Potent inhibition of arenavirus infection by a novel fusion inhibitor

Abstract

Several arenaviruses, including Lassa and Lujo viruses in Africa and five New World arenavirus (NWA) species in the Americas, cause life-threatening viral hemorrhagic fevers. In the absence of licensed antiviral therapies, these viruses pose a significant public health risk. The envelope glycoprotein complex (GPC) mediates arenavirus entry through a pH-dependent fusion of the viral and host endosomal membranes. It thus is recognized as a viable target for small-molecule fusion inhibitors. Here, we report on the antiviral activity and pre-clinical development of the novel broad-spectrum arenavirus fusion inhibitors, ARN-75039 and ARN-75041. In Tacaribe virus (TCRV) pseudotyped and native virus assays, the ARN compounds were active in the low to sub-nanomolar range with selectivity indices exceeding 1000. Pharmacokinetic analysis of the orally administered compounds revealed an extended half-life in mice supporting once-daily dosing, and the compounds were well tolerated at the highest tested dose of 100 mg/kg. In a proof-of-concept prophylactic efficacy study, doses of 10 and 35 mg/kg of either compound dramatically improved survival outcome and potently inhibited TCRV replication in serum and various tissues. Additionally, in contrast to surviving mice that received ribavirin or placebo, animals treated with ARN-75039 or ARN-75041 were cured of TCRV infection. In a follow-up study with ARN-75039, impressive therapeutic efficacy was demonstrated under conditions where treatment was withheld until after the onset of disease. Taken together, the data strongly support the continued development of ARN-75039 as a candidate therapeutic for the treatment of severe arenaviral diseases.

1. Introduction

The Mammarenavirus genus of the Arenaviridae family of viruses comprises a number of Old World and New World species, several of which cause severe, life-threatening arenaviral hemorrhagic fever (AHF) in humans^1,2. These pathogenic arenaviruses are primarily transmitted through inhalation or ingestion of virus-containing rodent excreta, although they may also be spread through human-to-human contact. While many infected individuals exhibit mild or no symptoms, others develop more severe disease following a typical incubation period of 6–21 days^3,4. The onset of disease is usually gradual, starting with fever, general weakness and malaise, after which headache, sore throat, muscle pain, chest pain, nausea, vomiting, diarrhea, cough and abdominal pain may follow. In severe cases, facial swelling and bleeding from the mouth, nose, vagina or gastrointestinal tract may develop along with shock, seizures, tremor, disorientation, low blood pressure, coma or death. The most significant AHF in terms of unmet medical needs is Lassa fever (LF), a disease endemic in regions of West Africa where annual Lassa virus (LASV) infections are estimated to exceed 300,000, with approximately 5000 deaths each year⁵. In hospitalized LF patients, there is a 15–20% mortality rate within 14 days of disease onset and deafness occurs in 25% of survivors⁶. Case fatality rates for the South American AHFs caused by Junín virus (JUNV), Machupo virus (MACV), Guanarito virus (GOTV), Sabia virus (SBAV) and Chapare virus (CHAPV) can be as high as 30–60%, depending on the outbreak^7,8. The Candid#1 vaccine is only approved for use in Argentina, where JUNV and Argentine hemorrhagic fever are endemic in agricultural areas of the country^9,10, and there are no antiviral drugs approved for the treatment of any of the AHFs. The nonspecific antiviral drug ribavirin has been utilized therapeutically despite the lack of definitive clinical data to support its effectiveness and the risk of potentially serious adverse effects, including thrombocytopenia and anemia¹¹. The development of safe and efficacious antivirals to treat LF and other AHFs is urgently needed.

In pursuit of the development of a potent, broad-spectrum arenavirus therapeutic, we identified a novel heterocyclic chemical series of cell entry inhibitors based on pharmacophore modeling of two previously reported arenavirus inhibitor chemical series¹², which target the GP2 glycoprotein subunit of AHF viruses: the 4-acyl-1,6-dialkylpiperazin-2-ones and 1,5- substituted benzimidazoles¹³. A number of these novel heterocyclic compounds demonstrated low to sub-nanomolar 50% effective concentration (EC₅₀) potency against Old World (LASV) and New World (JUNV and MACV) viruses¹². The 3,6-diphenyl substituted imidazopyridine, ARN-75039, and ARN-75041 (analogs 37 and 39, respectively, in reference ¹²), a 1,6-diphenyl substituted benzimidazole (Figure 1A), were further selected for their attractive drug-like properties, including metabolic half-lives > 1 h in liver microsomes across multiple species (human, mouse, dog, rat and guinea pig), as well as minimal hERG and CYP450 inhibition. Given their observed potency against LASV, JUNV and MACV, as well as their favorable in vitro absorption, distribution, metabolism and excretion (ADME) properties¹², they were advanced for further evaluation. Here, we report on the antiviral activity of ARN-75039 and ARN-75041 against additional New World arenaviruses (NWAs), including GTOV, CHAPV and Tacaribe virus (TCRV), and confirmation of their glycoprotein-targeted mechanism of action through drug-resistance studies with TCRV. In addition, we characterized the pharmacokinetics (PK) and tolerability of the compounds in mice and subsequently evaluated their antiviral efficacy in the AG129 mouse TCRV infection model¹⁴. The results of these studies confirm the drug-like properties of the novel ARN-75039 and ARN-75041 fusion inhibitors and highlight their potent antiviral activity in a lethal arenavirus infection model when administered either prophylactically or at extended post-exposure and therapeutic time points.

