Paired infection of PVs, each expressing a different luciferase affords the detection of two different PV infection in the same well, which improved HTS outcomes dramatically for reproducibility and by saving time and costs. We obtained 6 HTS assay outcomes in the same amount of plates and time as we would for 3 HTS campaigns thereby saving tens of thousands of dollars and approximately 6–12 months of time. The uHTS of approximately 650,000 compounds produced massive data, which efficiently eliminated non-specific compounds. To enhance one’s understanding of this impact, this multiplexed assay approach for e.g. LASV allowed 21,218 primary screening hits to be refined to 1,812 selective hits without further experimentation, such that only 1,812 LASV hits went into confirmation assays rather than 21,218. This exercise reduced our timelines by weeks and eliminated needless cherry-picking of compounds that would have been found to be non-specific at a later stage, both highly desirable attributes for HTS. After the primary screening, the list of compounds chosen to proceed with out of the ~650,000 collection was narrowed down to 0.40 %, 0.28 % and 0.23% for SARS2, LASV and MACV, respectively (Figure 3B). During the confirmation screening, we applied SARS2 F-luc and LASV N-luc, which were not used in the primary screen. The resulting data allowed us to exclude compounds that act as luciferase inhibitors. The list was then narrowed down further by virus specificity. To date, this was the largest screening effort directed at one mechanism (Entry) done at UF Scripps, whereby we have previously concluded over 450 full deck HTS runs. At the conclusion of the uHTS, a total of 202 compounds were evaluated in dose-response assays. In this step, an additional counter assay was included to explore the cytotoxicity of the selected compounds. Using this as a tool, we further down selected compounds that showed less than 10 μM IC₅₀, specifically inhibited SARS2, LASV, or MACV PV, but were not cytotoxic. These lead compounds and their analogues are currently under investigation for mode of action and improved potency.