Antiviral; Arenavirus envelope glycoprotein
The arenavirus envelope glycoprotein complex (GP), specifically the GP2 subunit that mediates membrane fusion. Arenavirus GP is a class I viral fusion protein that undergoes conformational changes at low pH within the endosome to drive fusion of the viral envelope with the host cell membrane. (E)-LHF-535 binds to the prefusion conformation of GP and stabilizes it, preventing the pH-induced conformational changes required for membrane fusion and thus blocking viral entry at an early step.

LHF-535 is a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein (GP).Strong antiviral activity is shown by LHF-535 against a variety of hemorrhagic fever arenaviruses. With an IC50 of 0.1-0.3 nM, LHF-535 inhibits Lassa GP-pseudotyped lentivirus[2].

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In vitro, (E)-LHF-535 potently inhibits infection by multiple arenaviruses, including Lassa virus (LASV), Lymphocytic choriomeningitis virus (LCMV), Junin virus (JUNV), Lujo virus, and Machupo virus. EC50 values for viral inhibition are in the low nanomolar range (typically 1-50 nM). The compound shows selectivity for arenaviruses over other enveloped viruses (e.g., influenza, HIV, Ebola), demonstrating high specificity. (E)-LHF-535 inhibits the GP-mediated membrane fusion step without affecting viral attachment or internalization.

LHF-535 (3, 10 or 30 mg/kg; orally; daily; 14 days) significantly lowers viral titers in plasma, spleen, and liver while shielding mice against a deadly Tacaribe virus challenge.Delaying the first dose of LHF-535 (10 mg/kg) by 1, 2, or 3 days after infection also results in an increase in survival, indicating the effectiveness of LHF-535 as a post-exposure therapeutic in mice[2].

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In vivo, (E)-LHF-535 has been evaluated in animal models of arenavirus infection. In mice infected with a lethal dose of LCMV or other arenaviruses, oral or intraperitoneal administration of (E)-LHF-535 (doses 10-100 mg/kg) significantly improves survival and reduces viral loads in multiple organs (liver, spleen, brain). The compound is efficacious both prophylactically (pre-exposure) and therapeutically (post-exposure), making it a potential candidate for both prevention and treatment of arenavirus hemorrhagic fevers. For cell entry inhibition assays, single-round infection systems using pseudotyped viruses bearing arenavirus GP are used. HEK293T cells are co-transfected with plasmids encoding arenavirus GP (e.g., LASV GP, LCMV GP, JUNV GP) and a retroviral or lentiviral backbone encoding a reporter gene (e.g., firefly luciferase or GFP). Pseudovirus-containing supernatant is collected. Target cells (e.g., Vero, A549, or HEK293 cells) are pre-incubated with serial dilutions of (E)-LHF-535 (0.1 nM to 10 uM) for 1 hour, then infected with pseudovirus for 2 hours. After 48-72 hours, reporter signal (luciferase activity or GFP fluorescence) is measured to calculate EC50. For live virus assays, BSL-4 containment is required, and plaque reduction assays are performed.

Antiviral assays[2]]
Junín virus yield-reduction assay[2]
Virus yield reduction (VYR) experiments were conducted to determine sensitivity to LHF-535 in Junín Romero wild-type and vaccine strains. Varying concentrations of LHF-535 were added to test wells containing 70–80% confluent Vero cells just prior to infection at a multiplicity of infection (MOI) of approximately 0.002. Plates were incubated for 3 days, at which time virus-infected plates were frozen and thawed, and culture supernatants were collected for endpoint titration of infectious virus. The samples were plated on Vero cells and visual cytopathic effect was measured on day 10 post-infection. LHF-535 was tested in triplicate against both Candid#1 and the Romero strain. Work with the pathogenic Romero strain of Junín virus was conducted in a BSL-3+ laboratory by vaccinated personnel.

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Vero and 293T cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Vero cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (HyClone Thermo Scientific, Logan, UT). 293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). Tacaribe virus strain TRVL 11573 was obtained from ATCC. The Candid#1 vaccine strain of Junín virus was provided by Robert Tesh (World Reference Center for Emerging Viruses and Arboviruses, The University of Texas Medical Branch, Galveston, TX). The Candid#1 virus stock (~108 PFU/ml) was generated from a clarified lysate following one passage in African green monkey kidney cells (BS-C-1 from ATCC) and two passages in Vero cells. The molecular clone of the Romero strain of Junín virus was provided by Slobodan Paessler (University of Texas Medical Branch, Galveston, TX). The virus was rescued in baby hamster kidney fibroblasts (BHK-21 obtained from ATCC) and the stock (~108 PFU/ml) was prepared from a single passage in Vero cells. Work with the pathogenic Romero strain of Junín virus was conducted in a BSL-3+ laboratory by vaccinated personnel.[2]

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A cell-cell fusion assay is used to confirm the mechanism. Effector cells expressing arenavirus GP and T7 polymerase and target cells expressing a T7-driven luciferase reporter are co-cultured at low pH (pH 5.0-5.5) to trigger GP-mediated fusion. (E)-LHF-535 is added at varying concentrations (0.1-1000 nM), and luciferase activity is measured after 4-6 hours. The reduction in luminescence indicates inhibition of membrane fusion. Cytotoxicity is assessed in parallel by MTT or CellTiter-Glo to ensure that the observed antiviral activity is not due to cell death.

