A potent Lassa virus antiviral targets an arenavirus virulence determinant.

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.

Anti-Ebola Activity of Diazachrysene Small Molecules.

Herein we report on a diazachrysene class of small molecules that exhibit potent antiviral activity against the Ebola (EBOV) virus. The antiviral compounds are easily synthesized, and the most active compounds have excellent in vitro activity (0.34-0.70 µM) and are significantly less lipophilic than their predecessors. The three most potent diazachrysene antivirals do not exhibit any toxicity in vivo and protected 70-90% of the mice at 10 mg/kg following EBOV challenge. Together, these studies suggest that diazachrysenes are a promising class of compounds for hit to lead optimization and as potential Ebola therapeutics.

Lead optimization of an acylhydrazone scaffold possessing antiviral activity against Lassa virus.

Previously we reported the optimization of antiviral scaffolds containing benzimidazole and related heterocycles possessing activity against a variety of arenaviruses. These series of compounds were discovered through an HTS campaign of a 400,000 small molecule library using lentivirus-based pseudotypes incorporated with the Lassa virus envelope glycoprotein (LASV GP). This screening also uncovered an alternate series of very potent arenavirus inhibitors based upon an acylhydrazone scaffold. Subsequent SAR analysis of this chemical series involved various substitutions throughout the chemical framework along with assessment of the preferred stereochemistry. These studies led to an optimized analog (ST-161) possessing subnanomolar activity against LASV and submicromolar activity against a number of other viruses in the Arenaviridae family.

Development of monoclonal antibody-based assays for the detection of Vibrio tubiashii zinc-metalloprotease (VtpA).

Vibrio tubiashii has been linked to disease outbreaks in molluscan species, including oysters, geoducks, and clams. In particular, oyster hatcheries in the Pacific Northwest have been plagued by intermittent vibriosis since 2006. Accurate detection of vibrios, including V. tubiashii, is critical to the hatcheries in order to allow for rapid remediation efforts. The current methods for detection of Vibrio spp. are not ideal for use at the hatchery. Plating samples require time and is not sensitive to extracelluar pathogenic products, such as the secreted zinc-metalloprotease, VtpA. Other sensitive methods to detect bacteria, such as qPCR, require a high level of laboratory skills and expensive supplies that are prohibitive for use at hatchery sites. Thus, hatcheries would benefit from a sensitive, simple method to detect V. tubiashii and its secreted toxin. Here, we describe the development of two inexpensive and highly specific tests for the shellfish-toxic zinc-metalloprotease secreted by V. tubiashii: enzyme-linked immunoassays (ELISA) and a lateral flow immunoassay (dipstick assay). Both technologies rely on a set of monoclonal antibodies used in a sandwich format, with the capture antibody recognizing a different epitope than the detection antibody on the mature VtpA protein. Both assays are quantitative and give colorimetric readouts. The sandwich ELISA was sensitive when VtpA was diluted into PBS, but was markedly less sensitive in conditions that correlate with the environment of hatchery-derived samples, such as in the presence of seawater, algae, or oyster larvae. In contrast, the dipstick assay remained very sensitive in the presence of these contaminants, is less work-intensive, and much more rapid, making this format the preferred assay method for detecting VtpA on site in a hatchery or environmental setting.

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole.

A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-throughput screening resulted in discovery of a hit compound (ST-37) possessing a benzimidazole core which led to a potent compound series. Herein, we report SAR studies which involved structural modifications to the phenyl rings and methylamino linker portion attached to the benzimidazole core. Many analogs in this study possessed single digit nanomolar potency against LASV pseudotypes. Compounds in this benzimidazole series also exhibited nanomolar antiviral activity against pseudotypes generated from other arenavirus envelopes indicating the potential for development of a broad-spectrum inhibitor. Ultimately, lead compound ST-193 was identified and later found to be efficacious in a lethal LASV guinea pig model showing superior protection compared to ribavirin treatment.

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole and related heterocycles.

A series of potent arenavirus inhibitors sharing a benzimidazole core were previously reported by our group. SAR studies were expanded beyond the previous analysis, which involved the attached phenyl rings and methylamino linker portion, to include modifications focused on the benzimidazole core. These changes included the introduction of various substituents to the bicyclic benzimidazole ring system along with alternate core heterocycles. Many of the analogs containing alternate nitrogen-based bicyclic ring systems were found to retain antiviral potency compared to the benzimidazole series from which we derived our lead compound, ST-193. In fact, 21 h, built on an imidazopyridine core, possessed a near tenfold increase in potency against Lassa virus pseudotypes compared to ST-193. As found with the benzimidazole series, broad-spectrum arenavirus activity was also observed for a number of the analogs discovered during this study.

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity.

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 µM) and La Crosse virus (EC(50)=0.27 µM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).

Virulence of metalloproteases produced by Vibrio species on Pacific oyster Crassostrea gigas larvae.

Vibrio tubiashii, a pathogen of shellfish larvae and juveniles, produces several extracellular products. Here, we document that culture supernatants of several marine Vibrio species showed toxicity to oyster larvae. Treatment of these supernatants with EDTA not only severely diminished proteolytic activities, but also dramatically reduced toxicity to the larvae. Culture supernatants of metalloprotease-deficient mutants of V. tubiashii, V. cholerae, and V. splendidus were impaired in their ability to cause larval death compared to the wild type strains. Culture supernatants of Pseudomonas aeruginosa, known to contain several secreted proteases, showed virtually no toxicity to oyster larvae. Purified V. tubiashii protease A (VtpA), but not the prototype metalloprotease, thermolysin from Bacillus thermoproteolyticus, was highly toxic to the larvae. In addition, toxicity of purified VtpA was much greater for 6-d-old oyster larvae than for 16-d-old larvae. Together, these results indicated that culture supernatants of a variety of Vibrio species are highly toxic to oyster larvae and that the production of a metalloprotease is required for this effect. We propose that there are, as yet uncharacterized, specific substrates contained in larval tissue that are degraded by VtpA as well as certain homologous metalloproteases produced by other marine Vibrio species which, in turn, may contribute to vibriosis.

Development of a quantitative real-time PCR assay for detection of Vibrio tubiashii targeting the metalloprotease gene.

Vibrio tubiashii has recently re-emerged as a pathogen of bivalve larvae, causing a marked increase in the mortality of these species within shellfish rearing facilities. This has resulted in substantial losses of seed production and thus created the need for specific as well as sensitive detection methods for this pathogen. In this project, quantitative PCR (qPCR) primers were developed and optimized based upon analysis of the V. tubiashii vtpA gene sequence, encoding a metalloprotease known to cause larval mortality. Standard curves were developed utilizing dilutions of known quantities of V. tubiashii cells that were compared to colony forming unit (CFU) plate counts. The assay was optimized for detection of vtpA with both lab-grown V. tubiashii samples and filter-captured environmental seawater samples seeded with V. tubiashii. In addition, the primers were confirmed to specifically detect only V. tubiashii when tested against a variety of non-target Vibrio species. Validation of the assay was completed by analyzing samples obtained from a shellfish hatchery. The development of this rapid and sensitive assay for quantitative detection of V. tubiashii will accurately determine levels of this bacterium in a variety of seawater samples, providing a useful tool for oyster hatcheries and a method to assess the presence of this bacterium in the current turbulent ocean environment.