An oral non-covalent non-peptidic inhibitor of SARS-CoV-2 Mpro ameliorates viral replication and pathogenesis in vivo

Abstract

Safe, effective, and low-cost oral antiviral therapies are needed to treat those at high risk for developing severe COVID-19. To that end, we performed a high-throughput screen to identify non-peptidic, non-covalent inhibitors of the SARS-CoV-2 main protease (Mpro), an essential enzyme in viral replication. NZ-804 was developed from a screening hit through iterative rounds of structure-guided medicinal chemistry. NZ-804 potently inhibits SARS-CoV-2 Mpro (0.009 μM IC₅₀) as well as SARS-CoV-2 replication in human lung cell lines (0.008 μM EC₅₀) and primary human airway epithelial cell cultures. Antiviral activity is maintained against distantly related sarbecoviruses and endemic human CoV OC43. In SARS-CoV-2 mouse and hamster disease models, NZ-804 therapy given once or twice daily significantly diminished SARS-CoV-2 replication and pathogenesis. NZ-804 synthesis is low cost and uncomplicated, simplifying global production and access. These data support the exploration of NZ-804 as a therapy for COVID-19 and future emerging sarbecovirus infections.

Keywords: CP: Microbiology; Paxlovid; SARS-CoV-2; antiviral; broad-spectrum; coronavirus; emerging viruses; protease; therapeutics.

Figure 1:. Discovery and development of NZ-804.

(A) Screening hit 1 bound to the active site of SARS-CoV-2 Mpro. Two orientations are built to account for the electron density. 2Fo-Fc difference electron density of the sigma-A weighted refined map contoured at 1 σ displayed as blue mesh. (B) Structure activity relationship (SAR) showing representative molecules of developing initial hit (1) into potent lead (5). (C) The surface view colored by hydrophobicity of the binding pocket for the lead molecule 5. (D) The Mpro inhibitory activities (IC₅₀) of 118 compounds synthesized in the current study are plotted against the antiviral activities on SARS-CoV-2 infected HeLa-hACE2 cells (EC₅₀). The Spearman’s rank correlation coefficient indicates the inhibitors are probably on-target. The binding modes of two conditionally/locally approved Mpro inhibitors: (E) Nirmatrelvir (PDB id: 7RFW) and (F) Ensitrelvir (PDB id: 8HBK). The Mpro subsites are oriented the same way as in (A)/(C) for molecules 1 and 5 in the current study, revealing dramatically different binding modes of the three classes of Mpro inhibitors.

Figure 2:. NZ-804 has potent and broad activity against the CoV family.

(A) Sarbecovirus panel antiviral assays. A549-hACE2 cells were infected at an MOI of 0.08 with SARS-CoV-2 D614G variant, a SARS-like pangolin virus PgCoV, or two SARS-like Bat CoV (WIV1 and SHC014) recombinant nanoluciferase reporter viruses for 1hr after which virus was removed, monolayers washed and a dose response of NZ-804 or nirmatrelvir was added. NZ-804 was tested in duplicate in a single study. Nirmatrelvir was tested in at least quadruplicate in two independent studies. (B) Merbecovirus panel antiviral assays. Huh7 cells were infected at an MOI of 0.08 with MERS-CoV or a MERS-like Bat CoV BtCoV/Ii/GD/2014–422 recombinant nanoluciferase reporter viruses for 1hr after which virus was removed, monolayers washed and a dose response of NZ-804 or nirmatrelvir was added. NZ-804 and Nirmatrelvir were tested in at least triplicate in two independent studies. For A and B, viral replication was assessed 24hr after infection. (C) Endemic human CoV OC43 antiviral assay. Huh7 cells overexpressing IFITM3 were infected at an MOI of 0.03 with recombinant nanoluciferase reporter HCoV-OC43 in the presence of a dose response of NZ-804 or nirmatrelvir in duplicate. Viral replication was assessed 48hr after infection. Data is representative of two independent studies. For A-C, IC₅₀ values were generated from log transformed percent inhibition subjected to non-linear regression and symbols represent the mean. (D) Antiviral activity in primary human airway epithelial (HAE) cells. HAE cells were exposed to a dose response of the indicated drugs in quadruplicate and then infected with SARS-CoV-2 WA1 at an MOI of 0.5 for 1.5hr after which input virus was removed and cultures were washed. After 72hr at 37°C, infectious virus released was collected through apical wash which titered by plaque assay. Data presented is combined from two independent studies using cells from two independent human donors. The boxes represent the 25^th-75^th percentile, the line is at the median and the whiskers represent the range. P values were generated by Kruskall-Wallis test.

