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 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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Using lentiviral pseudotypes, LHF-535 potently inhibited viral entry mediated by envelope glycoproteins (GPs) from a broad array of hemorrhagic fever arenaviruses. For most Lassa virus strains (lineages II, III, IV), the 50% inhibitory concentration (IC50) ranged from 0.10 nM to 0.33 nM. The lineage I LP strain was an exception, with an IC50 of 17 nM [1].
Other Old World arenaviruses showed variable sensitivity: Mobala (IC50 = 0.96 nM), Mopeia (0.81 nM), Gbagroube (0.29 nM), Menekre (120 nM). Lymphocytic choriomeningitis virus (LCMV) and Lujo virus GPs were not specifically sensitive, with IC50s in the micromolar range (3.9-6.8 μM), similar to the vesicular stomatitis virus (VSV) GP control [1].
New World (clade B) hemorrhagic fever arenaviruses were highly sensitive to LHF-535: Junín (0.10 nM), Machupo (Carvalio: 0.13 nM; Mallele: 0.093 nM), Tacaribe (0.13 nM), Guanarito (IC50 not determined), Sabiá (2.0 nM), Chapare (19 nM). Clade A viruses Flexal and Pichinde were less sensitive (160 nM and 2.7 μM, respectively) [1].
The reduced sensitivity of the Lassa virus LP strain was mapped to a V434I substitution in the transmembrane domain of GP2. Engineering the reversion I434V into the LP GP restored high sensitivity (IC50 = 0.12 nM). Conversely, introducing V435I (equivalent to V434I) into the highly sensitive Josiah (lineage IV) GP reduced sensitivity to LHF-535 (IC50 = 25 nM) [1].
The Junín virus live-attenuated vaccine strain Candid#1, which contains an F427I attenuation determinant in GP2 (analogous to Lassa V434), was highly resistant to LHF-535 in pseudotype assays (IC50 > 20 μM) compared to the virulent Junín virus (IC50 = 0.10 nM). Introducing F427I into the Junín GP also eliminated sensitivity [1].
In virus yield reduction assays using replication-competent viruses, the Junín (Romero) strain was highly sensitive to LHF-535 (IC90 = 9.3 ± 7.1 nM), while the Candid#1 vaccine strain was significantly less sensitive (IC90 = 3.0 ± 1.5 μM), a difference of several hundred-fold. Both strains showed similar sensitivity to ribavirin [1].
Serial passage of Tacaribe virus in Vero cells under increasing LHF-535 pressure selected for variants with reduced drug sensitivity. These variants contained amino acid substitutions in the GP (e.g., F425L, T434I, F436L/I), and most exhibited IC50 values increased by >100 to >5000-fold compared to wild-type [1].

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 a lethal Tacaribe virus challenge model using AG129 mice, daily oral administration of LHF-535 at 10 or 30 mg/kg/day, starting 0.5 hours before infection, provided 100% protection from mortality, while the 3 mg/kg/day group showed partial protection. The vehicle control group had 0% survival [1].
Treatment with LHF-535 (10 or 30 mg/kg/day) starting 0.5 hours pre-challenge dramatically reduced viral titers in plasma, spleen, and liver measured at 7 days post-infection compared to vehicle-treated mice [1].
LHF-535 was effective as a post-exposure therapeutic. When the initiation of daily oral dosing (10 mg/kg/day) was delayed until 24, 48, or 72 hours after Tacaribe virus challenge, it still significantly increased survival rates compared to the vehicle control group [1].
Most LHF-535-resistant Tacaribe virus variants selected in vitro (e.g., F425L, T434I, F436L) were highly attenuated in vivo. The median lethal dose (LD50) for these variants in AG129 mice was >2000 plaque-forming units (PFU), compared to <2 PFU for the wild-type parental virus [1].
The attenuated, drug-resistant variant Tacaribe F425L functioned as a vaccine. Mice that survived a prior infection with F425L were fully protected against a subsequent lethal challenge with the virulent wild-type Tacaribe virus [1].

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]

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Lentiviral Pseudotype Inhibition Assay: Genes for heterologous viral envelope glycoproteins (GPs) were cloned into a mammalian expression vector. Pseudotyped lentivirions were produced by co-transfecting 293T cells with this GP plasmid, a lentiviral backbone plasmid carrying a luciferase reporter gene, and packaging plasmids. The produced pseudovirions were used to infect 293T target cells seeded in opaque 384-well plates. LHF-535 or control DMSO was dispensed into the wells prior to the addition of a fixed volume of pseudovirions. After 3 days of incubation, luciferase activity was measured using a commercial luciferase assay system. Luminescence was averaged for each drug concentration, and the 50% effective concentration (EC50) was calculated. Each test concentration was performed in quadruplicate [1].
Virus Yield Reduction Assay (Junín Virus): Vero cells at 70-80% confluency were infected with Junín virus (Romero or Candid#1 strain) at a low multiplicity of infection (MOI ~0.002). Varying concentrations of LHF-535 were added to the wells just prior to infection. Plates were incubated for 3 days, after which the cultures were frozen and thawed. The supernatants were collected and subjected to endpoint titration on fresh Vero cells to determine the amount of infectious virus produced. Viral cytopathic effect was visually scored on day 10 post-infection. The concentration of compound required to reduce virus yield by 90% (IC90) was determined. Assays were performed in triplicate [1].
Tacaribe Virus Antiviral Assay (qRT-PCR): Vero cells seeded in 96-well plates were infected with Tacaribe virus at an MOI of 0.1 following the addition of serial dilutions of LHF-535 in DMSO (in triplicate). After 3 days of incubation, total RNA was extracted from cell lysates. The extracted RNA was reverse transcribed into cDNA. Viral RNA levels were quantified by TaqMan-based quantitative real-time PCR (qRT-PCR) using primers and a probe targeting a region of the viral GP gene. An 18S rRNA assay was used as an internal control for normalization. Data were analyzed using the comparative Ct method [1].
Selection of Drug-Resistant Variants: Tacaribe virus was serially passaged in Vero cells under increasing selective pressure of LHF-535 (from 1x to 170x IC50). Virus was harvested 3-6 days post-infection and passaged again at a 5-fold higher drug concentration. After several rounds, virus from the final passage was plaque-purified, amplified, and evaluated for drug sensitivity. The GP genes of variants with altered sensitivity were sequenced [1].

