Antiviral; Arenavirus envelope glycoprotein

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].

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].

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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Tacaribe virus antiviral assay [2]
Vero cells seeded in 96-well plates (5,000 cells per well) were infected with Tacaribe virus at an MOI of 0.1 following addition in triplicate of serial compound dilutions in DMSO. After 3 days, RNA was extracted from cell lysates (Promega SV 96 Total RNA Isolation System) for evaluation of Tacaribe virus RNA via qRT-PCR and the comparative CT method [40]. Briefly, extracted RNA is used to generate cDNA with the High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific). For TaqMan based qPCR, reactions were performed with the cDNA using the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) along with primers and a dual-labeled TaqMan probe set targeting a ~100 nucleotide region of GP (nt 809–912). 18S rRNA (VIC/MGB probe) from Thermo Fisher Scientific was used as internal control.

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Pseudotype virus inhibition [2]
293T cells were seeded in opaque 384-well plates (4,000 cells per well). The following day, LHF-535 (dissolved in DMSO) and DMSO alone were dispensed via an HP D300e Digital Dispenser to a final concentration of 0.2% DMSO in all wells. This was followed by addition of a fixed volume of lentiviral pseudovirions, 3 days incubation at 37°C, and measurement of luciferase activity (Promega Bright-Glo Luciferase Assay System). Test concentrations were performed in quadruplicate. Luminescence was averaged for each concentration or control (positive controls received DMSO alone, negative controls were mock-infected) and the 50% effective concentration was calculated using XLfit. Experiments were repeated multiple times to establish an average (geometric mean) EC50; experiments were repeated until the standard error of the mean across multiple experiments was less than one-quarter of the average.

Cells and viruses
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]

Animal Model: IFN-α/β and-γ receptor-deficient AG129 mice[2]
Dosage: 3, 10, or 30 mg/kg/day
Administration: Orally; daily; 14 days
Result: Effective as a post-exposure therapeutic.

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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

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

[2]. A potent Lassa virus antiviral targets an arenavirus virulence determinant. PLoS Pathog. 2018 Dec 21;14(12):e1007439.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses.[2]

C27H28N2O2

412.523427009583

412.22

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

1450929-77-7

(E)-LHF-535

71711529

Light yellow to yellow solid powder

5.8

1

3

6

31

588

0

CC(C)OC1=CC=C(C=C1)N2C=NC3=C2C=CC(=C3)/C=C\C4=CC=C(C=C4)C(C)(C)O

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-

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

LHF535; LHF 535; LHF-535

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 : ~150 mg/mL (~363.62 mM)

Solubility in Formulation 1: ≥ 2.42 mg/mL (5.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 24.2 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.42 mg/mL (5.87 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 24.2 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.)