Figure 1. ARN compound structures and mutations in the GP2 subunit that confer resistance.

A) Structures of ARN-75039, ARN-75041, ARN-74818 and the 1,5-substituted benzimidazole, ST-193. B) TCRV GPC open-reading frame comprising the stable signal peptide (SSP), GP1 and GP2 subunits. Cleavage sites for signal peptidase (SPase) and the proprotein convertase subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) are indicated, as is the transmembrane domain (TM) within the GP2 subunit (amino acids 418–438). GP2 amino acids 409–438 are shown with wild-type (top and bold) and mutant sequences (below) generated by ARN-74818 selection. ARN-74818 unique mutations (cyan highlight). Mutations similar to published ST-193 resistance mutations (grey highlight)¹⁸. TCRV GP2 amino acid residue (F425) analogous to the F427I resistance mutation in the JUNV Candid#1 vaccine strain (orange highlight).

2. Materials and Methods

2.1. Pseudotyped virus generation.

A vesicular stomatitis virus (VSV) pseudotype system expressing arenavirus envelope glycoproteins (GPCs) and the Renilla luciferase reporter gene was used to generate pseudotyped viruses in cultured HEK-293T cells (ATCC^® CRL-3216™), which were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Cytiva) supplemented with 10% FBS, 1% MEM non-essential amino acids solution, Penicillin-Streptomycin solution (100 units/mL Penicillin/100ug/mL Streptomycin), 1mM Sodium Pyruvate and 2mM L-Glutamine (DMEM-S). Cells grown in 10 cm dishes were transfected with a mixture of 15 μg of the pCAGGS plasmid encoding GPC sequence from TCRV (strain TRVL 11573), GTOV (strain INH-95551), or CHAPV (strain 810419), and 45 μL of Polyethylenimine “MAX” transfection reagent (Polysciences, Inc.) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific). The TCRV GPC gene was modified to contain a deletion of amino acid residues 121–132 and a glutamic acid to arginine substitution at position 458¹⁵. The cells were incubated with the solution for 5 h at 37°C and 5% CO₂, then washed and the mixture replaced with DMEM-S for additional incubation of 18 h. Subsequently, cells were infected with 50 μL of VSV reporter virus, whereby the VSV glycoprotein gene was replaced with a luciferase reporter gene. The cells were infected for 1 h, then washed once with phosphate-buffered saline (PBS) and incubated in DMEM-S. At 24 h post-infection (p.i.), culture supernatant was collected, clarified by centrifugation, passed through a 0.45 μm filter, aliquoted and stored at −80°C. The GTOV, CHAPV and TCRV pseudotyped viruses are hereto referred to as pGTOV, pCHAPV and pTCRV, respectively. The newly generated pseudotyped viruses were titrated for luminescence activity in Vero cells as described in the luciferase assay protocol described in 2.2.

2.2. Pseudotyped virus assay.

Vero cells (ATCC^® CCL-81™) grown in clear 384-well plates were exposed to varying concentrations of ARN-75039 and ARN-75041 and pseudotyped virus in assay media consisting of 50% Opti-MEM, 50% Minimum Essential Medium (MEM; Cytiva), 1% FBS, 1% MEM non-essential amino acids, Penicillin-Streptomycin (100 units/mL Penicillin/100 μg/mL Streptomycin), 1 mM Sodium Pyruvate and 2 mM L-Glutamine. Each of the pseudotyped virus preparations generated was diluted to give a similar luminescence signal to background value of > 200. The assay volume was 50 μL/well with the final DMSO concentration at or below 1%. Control wells were treated with assay media and 1% DMSO. Cells were incubated for 24 h at 37°C and 5% CO₂ and assessed for luciferase activity according to the Pierce Renilla Luciferase Glow Assay Kit (Thermo Fisher Scientific). Cells were lysed with 20 μL of lysis buffer and 5 μL of cell lysate was transferred to an opaque white plate and mixed with 12.5 μL of coelenterazine diluted in buffer. The mixture was incubated at room temperature for 10 min on a shaker before luminescence detection (Beckman Coulter DTX 880 multimode detector; Beckman Coulter). Luminescence signals for compound-treated and control wells were used to determine the antiviral activity (percent inhibition of the luciferase signal) for each compound.

2.3. Native replicating TCRV assay.

Compounds were tested against native, replication-competent TCRV, strain TRVL 11573 (BEI Resources, Manassas, VA), using an ELISA-based assay. Vero cells in 96-well plates were exposed to a mixture of TCRV (MOI of 0.05) and varying concentrations of ARN-75039 and ARN-75041, prepared in MEM with 1% FBS, 1% MEM non-essential amino acids, Penicillin-Streptomycin, 1 mM Sodium Pyruvate and 2 mM L-Glutamine. Final DMSO concentration in the compound testing wells was less than or equal to 1% and control wells were exposed to TCRV with 1% DMSO. After 5 days of incubation at 37°C and 5% CO₂, cells were fixed with 2% paraformaldehyde for 45 min and permeabilized with 0.25% Triton-X prior to addition of 1 μg/mL of a monoclonal anti-JUNV antibody (Clone MA03-BE06, NR-41860; BEI Resources), which cross-reacts with the TCRV nucleoprotein. After washing, cells were treated with goat anti-mouse IgG1 heavy chain antibody (ab97238; Abcam, Cambridge, MA) for 1 h and subsequently, Pierce Streptavidin-HRP (Thermo Fisher Scientific) for 1 h. TMB substrate (Thermo Fisher Scientific) was added to the wells and the reaction was stopped using 2 M sulfuric acid. The absorbance at 450 nm was measured using the Beckman Coulter DTX 880 multimode detector. Antiviral activity was measured as the percent decrease in absorbance in test wells that received ARN-75039 or ARN-75041 compared to control wells that did not receive either compound.