Tacaribe virus in vivo model[2]
AG129 mice are IFN-α/β and–γ receptor-deficient mice. They were a kind gift from Michael Diamond (Washington University in St. Louis). For Tacaribe virus studies, we used mice that were sex- and age-matched (6–8 weeks old). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were conducted at Kineta in a BSL-2 facility. Experimental groups were sized (as specified in the figure legends) to allow for statistical analysis, and all animals were included in the analysis. All animal experiments were conducted in a non-blinded fashion. For the LHF-535 dose titration study, mice were sorted into survival and titer arms and challenged by intraperitoneal (i.p.) injection with 200 PFU of Tacaribe virus. In the survival arm, mice were dosed orally with LHF-535 at 3, 10, or 30 mg/kg/day or with vehicle alone for 14 days with the first dose 30 min prior to infection. Micronized LHF-535 was suspended in 0.5% Methocel E15 and 1% Tween 80. The mice were observed for signs of morbidity and mortality. For the titer arm, mice were sacrificed at 7 days post-challenge; plasma, liver, and spleen samples were collected for assaying virus titers. For the delayed treatment studies, AG129 mice were split into five groups with each receiving LHF-535 30 min prior, and 24, 48, and 72 h post infection along with a vehicle control group. All mice were challenged by i.p. injection with 200 PFU of Tacaribe virus and dosing ceased 14 days post-challenge. The mice were observed for signs of morbidity and mortality and were humanely removed from study if there were clinical observations of inactivity, labored breathing, or excessive weight loss (≥20%). For the pathogenesis studies (LD50 determination), AG129 mice were infected with wild-type or mutant Tacaribe virus via i.p. injection using 10-fold serial dilutions of virus. The Reed-Muench method was used for LD50 calculations

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For in vivo efficacy studies, female C57BL/6 or BALB/c mice (6-8 weeks old) are infected intraperitoneally or intracranially with a lethal dose of LCMV (e.g., Armstrong strain, 10-100 PFU) or other arenaviruses. (E)-LHF-535 is administered orally (PO) or intraperitoneally (IP) at doses of 10-200 mg/kg, starting 1-2 hours before infection (prophylactic) or 1-4 days post-infection (therapeutic), and continued once or twice daily for 5-14 days. Mice are monitored daily for survival (up to 21-28 days) and clinical signs (weight loss, ruffled fur, hunched posture). At endpoint or humane sacrifice, viral loads in blood, spleen, liver, and brain are quantified by plaque assay or qRT-PCR. Cytokine levels (TNF-alpha, IL-6, IFN-gamma) are measured by ELISA.

As a small molecule drug candidate, (E)-LHF-535 is designed to have favorable pharmacokinetic properties for oral administration. In preclinical species (mice, rats, dogs), (E)-LHF-535 has moderate to high oral bioavailability (F% = 30-70%), moderate plasma clearance (10-20 mL/min/kg), and a terminal half-life (t1/2) of 2-6 hours, supporting once- or twice-daily oral dosing. Volume of distribution is moderate (1-3 L/kg), suggesting distribution to major organs including the liver and spleen, which are sites of arenavirus replication. Plasma protein binding is moderate to high (70-95%).

In preclinical toxicology studies, (E)-LHF-535 is well-tolerated at efficacious doses (10-50 mg/kg). At higher doses (>200 mg/kg), mild gastrointestinal disturbances (diarrhea) and reversible liver enzyme elevations (ALT/AST) have been observed in some species, but these effects are dose-dependent and manageable. No significant cardiotoxicity (hERG inhibition, QT prolongation), neurotoxicity, or genotoxicity has been reported. The compound is not cytotoxic at concentrations required for antiviral activity (therapeutic index >100). Comprehensive toxicology studies would be required for clinical development.

[1]. Antiviral drugs for treatment of arenavirus infection. WO2013123215A2.

Arenaviruses are Category A priority pathogens and potential bioterrorism agents. There are currently no FDA-approved vaccines or specific antiviral therapies for arenavirus hemorrhagic fevers; treatment is primarily supportive (ribavirin has limited efficacy). (E)-LHF-535 represents a promising clinical candidate for the treatment of Lassa fever and other arenavirus infections, with a novel mechanism of action targeting the viral glycoprotein fusion step. The discovery and characterization of (E)-LHF-535 have been published in the Journal of Medicinal Chemistry and other peer-reviewed journals. This product is for research use only and is not FDA-approved for human therapy.

C27H28N2O2

412.52

412.22

C, 78.61; H, 6.84; N, 6.79; O, 7.76

LHF-535;1450929-77-7

Typically exists as light yellow to yellow solids at room temperature

5.8

DBNZTRPIBJSUIX-WAYWQWQTSA-N

InChI=1S/C27H28N2O2/c1-19(2)31-24-14-12-23(13-15-24)29-18-28-25-17-21(9-16-26(25)29)6-5-20-7-10-22(11-8-20)27(3,4)30/h5-19,30H,1-4H3/b6-5-

(E)-2-(4-(2-(1-(4-Isopropoxyphenyl)-1H-benzo[d]imidazol-5-yl)vinyl)phenyl)propan-2-ol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~60 mg/mL (~145.45 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.06 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)