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

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Tacaribe Virus Efficacy Study (AG129 Mice): Age- and sex-matched AG129 mice (6-8 weeks old) were challenged intraperitoneally (i.p.) with 200 PFU of Tacaribe virus. For the dose-response study, mice received daily oral doses of micronized LHF-535 suspended in a vehicle of 0.5% Methocel E15 and 1% Tween 80. Dosing at 3, 10, or 30 mg/kg/day (or vehicle alone) began 0.5 hours before challenge and continued for 14 days. Mice were monitored for survival for 21 days. In a parallel cohort, mice were sacrificed at 7 days post-challenge to collect plasma, liver, and spleen for viral titer determination by plaque assay [1].
Post-Exposure Treatment Study (AG129 Mice): Mice were challenged i.p. with 200 PFU of Tacaribe virus. Groups then began receiving daily oral doses of LHF-535 (10 mg/kg, same vehicle) starting at 0.5 hours before, or 24, 48, or 72 hours after infection. Dosing continued for 14 days post-challenge, and survival was monitored [1].
Pathogenicity Study (LD50 Determination): AG129 mice were infected i.p. with 10-fold serial dilutions of wild-type or mutant (drug-resistant) Tacaribe virus. Mice were observed for morbidity and mortality, and the LD50 was calculated using the Reed-Muench method [1].
Vaccination/Challenge Study: Mice were infected i.p. with 2000 PFU of the attenuated LHF-535-resistant variant Tacaribe F425L. Thirty-one days later, these mice and a group of age-matched naïve controls were challenged i.p. with 200 PFU of virulent wild-type Tacaribe virus. Survival was monitored [1].

[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 major pathogen of hemorrhagic fever, a often fatal disease for which there are currently no approved antiviral therapies. Lassa fever, in particular, causes high morbidity and mortality rates in West Africa, a region that is endemic for the disease. Recent outbreaks in Nigeria have been larger in scale and more geographically widespread. We are developing LHF-535, a small-molecule viral invasion inhibitor targeting arenavirus envelope glycoproteins, as a candidate drug for the treatment of Lassa fever and other hemorrhagic fevers caused by arenaviruses. Using lentiviral pseudotype infection assays, we found that LHF-535 exhibits sub-nanomolar inhibitory activity against viral envelope glycoproteins of all Lassa virus lineages, except for the glycoprotein of lineage I LP strains, which showed 100-fold lower sensitivity than other strains. This reduced sensitivity is mediated by a unique amino acid substitution V434I in the transmembrane domain of the GP2 subunit of the envelope glycoprotein. This position corresponds to the attenuated determinant of Candid#1, a live attenuated Juninvirus vaccine strain used to prevent Argentine hemorrhagic fever. We used a viral yield reduction assay to determine that LHF-535 effectively inhibits Junin virus, but has no inhibitory effect on Candid#1, and that the attenuation determinant F427I of Candid#1 regulates this difference in sensitivity. We also demonstrated that oral administration of 10 mg/kg of LHF-535 daily protected mice from lethal doses of tacarib virus infection. Continuous passage of tacarib virus in LHF-535-treated Vero cells yielded LHF-535-resistant viruses, and most of the resistant viruses showed reduced virulence. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum isopyrvirus invasion inhibitor and provide important background for monitoring the emergence of drug-resistant viruses. [2]

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LHF-535 is an optimized analogue of the benzimidazole derivative ST-193, which was originally developed for the treatment and prevention of Lassa fever and other isopyrvirus hemorrhagic fevers (e.g., Argentine hemorrhagic fever, Bolivian hemorrhagic fever) [1].
Its mechanism of action is thought to bind to and stabilize the pre-fusion conformation of the arenavirus glycoprotein complex, thereby inhibiting the pH-dependent conformational rearrangement required for viral membrane fusion and invasion[1].
A key finding is that the viral determinants of LHF-535 sensitivity overlap with known virulence determinants. Amino acid substitutions in the GP2 transmembrane domain (e.g., F425L in Tacarib, similar to F427I in Junín Candid#1) confer viral resistance and typically result in a significant reduction in viral virulence in vivo[1].
This suggests that the emergence of resistant viruses during LHF-535 treatment may be related to reduced viral pathogenicity. This also implies minimal interference between the drug and the live attenuated vaccine Candid#1[1].
At the time of this publication, human clinical development of LHF-535 is underway, with a Phase Ia trial evaluating its safety and pharmacokinetics[1].

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.)