2.4. Cytotoxicity assay.

ARN-75039 or ARN-75041 were serially diluted and added to Vero cells in 96-well plates with a final DMSO concentration of 1% in MEM with 1% FBS, 1% MEM non-essential amino acids, Penicillin-Streptomycin, 1 mM Sodium Pyruvate and 2 mM L-Glutamine. Control wells were treated with 1%DMSO in the same medium. The plates were incubated at 37°C and 5% CO₂ for 5 days and dead cells were removed by washing with PBS. Cell viability was measured by staining with Neutral Red dye (40 μg/mL) for 1 h, followed by extraction of the dye with a solution of 50% ethanol and 1% acetic acid. The absorbance was read at 540 nm and 690 nm on a Spectramax Plus 384 spectrophotometer (Molecular Devices). Adjusted absorbance values (540 nm measurement minus the 690 nm reference reading) were used to calculate the 50% cell cytotoxic (CC₅₀) concentrations by linear regression analysis.

2.5. Generation of TCRV-resistant variants.

Vero cells were infected with TCRV (MOI of 0.1) in MEM supplemented with 2% FBS in the presence of increasing concentrations of ARN-74818. The cells were incubated for 7 days and the supernatant containing virus was used to infect a new flask of Vero cells. Four passages were performed in the presence of compound concentrations of 1 μM, 1.2 μM, 1.5 μM and 1.8 μM, respectively. After the fourth passage, the virus was propagated again with 1.8 uM ARN-74818 and the resulting supernatant was used to isolate viral RNA using the QIAamp Viral RNA Mini Kit (Qiagen), converted to cDNA using the Qiagen one-step RT-PCR Kit (Qiagen) and cloned into the TOPO vector using pcDNA™ 3.4−TOPO^® TA Cloning Kit (Thermo Fisher Scientific). Primers specific for TCRV GPC used for PCR: Forward 5’-CCACCATGGGGCAATTCATTAGTTTC-3’ and Reverse 5’-CTAATGTCTACGATGCCAAATAGT-3’. The clones were sequenced by the Sanger method (Eton Bioscience) and subcloned into the pCAGGS vector to generate pseudotyped VSV according to the protocol described in 2.1.

2.6. Pharmacokinetics and tolerability.

Six to eight-week-old S129 male mice were dosed by oral gavage with a formulated suspension of either ARN-75039 or ARN-75041 at 30 mg/kg. The in-life portion of the study was conducted at Explora BioLabs (San Diego, CA). Blood was drawn at several time points up to 24 h post-dosing, with 3 mice sampled at each time point. Plasma was obtained, spiked with a related analog serving as an internal standard, deproteinized in acetonitrile and analyzed by LC/MS/MS. Compound concentrations were extrapolated from a standard curve from normal mouse plasma spiked with the same internal standard and different concentrations of either ARN-75039 or ARN-75041. For the 3-day dosing studies, equal numbers of male and female 6 to 8-week-old S129 mice were orally gavaged with 100 mg/kg/day of either ARN-75039 or ARN-75041. Mice were observed for clinical signs of distress and monitored daily for body weight over the 3 days. Twenty-four h following the final dosing on day 3, terminal blood samples were collected along with livers that were flash-frozen. Plasma was obtained, deproteinized in acetonitrile and analyzed by LC/MS/MS. Frozen livers were weighed and after thawing they were flushed several times with PBS to remove any excess blood. An equal volume (mL) of PBS to weight (g) was used to homogenize the liver samples, followed by centrifugation at 14,000 rpm, for 10 minutes, to pellet the debris. The remaining supernatant was spiked with an internal standard, deproteinized with acetonitrile, centrifuged again at 14,000 rpm to pellet protein and the remaining supernatant analyzed by LC/MS/MS. Each compound’s plasma and liver concentrations were extrapolated from a standard curve generated from known spiked concentrations of the respective compounds.

2.7. ARN compound antiviral efficacy vs. TCRV infection in mice.

All animal procedures complied with guidelines set by the USDA and Utah State University Institutional Animal Care and Use Committee. The TCRV (strain TRVL 11573) used for the in vivo efficacy studies was obtained from American Type Culture Collection (ATCC; Manassas, VA). The virus stock was prepared from clarified liver homogenates from AG129 mice challenged with TCRV (2 passages in Vero 76 cells). Virus stock was diluted in sterile MEM and approximately 500 fifty percent cell culture infectious doses (CCID₅₀) were inoculated by a 0.2 mL intraperitoneal (IP) injection for prophylactic and therapeutic efficacy studies.

