Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses

Abstract

Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67-78% identical to Junín virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junín, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

Fig. 1

Chemical structure, formula, and molecular weight of ST-336.

Fig. 2

Effect of the time of addition of ST-336 on Tacaribe virus yield and plaque formation. (A) Vero cells were infected with Tacaribe virus at a MOI = 0.01. ST-336 was added prior to or during Tacaribe infection (−1, 3, 6, 9, 12, 15, 18 or 21 h p.i.). At 24 h p.i. virus yields were determined by plaque assay. (B) Vero cells were infected with 400 pfu Tacaribe virus. ST-336 was added for 1 h before the infection (−1), for 1 h during adsorption (0), and for 1 h after the infection (+1). Infected monolayers were washed with PBS and overlaid with medium containing agarose. Five days post-infection, cells were glutaraldehyde fixed and crystal violet stained prior to plaque counting.

Fig. 3

ST-336 binds with slow Koff to intact Tacaribe virion in absence of cells. (A) Diagram of virus dilution scheme prior to plating. Virus mixed with ST-336 and diluted (left side) or virus diluted and ST-336 added after dilution (right side). (B) Picture of plaques that resulted after plating each dilution shown in A on Vero cells.

Fig. 4

Mapping of ST-336 drug resistant variants (DRV's). (A) Linear map of the glycoprotein precursor (GPC) showing the location of the signal peptide (SP), transmembrane domain (TM), the cleavage site between GP1 and GP2 (K261-A262) and the location of the four ST-336 resistant mutants (DRV#1–4) and the amino acid change for each. (B) Amino acid sequence alignment of GP2 from wild type NWA and ST-336 DRV's. Shown is the amino acid sequence of the C-terminal portion of GP2 (aa397–457) containing the transmembrane domain (marked by vertical lines), the location of the mutations for DR#1–4 (underlined) and the amino acid difference in Amapari (bold).

Fig. 5

Chemical structure, formula, and molecular weight of ST-294.

Fig. 6

Effect of ST-294 in newborn mice challenged with Tacaribe virus. Four day old BALB/c mice were infected IP with 30XLD₅₀ Tacaribe virus and treated daily for 10 days with vehicle (control), ribavirin at 25 mg/kg, ST-294 twice a day (BID) at 50 mg/kg or once a day (SID) at 100 mg/kg. Shown are the percent survivors in each treatment group on days 9 and 10 after infection. Significance (P values) was determined using the Fisher exact test.