Fig 1.
The chemical structures of 3CLpro inhibitors and their antiviral activity against feline coronavirus in cell culture.
The chemical structures of GC376 and NPI64 are shown. The 50% effective concentration (EC50) values of GC376 or NPI64 against FIPV 3CLpro [28, 30] and the 50% cytotoxic concentration (CC50) values of GC376 or NPI64 determined in various cell lines were previously reported [28, 30] and summarized in a table.
Fig 2.
Changes in plasma drug concentrations after administration of 3CLpro inhibitors via a subcutaneous route.
(A) In the single-dose pharmacokinetics study, two healthy specific pathogen free (SPF) cats were subcutaneously injected with GC376 at 10 mg/kg/dose or NPI64 at 5 mg/kg for the determination of serial plasma drug concentrations. GC376 and NPI64 are readily converted into aldehyde forms in the blood [28]. The red triangles and black circles indicate the plasma concentrations of the aldehyde forms of GC376 and NPI64, respectively (means and standard error of the means are shown). (B) In the safety study, four healthy SPF cats were subcutaneously given GC376 at 10 mg/kg/dose daily at 9 AM and 5 PM for 4 weeks. During that time, plasma drug concentrations were measured at 2 and 16 hr post-injection for the first three days and weekly thereafter (red and black triangles, respectively, means are shown). The dotted red line indicates the EC50 value of GC376. The 50% cytotoxic concentration (CC50) value of GC376 (>150 μM) is greater than the dotted blue line.
Fig 3.
Antiviral treatment of symptomatic cats with FIP.
(A) In two independent studies, cats were inoculated with FIPV at day 0 and GC376 treatment was started after they developed lymphopenia and clinical symptoms. In the 2nd study, cats received supportive treatment for five days (shaded boxes), which was discontinued prior to antiviral treatment. The arrows and forward slashes indicate antiviral treatment duration and euthanasia, respectively. dpi, days post infection. (B-D) Responses of cats with FIP to antiviral treatment: body temperature (B), percent body weight changes (C) and lymphocyte counts (D) over time. The shaded areas indicate the normal range of values. Colored arrows located between panels B and D indicate the treatment duration for each cat.
Table 1.
Clinical and laboratory findings in cats challenged with FIPV prior to antiviral treatment.
Fig 4.
Changes in the viral RNA levels in P15 and P16 before and during antiviral treatment.
(A and B) The viral RNA fold changes in the macrophages from the ascites of P15 (A) and P16 (B) over time are shown. The Ct values from viral RNA real-time qRT-PCR were normalized to β-actin and the 2-ΔΔCt method was used to calculate the relative change in viral RNA level, compared to the pre-treatment value. (C) The viral RNA level (2-ΔCt) in the omentum of P15 and P16 which are collected after 4 and 7 days of antiviral treatment, respectively. The bar graph shows the 2-ΔCt values calculated by normalizing the Ct values from viral RNA real-time qRT-PCR to β-actin. (D) The 2-ΔCt values for each viral RNA in the macrophages from the ascites of P15 and P16 at pre-treatment and during treatment are listed in the table. N/A, not available.
Fig 5.
Activity of GC376 against 3CLpro of various coronaviruses in a fluorescence resonance energy transfer (FRET) assay.
The upper graph shows the percent activity of 3CLpro of FIPV, SARS-CoV, and MERS-CoV in the presence of GC376, determined by a FRET assay. The lower table summarizes the 50% inhibitory concentration (IC50) values of GC376 against 3CLpro of FIPV, SARS-CoV, and MERS-CoV. Asterisks indicate the previously published value [28].