For the prophylactic dosing study, male and female 6 to 8-week-old AG129 (IFN-α/β and γ receptor-deficient) mice were obtained from the Utah State University breeding colony (Logan, UT) and fed irradiated Harlan Lab Block and autoclaved tap water ad libitum. The mice were weighed the day before the infection and assigned to treatment groups so that sex and weight were evenly distributed between the groups. A total of fifteen animals were assigned to each treatment group, which included vehicle placebo, 100 mg/kg ribavirin or 10 or 35 mg/kg of ARN-75039 or ARN-75041. The ARN compounds were prepared as a suspension at 8.75 and 2.5 mg/mL (4 mL/kg dose volume) to provide the low and high-dose formulations. The same oral suspension vehicle was used as the placebo. Both ARN compounds and the placebo were administered by oral gavage, once daily for 12 days. Ribavirin, obtained from ICN Pharmaceuticals (Costa Mesa, CA), was prepared in sterile saline for twice-daily dosing by oral gavage for 10 days. Treatments were initiated 2 h before TCRV challenge. Preselected cohorts of 4 mice/group were sacrificed on day 9 to assess serum, liver and spleen viral titers. The remaining eleven animals per group were observed for 27 days for morbidity and mortality. Sham-infected normal controls were included for comparison. Several deceased, moribund and surviving animals were necropsied to assess serum, liver, spleen and brain viral titers.

For the therapeutic dosing study, male and female 7 to 11-week-old AG129 mice were managed and distributed into experimental groups as described in the prophylactic efficacy study, unless otherwise noted. A total of fifteen animals per treatment group were dosed once daily for 12 days by oral gavage with 20 mg/kg of ARN-75039 beginning on days 1, 3, 5 or 7 p.i., with the following exceptions. The day 5 treatment initiation group was given a 50 mg/kg loading dose on day 5 and 20 mg/kg thereafter. The day 7 group received 20 mg/kg on day 7, a 50 mg/kg loading dose on day 8, and 20 mg/kg thereafter. Vehicle and formulated ARN-75039 suspensions were prepared as described above at 5 mg/mL (20 mg/kg/day treatment) and 12.5 mg/mL (50 mg/kg loading dose). To help prevent late-onset disease thought to be associated with secondary bacterial infections, all animals were given a course of 5 mg/kg/day of Baytril administered by subcutaneous injection, twice daily for 7 days, beginning 24 h after the final antiviral drug treatment. The day 7 treatment initiation group received its first Baytril treatment 12 h after the final ARN-75039 dose. Preselected animals from each treatment group were sacrificed on day 9 to assess serum, liver, spleen and brain viral titers. The remaining animals were observed 28 days for morbidity and mortality. Several deceased, moribund and surviving mice were necropsied to assess viral titer in serum and selected tissues. Tissues with visible pyogranulomas were sent to the Utah Veterinary Diagnostic Laboratory (Logan, UT) for bacterial cultures.

2.8. Measurement of serum and tissue virus titers.

Virus titers were assayed using an infectious cell culture assay as previously described¹⁴. Briefly, a specific volume of serum or tissue homogenate was serially diluted and added in quadruplicate to wells of Vero cell monolayers in 96-well microtiter plates. The viral cytopathic effect (CPE) was determined 8–10 days after plating and the 50% endpoints were calculated as described¹⁶. The assay lower limits of detection were 1.67 log₁₀ CCID₅₀/mL serum and ranged from 2.5–2.8 log10 CCID₅₀/g tissue. In samples presenting with virus below the limit of detection, a value representative of the limit of detection was assigned for statistical analysis.

2.9. Statistical analysis.

The log-rank test was used for the analysis of Kaplan-Meier survival curves. A one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was used to assess differences in virus titers. All statistical evaluations were performed with Prism 9 (GraphPad Software, La Jolla, CA).

3. Results

3.1. Antiviral activity of ARN compounds against additional NWAs.

ARN-75039 and ARN-75041 (Figure 1A) were previously shown to exhibit low to sub-nanomolar EC₅₀ values against pathogenic Old World and NWAs, including LASV, JUNV and MACV, in pseudotyped virus assays and against native replicative LASV and JUNV¹². To determine whether the inhibitory activity of the compounds extended to additional South American hemorrhagic fever and related NWAs, VSV pseudotyped viruses expressing Guanarito (pGTOV), Chapare (pCHAPV) or Tacaribe (pTCRV) virus GPCs were incubated with escalating concentrations of compound and virus entry into Vero cells was measured. As shown in Table 1, ARN-75039 was more potent than ARN-75041 at inhibiting entry of pGTOV and pCHAPV, while both compounds were less effective against pCHAPV relative to the other two pseudoviruses. Both compounds equally demonstrated sub-nanomolar EC₅₀ activity against pTCRV that further translated to native wild-type TCRV along with impressive selectivity indices near to or exceeding 10,000 (Table 1).

Table 1.

ARN compound activity against additional NWAs

Compound	Pseudotyped virus EC50 (nM)			Native virus EC50 (nM)	Native virus SI
	pGTOV	pCHAPV	pTCRV	TCRV
ARN-75039	a0.13 ± 0.01	a4.3 ± 1.9	a0.22 ± 0.09	a0.75 ± 0.26	11,150
ARN-75041	b1.7 ± 0.05	b15.3 ± 0.67	a0.29 ± 0.17	c0.89	9,146

^aEC₅₀ data represent the mean and standard deviation of 3 or more independent experiments.

