Figure 6. Specific inhibition of TMP1 against coronavirus M^pro and its antiviral efficacy against nirmatrelvir-resistant SARS-CoV-2 escape mutant.

(a) Crystal structure of TMP1 in complex with SARS-CoV-2 Omicron M^pro. Left panel, The co-crystal structure (PDB: 9IZB) of TMP1 (orange) in complex with SARS-CoV-2 Omicron M^pro (grey). The H41 (blue) and C145 (yellow) catalytic dyad was shown. The S1′, S1, and S2 pockets of M^pro are labelled in red. The Fo-Fc electron density map of TMP1 is shown in gray mesh (σ = 2.5). Right panel, close-up view of TMP1 with the substrate binding pocket of M^pro. The residues of M^pro involved in TMP1 binding were displayed by sticks. The hydrogen bonds were displayed as red dashed lines. The covalent-bond between Cys145 and TMP1 warhead was indicated by a black arrow.

(b) Superimposition of the TMP1 in complex with M^pro from 9 coronaviruses including SARS-CoV-2 (Omicron, PDB: 9IZB), HCoV-229E (PDB: 2ZU2), -NL63 (7E6M), -OC43, -HKU1, SARS-CoV-1 (PDB: 1WOF), MERS-CoV (PDB: 4RSP), RaTG13 and GX/P3B.

(c) Enzymatic activity of recombinant SARS-CoV-2 M^pro with TMP1 treatment. Enzymatic activity of the recombinant SARS-CoV-2 M^pro was measured by fluorescence resonance energy transfer (FRET) assays (n=4). Fluorescence signals were normalized to the readouts of mock-treated wells.

(d) Quantification of the sgE gene in VeroE6 cells (n=6) infected with wildtype SARS-CoV-2 and Alpha, Beta, Delta, Omicron (BA.1 and XBB1.5) variants, followed by treatment with TMP1 or vehicle only at 1 hpi.. Lysates were harvested at 24 hpi. for one-step reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis.

(e) Quantification of the N gene of SARS-CoV-1 and MERS-CoV in VeroE6 cells (n=6) infected with SARS-CoV-1 or MERS-CoV, followed by treatment with TMP1 or vehicle only at 1 hpi.. Lysates were harvested at 24 hpi. for one-step reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis.

(f) Sensitivity of recombinant SARS-CoV-2 M^pro mutants to TMP1 treatment. Inhibition of TMP1 against the recombinant SARS-CoV-2 M^pro mutants carrying reported nirmatrelvir-resistant mutations was measured by fluorescence resonance energy transfer (FRET) enzymatic assays (n=3). Fold change in the IC₅₀ was obtained by comparing with that of the wildtype M^pro.

(g) Schematic illustration of characterizing the in vitro and in vivo antiviral efficacy of TMP1 against nirmatrelvir-resistant recombinant SARS-CoV-2. Recombinant SARS-CoV-2 was constructed with NSP5-E166V mutation in the background of ancestral SARS-CoV-2 with D614G mutation in the spike (rSARS-CoV-2-NSP5-E166V). For in vitro infection, Calu3 cells were pretreated with TMP1 for 1 hour followed by infection with rSARS-CoV-2-NSP5-E166V (n=4). Lysates were harvested at 24 hpi. for RNA extraction. For in vivo infection, 8- to 12-week-old K18-hACE2 transgenic mice were challenged with 5000 PFU rSARS-CoV-2-NSP5-E166V. One day prior to infection, mice were orally treated with 100 mg/kg/dose TMP1 in combination with 20 mg/kg/dose RTV (n=4). Control mice were treated with vehicle only (n=4). Mice were treated twice per day until sample harvest at 3 dpi.

(h) Quantification of the sgE gene in Calu3 cells (n=6) infected with rSARS-CoV-2-NSP5-E166V, followed by treatment with TMP1 or vehicle only at 1 hpi.. Lysates were harvested at 24 hpi. for one-step reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis.

(i) Quantification of SARS-CoV-2 sgE gene in the nasal turbinate and lung tissues of the rSARS-CoV-2-NSP5-E166V infected mice at 3 dpi by RT-qPCR analysis.

(j) Quantification of the infectious viral titres in the nasal turbinate and lung tissues of the rSARS-CoV-2-NSP5-E166V infected mice at 3 dpi by plaque assays.

Each data point represents one biological repeat. Data represents mean ± SD from the indicated number of biological repeats. Statistical significances were determined using one way-ANOVA with Dunnett’s multiple comparisons test (i-j) and two-tailed Student’s t-test (f). Data were obtained from three independent experiments. * represented p < 0.05, ** represented p < 0.01, *** represented p < 0.001, ns, not statistically significant. Veh, vehicle; NRV, nirmatrelvir; PAX, Paxlovid.