Figure 3.. NZ-804 sensitivity following in vitro passage.

(A) Assessing the phenotypic sensitivity of passage-populations to NZ-804 relative to vehicle-passaged controls. The sensitivity of passaged lineages was assessed in antiviral dose response assays in A549-hACE2 cells. Change in genome copies was measured via qRT-PCR over the dose response (Top) and EC₅₀ curves were generated (bottom). (B) Evaluating sensitivity of engineered Mpro Q83P mutation relative to WT infectious clone control. Mutations in Mpro were only observed in one lineage after 10 passages. Q83P, found in lineage 1, was introduced into SARS-CoV-2 WA1 via reverse genetics. Change in WT and Q83P virus genomic RNA measured via qRT-PCR over the dose response (Top) and EC₅₀ (bottom). (C) Direct comparison of NZ-804 sensitivity in lineage 1 passage population and Mpro Q83P engineered mutant relative to infectious clone and vehicle-passaged controls. Sensitivity was determined by measuring change in genome copies via qRT-PCR (Top) and EC₅₀ dose response curves (Bottom). (D) Symbol key provided for graphical reference. (E) Calculated EC₅₀ and EC₉₀ values for all passage-population and infectious clone viruses. Data represents mean ± standard deviation from three to six independent experiments, each with two biological replicates.

Figure 4:. NZ-804 once or twice daily diminishes SARS-CoV-2 replication and pathogenesis in mice.

(A) Mouse lung pharmacokinetics. BALB/c mice were gavaged with 50mg/Kg NZ-804 in 10% EtOH, 30% PEG-400 and 60% Phosal. At the indicated times, concentration of NZ-804 was determined by mass spectrometry (n = 3 mice/time). This experiment was performed only once. (B) Study design. 10-week-old female BALB/c mice were infected with 1×10⁴ PFU SARS-CoV-2 MA10. Therapy was initiated 12hr after infection. Dose groups: NZ-804 vehicle (n = 11), nirmatrelvir vehicle (n = 10), NZ-804 at 200 mg/kg dosed once daily (n = 11), NZ-804 at 200 mg/kg dosed twice daily (n = 11), and nirmatrelvir (Nirm.) at 200 mg/kg dosed twice daily (n = 10). (C) Body weight loss. Percent starting weight over time is shown for the dose groups noted in A. The boxes represent the 25^th-75^th percentile, the line is at the median and the whiskers represent the range. Asterisks indicate statistical significance by Two-way ANOVA Tukey’s multiple comparisons test. (D) Gross lung discoloration (GLD) score. GLD is associated with CoV pathogenesis in mice where the normal pink lung tissue appears a hemorrhaged dark red color and is score on a scale of 1 (normal, pink) to 4 (100% lung tissue is dark red). The line is at the median and each symbol represents the score from a single animal. SARS-CoV-2 MA10 infectious titers were determined at 2dpi (All n = 4/group) (E) and 4dpi (n = 6–7/group) (F) in clarified lung homogenate via plaque assay. The line is at the median and each symbol represents the titer from a single animal. Veh.= Vehicle. Nirm. = Nirmatrelvir. For D, E and F, asterisks indicate statistical significance by Mann-Whitney test. NZ-804 Vehicle and BID NZ-804 therapy conditions were repeated in prior independent studies. (G) SARS-CoV-2 antigen labeling. Lung tissue sections from 2dpi were labeled for SARS-CoV-2 nucleocapsid antigen (brown) and nuclei (blue).

Figure 5:. NZ-804 therapy diminishes SARS-CoV-2 replication in Syrian Hamster.

Three-week old, female Syrian hamsters (Envigo) infected with 1 × 10⁵ PFU of the Omicron subvariant, EG.5.1, by intranasal inoculation. 24h after infection, oral twice daily therapy was initiated vehicle for nirmatrelvir, nirmatrelvir (200mg/kg), vehicle for NZ-804, or NZ804 at either 200 or 400 mg/kg and continued through day 3. n = 4 per group. On day 4, animals were humanely sacrificed, lung tissue was removed, and virus titers were generated by plaque assay. Each symbol represents titer data from single mice, the apex of the bar is at the mean and the error bars represent the standard error of the mean. This study was performed once. Asterisks indicate statistical significance (P < 0.05) by Mann Whitney test (nirmatrelvir) or Kruskall-Wallis test (NZ-804). Limit of Detection (LOD) is 1.2.