^bEC₅₀ data represent the mean and standard deviation of 2 independent experiments.

^cData from a single experiment

Selectivity index (SI)=CC₅₀/EC₅₀

3.2. Mapping determinants of ARN compound sensitivity.

ARN-75039 and ARN-75041 belong to a chemical series of diphenyl substituted heterocyclic compounds generated through pharmacophore modeling of two previously reported arenavirus inhibitor chemical series, the 4-acyl-1,6-dialkylpiperazine-2-one and 1,5-substituted benzimidazoles, both of which had been suggested to bind to an overlapping site near the transmembrane domain of the GP2 subunit of arenavirus GPC^13,17. To further evaluate and confirm the binding site and mechanism of action of compounds from our novel heterocyclic chemical series, drug-resistant TCRV variants were selected for (Figure 1B) in escalating concentrations of ARN-74818, an early chemical series analog (3a in reference ¹²) that also exhibits low nanomolar potency against TCRV (EC₅₀=1.8 nM)¹². As anticipated, ARN-74818 selection induced mutations near the GP2 transmembrane region (Figure 1B) similar to that observed for the 1,5-substituted benzimidazole analog ST-193¹⁸. pTCRV variants expressing the identified GP2 mutations were subsequently generated and tested in pseudotyped virus assays to assess resistance to ARN compounds and ST-193. As shown in Table 2, the mutations conferred resistance to each of the ARN compounds confirming the target and expected mechanism of action. Interestingly, the T434I pTCRV variant displayed greater resistance to ST-193 than the ARN compounds, indicating differences in binding interactions at GP2.

Table 2.

Mutations identified in ARN-74818-resistant TCRV variants abrogate pTCRV sensitivity to ARN-75039, ARN-75041 and ST-193

pTCRV variant	Compound EC50 (nM)
	ARN-74818	ARN-75039	ARN-75041	ST-193
Wild-type	1.7	0.2	0.4	2.0
E411K, H438R	>1000	>1000	>1000	>1000
I418N	>1000	>1000	>1000	>1000
S433G, H438R	>1000	>1000	>1000	>1000
T434I	600	29	26	>1000

3.3. Oral PK and tolerability of ARN-75039 and ARN-75041 in S129 mice.

To determine the PK properties and suitability of ARN-75039 and ARN-75041 for oral dosing, S129 mice were administered a dose of 30 mg/kg by oral gavage and plasma concentrations for each compound determined at various time points post-treatment (Figure 2). The C_max for ARN-75039 and ARN-75041 was 5.5 μg/mL (13.3 μM) and 20.4 μg/mL (49.2 μM), respectively. These values were 1000 times greater than the EC₅₀ for both compounds against TCRV (Table 1). The half-life for both compounds was 13.2 and 17.6 h, respectively. Based on parallel intravenous dosing at 3 mg/kg, oral bioavailability was 30% and 35%, respectively. Given these results, the oral suspension formulation and exposure appeared suitable for once-daily dosing in the mouse TCRV challenge model.

Figure 2. Oral PK of ARN compounds in S129 mice.

ARN-75039 and ARN-75041 were dosed at 30 mg/kg by oral gavage. Plasma concentrations are shown over 24 h and each data point represents the mean and standard deviation from 3 mice.

Multiple organs, including the liver, are known sites of arenavirus replication and pathogenesis^1,19, and therefore compound exposure in target tissues is critical for efficacy. In the mouse TCRV infection model, the virus replicates to high titers in the liver and other tissues¹⁴. To further evaluate the PK and tolerability of ARN compounds, and assess liver exposure, higher doses (100 mg/kg) of ARN-75039 and ARN-75041 were administered by oral gavage for 3 consecutive days. Blood and liver were collected 24 h after the final dosing to measure compound trough levels. Table 3 shows trough levels for plasma and liver from mice dosed with either ARN-75039 or ARN-75041. The concentration of the compounds in liver was 3.5-fold and greater than 12-fold higher than plasma concentrations of ARN-75039 and ARN-75041, respectively, indicating good tissue penetration. Interestingly, a sex-related difference in both plasma and liver tissue concentration was noted for male and female mice dosed with ARN-75041. During the 3-day study, there was no evidence of toxicity or weight loss with either compound at the 100 mg/kg daily dose.

Table 3.

Mouse plasma and liver trough levels 24 h after 3^rd 100 mg/kg dose

Compound	Plasma (μM)a		Liver (μM)a
	Male	Female	Male	Female
ARN-75039	21.1 ± 5.5	22.1 ± 14.1	71.8 ± 19.4	79.2 ± 66.0
ARN-75041	10.8b	21.2b	150.0 ± 15.1	>200c

^aData points represent the mean and standard deviation of 3 mice per sex

^bPlasma samples pooled from 3 mice

^cMeasured concentration above the upper limit of the standard curve

3.4. Prophylactic efficacy of ARN-75039 and ARN-75041 in the AG129 mouse TCRV challenge model.

Given the favorable PK and liver exposure data and the lack of toxicity, both compounds were advanced to evaluate prophylactic efficacy in the AG129 mouse TCRV challenge model. Selected doses of ARN-75039 and ARN-75041 were administered once daily by oral gavage for 12 days, beginning 2 h before infection. Animals treated with either compound at doses of 10 or 35 mg/kg were protected against mortality associated with TCRV infection (Figure 3A). The positive control ribavirin-treated group had 82% survival, while only 18% of the animals receiving the placebo survived. In addition to significantly improved survival outcomes, mice treated with ARN-75039 or ARN-75041 displayed little to no weight loss during the acute infection (days 7–13 p.i.; Figure 3B). Notably, there were several late deaths in all of the drug-treated groups. This late-onset disease and mortality were not believed to be due to viral infection and were most likely a consequence of a secondary opportunistic bacterial infection in some of the immunocompromised mice. The highly variable late-stage weight loss in the ARN compound treatment groups was due to the few mice per group that were affected by the secondary bacterial infections.

Figure 3. Prophylactic efficacy of ARN compounds in AG129 mice challenged with TCRV.

Mice (n=11/group) were treated with the indicated dose of ARN-75039 or ARN-75041, or the vehicle placebo, once daily for 12 days, beginning 2 h prior to TCRV challenge. Twice-daily ribavirin treatments also began 2 h before virus challenge. A) Survival outcomes and B) change in body weights are shown. The weight data are represented as the group mean and standard error of the percent change in weight of surviving animals relative to their starting weights on the day of TCRV challenge. Sham-infected normal control mice are shown for comparison. **p<0.01 and ***p<0.001 compared to animals treated with the placebo. Tx; treatment duration.

The impact of ARN-75039 and ARN-75041 treatments on reducing viral titers in predetermined subsets of mice sacrificed on day 9 p.i. was also assessed (Figure 4). Collectively, treatment with the ARN compounds resulted in undetectable levels of TCRV in serum, liver and spleen samples, with the exception of a low concentration of virus measured in the spleen of a single animal in the 10 mg/kg ARN-75041 group. In contrast, ribavirin only modestly reduced viral loads compared to the placebo treatment (Figure 4).

Figure 4. Prophylactic treatment with ARN compounds prevents TCRV viremia and replication in target organs.

A) Serum, B) liver and C) spleen viral titers measured in blood and tissue samples collected from subsets of AG129 mice (n=4/group) on day 9 p.i. of the prophylactic efficacy study. One mouse in the placebo group succumbed just prior to the time of sampling and therefore serum could not be obtained. Unique symbols in each treatment group represent values for the same animal across all parameters. The grey dotted lines represent the assay lower limits of detection. **p<0.01 and ***p<0.001 compared to animals treated with the placebo.

To further investigate the morbidity and mortality observed late in the study, six ARN-treated mice that were moribund or had expired beyond the acute TCRV infection phase were necropsied and found to have undetectable serum and tissue (liver, spleen, brain) viral titers (Table 4). In addition, TCRV could not be detected in serum or tissues in randomly selected surviving animals from each ARN treatment group (n=2/group) necropsied at the termination of the experiment. Thus, regardless of survival outcome beyond the acute infection, mice treated with ARN-75039 or ARN-75041 were cleared of the TCRV infection. In contrast, survivors from both placebo and ribavirin-treated groups (n=2/group) were found to have moderate to high viral loads in the liver, spleen and brain (Table 4). The measurable virus in the tissues of these four apparently healthy animals suggests a transition to a carrier state in ribavirin and placebo-treated survivors. Viral persistence in surviving animals following experimental drug treatment has been reported previously with this model²⁰.

Table 4.

Detection of infectious TCRV in deceased, moribund and surviving animals from the prophylactic efficacy experiment

			Infectious TCRVa
Animal #	Treatment, dose	Status	Serum	Liver	Spleen	Brain
963	ARN-75041, 35 mg/kg	Moribund day 18	ND	ND	ND	ND
972	ARN-75039, 35 mg/kg	Expired day 19	ND	ND	ND	ND
932	ARN-75039, 35 mg/kg	Moribund day 22	ND	ND	ND	ND
945	ARN-75039, 10 mg/kg	Moribund day 22	ND	ND	ND	ND
962	ARN-75041, 35 mg/kg	Moribund day 25	ND	ND	ND	ND
980b	ARN-75041, 10 mg/kg	Expired day 27	ND	ND	ND	ND
931, 998	ARN-75039, 35 mg/kg	Day 27 survivors	ND, ND	ND, ND	ND, ND	ND, ND
943, 950	ARN-75039, 10 mg/kg	Day 27 survivors	ND, ND	ND, ND	ND, ND	ND, ND
955, 956	ARN-75041, 35 mg/kg	Day 27 survivors	ND, ND	ND, ND	ND, ND	ND, ND
939, 982	ARN-75041, 10 mg/kg	Day 27 survivors	ND, ND	ND, ND	ND, ND	ND, ND
965, 968	Vehicle placebo	Day 27 survivors	2.0, ND	3.3, 5.8	5.5, 6.1	7.3, 6.8
975, 991	Ribavirin, 100 mg/kg/day	Day 27 survivors	2.5, ND	4.3, 3.5	6.1, 5.5	7.1, 7.1

^aNot detected (ND) or TCRV measured in log₁₀ CCID₅₀/mL

^bHistopathology consistent with bacterial infection

During the necropsies of deceased, moribund and surviving mice, gross pyogranulomatous lesions were observed on the serosal surface of several internal organs, including the livers, spleens, and intestines of animals treated with either test compound. Tissue sections from two of the ARN-75041-treated moribund mice were also preserved in formalin and evaluated for histopathology (Table 4). Based on histologic analysis by a board-certified veterinary pathologist, the observed lesions are not characteristic of viral pathology and are most likely due to bacterial infection. These types of lesions have also been previously observed with other antiviral agents evaluated in this model and are not specific to the ARN compounds. Rather, the presence of the lesions was most likely due to the immunocompromised IFN-α/β and γ receptor-deficient AG129 mouse background in which a combination of factors results in further immunosuppression and greater susceptibility to opportunistic secondary bacterial infections.

3.5. Therapeutic efficacy of ARN-75039 vs. TCRV infection in AG129 mice.

Based on the encouraging results of the prophylactic efficacy study, the ability of ARN-75039 to protect mice with advanced infections was pursued. ARN-75039 was selected as the lead candidate based on broad-spectrum potency, the lack of PK sex differences in mice (Table 3), its superior in vitro human liver microsome metabolic stability and a simpler, high-yield 3-step synthesis¹². As observed in the prophylactic efficacy study, AG129 mice treated with placebo had considerable weight loss starting on day 6 p.i. (Figure 3B), with animals succumbing as early as day 9 p.i. (Figure 3A). To assess the therapeutic potential of ARB-75039 when administered at later stages of TCRV infection, we began post-exposure interventions with 20 mg/kg of the compound at 1, 3, 5 and 7 days p.i. A loading dose strategy was implemented for the day 5 and day 7 treatment groups to help control the higher viral loads expected at the initiation of treatment in those groups. As shown in Figure 5A, all groups of mice treated with ARN-75039 were significantly (p<0.001) protected from mortality compared to the placebo-treated animals. Treatments initiated on days 1 or 3 p.i. were the most effective (100% survival), with therapy initiated on day 5 (91% survival) and day 7 (45% survival) also dramatically improving survival outcome and extending the time of death. Weight loss was the most abrupt in mice treated with placebo (Figure 5B). Notably, mice in the day 7 ARN-75039 group, in which treatment was withheld until after the mice were losing weight, plateaued in their weight loss for several days after the loading dose was administered on day 8. The subsequent weight loss in the day 7 group mice may have been contributed to by concomitant bacterial infection. The mice that were started on ARN-75039 on day 5 or earlier had weight trajectories similar to that of the sham-infected normal control mice (Figure 5B).

Figure 5. Therapeutic efficacy of ARN-75039 in AG129 mice challenged with TCRV.

Mice (n=11/group) were treated with 20 mg/kg ARN-75039 or the vehicle placebo, once daily for 12 days, starting at the indicated time p.i. Animals in the day 5 p.i. group were administered a 50 mg/kg loading dose on the first day of treatment. Animals in the day 7 p.i. group were administered a 50 mg/kg loading dose on the second day of treatments. A) Survival outcomes and B) change in body weights are shown. The weight data are represented as the group mean and standard error of the percent change in weight of surviving animals relative to their starting weights on the day of TCRV challenge. Sham-infected normal control mice are shown for comparison. **p<0.01 and ***p<0.001 compared to animals treated with the placebo.

Overall, the administration of Baytril appeared to reduce the secondary bacterial infections suspected in the prophylactic efficacy study. However, all but one of the deaths in the day 5 and day 7 ARN-75039-treated groups occurred beyond the acute infection when the placebo-treated mice had succumbed. Because the administration of low-dose Baytril was initiated on days 17 and 18 p.i. in the day 5 and day 7 treatment groups, respectively, the late-stage deaths were likely associated with secondary opportunistic bacterial infections in the immunocompromised AG129 mice, brought on by additional immunosuppressive effects associated with TCRV infection.

ARN-75039 treatments significantly reduced viral titers in mice sacrificed on day 9 p.i. in a treatment initiation time-dependent manner (Figure 6). Consistent with the prophylactic efficacy study, no viremia was detected in serum or tissues of animals dosed with ARN-75039 starting 2 h p.i., with the exception of single liver and spleen samples wherein low concentrations of virus were present. Mice that began treatments starting on days 1 or 3 p.i. had nearly undetectable levels of TCRV in serum, spleen and brain but substantial viral titers in the liver. Groups wherein treatment was initiated on day 5 or day 7 p.i. had more variable reductions in viral burden. Collectively, TCRV was detected at higher concentrations in the liver for all experimental groups, with only the day 1 and 2 h p.i. groups having a significant reduction in hepatic viral loads relative to the placebo group (p<0.01 and p<0.001, respectively; Figure 6B).

Figure 6. Post-exposure intervention with ARN-75039 limits TCRV viremia and replication in target organs.

A) Serum, B) liver, C) spleen and D) brain viral titers measured in blood and tissue samples collected from subsets of AG129 mice (n=4/group) on day 9 p.i. of the therapeutic efficacy study. One mouse in the placebo group succumbed just prior to the time of sampling and therefore serum could not be obtained. Unique symbols in each treatment group represent values for the same animal across all parameters. The grey dotted lines represent the assay lower limits of detection. *p<0.05, **p<0.01 and ***p<0.001 compared to animals treated with the placebo.

Sera, livers and spleens were collected from five mice from the day 5 and day 7 ARN-75039 treatment groups found either deceased or moribund to evaluate the TCRV infection status beyond the acute phase of infection. Samples were also collected from 2 or more randomly selected surviving animals from day 5, day 7 and 2 h p.i. groups. TCRV was not detected in the serum or any tissue in any of the mice treated with ARN-75039 (Table 5). During necropsies of deceased, moribund and surviving mice, gross pyogranulomatous lesions were observed on the serosal surface of some livers and spleens. The tissues containing pyogranulomas were submitted for analysis of bacterial growth. Confirming our suspicion, several mice were found to be infected with Lactobacillus, with a single animal co-infected with Streptococcus and Staphylococcus (Table 5).

Table 5.

TCRV and bacterial infection status in deceased, moribund and surviving animals from the therapeutic efficacy experiment.

Animal #	Treatments	Status	Infectious TCRV	Bacterial infection
985	ARN-75039 day 5–16 p.i. Baytril day 17–23 p.i.	Expired day 28	NDa	Lactobacillus Streptococcus Staphylococcus
987		Day 28 survivor	ND	Lactobacillus
930		Day 28 survivor	ND	No growth
935	ARN-75039 day 7–18 p.i. Baytril day 18–24 p.i.	Moribund day 19	ND	Not tested for bacterial growth No pyogranulomas present in any of the mice
923		Moribund day 23	ND
937		Moribund day 26	ND
938		Moribund day 26	ND
939		Day 28 survivor	ND
988		Day 28 survivor	ND
936		Day 28 survivor	ND
980	ARN-75039 day 0–11 p.i. Baytril day 12–18 p.i.	Day 28 survivor	ND	Lactobacillus
901		Day 28 survivor	ND	Not tested / No pyogranulomas
927		Day 28 survivor	ND	No growth

^aND = Not detected

4. Discussion

In a separate report, we described the discovery of a novel chemical series of arenavirus inhibitors that was identified utilizing the comparative structure-activity relationship (SAR) and pharmacophore modeling of two distinct chemical series of arenavirus cell entry inhibitors¹². Several analogs, including ARN-75039 and ARN-75041, were shown to exhibit potent activity against both Old World (LASV) and New World (JUNV and MACV) arenaviruses. Here, we show that ARN-75039 and ARN-75041 exhibit similar activities against other pseudotyped (pGTOV, pCHAPV and pTCRV) and native (TCRV) NWAs. We also confirmed the GP2 binding site and mechanism of action for the chemical series through drug resistance studies with TCRV and testing against the mutant pTCRV variants. Of particular note, it has been reported that several mutations that induce resistance to compounds interacting with the GP2 domain, such as the F427I mutation of the JUNV Candid#1 strain, are strongly associated with attenuated virulence^12,13,17,21. Thus, the identification and characterization of ARN-75039-specific drug-resistant viruses, both in vitro and in vivo, is of great interest.

Oral PK and tolerability were favorable for the two lead compounds, ARN-75039 and ARN-75041, which led to their evaluation in the mouse AG129 TCRV infection model. Because immune-competent strains of mice are refractory to disease following TCRV challenge, AG129 mice deficient in type I and type II IFN responses provide a relevant and lethal biosafety level 2 NWA infection model¹⁴. In the initial prophylactic efficacy study, both compounds completely blocked TCRV replication in the blood and target organs and significantly improved survival outcomes. However, a late-onset disease associated with opportunistic bacterial infections in the immunocompromised AG129 mice can complicate determining the level of antiviral protection afforded by the antiviral agent under consideration. In the prophylactic efficacy study, we found that in the absence of TCRV in blood or tissues evaluated, a limited number of ARN-treated mice succumbed to the late-onset disease. Upon necropsy, the observed gross pyogranulomatous lesions prompted further investigation to assess histopathology and the presence of bacterial agents. The findings of these analyses suggested that secondary bacterial infections were underlying the late-onset disease development as there was no evidence of characteristic viral histopathology and TCRV could not be detected in the serum, liver, spleen or brain in any ARN compound-treated mice at any time beyond the 13 to 16-day acute infection period. In contrast to the lack of virus in moribund, deceased or surviving mice treated with ARN-75039 and ARN-75041, surviving animals treated with placebo or ribavirin had significant tissue viral loads. The presence of TCRV in these mice at the end of the study suggests a transition to a state of viral persistence, which we have previously reported²⁰. Treatment with the ARN compounds effectively eliminated the virus, curing the animals of the infection.

More notably, ARN-75039 provided highly significant post-exposure protection when initiating treatment 5 days p.i. with evidence of therapeutic efficacy when treatment was started 7 days after virus challenge when mice were losing weight due to advanced TCRV infection and disease progression. For the most part, the Baytril antibacterial treatments appeared to mitigate the late-onset disease associated with secondary infections in all but the one day 5 ARN-75039-treated mouse that expired on day 28 and was found to be infected with multiple bacterial genera. Future studies employing the TCRV model should strongly consider overlapping Baytril treatment with antiviral therapy, as in a clinical setting it is not uncommon to have broad-spectrum antibiotics co-administered with antiviral drugs²². In summary, the drug-like properties and potent antiviral activity of ARN-75039 firmly support its continued development as an orally bioavailable and broad-spectrum arenavirus therapeutic.