Fig. 1
The inhibitory activity and mechanism of ginkgolic acid (GA) and anacardic acid (AA) against SARS-CoV-2 3CLpro. a, b Dose-dependent inhibition of SARS-CoV-2 3CLpro by GA (a) and AA (b) using sandwich-like fluorescence polarization (FP) assay. As described previously [5], the mixture of SARS-CoV-2 3CLpro (0.4 μM) and GA or AA with concentrations ranging from 2.5 to 80 μM was preincubated for 35 min at room temperature (RT), then 40 nM FP tracer (FITC-AVLQSGFRKK-Biotin) was added into the mixture to initiate proteolytic reaction. After addition of avidin, the millipolarization unit (mP) value was measured to calculate the IC50 using GraphPad Prism 8.0. Three independent experiments were performed. c, d IC50 plots from in vitro fluorescence resonance energy transfer (FRET)-based enzymatic assay against SARS-CoV-2 3CLpro of GA (c) and AA (d). SARS-CoV-2 3CLpro was incubated in the reaction buffer with various concentrations of GA or AA at RT for 30 min. Then the enzymatic reaction was initiated by adding MCA-AVLQSGFRLys(Dnp)-Lys-NH2 as the fluorescently labeled substrate. After the RFU value monitored by a microplate reader (BioTek), the efficacy of two protease inhibitors was evaluated in GraphPad Prism 8.0. The results are average ± SD of three repeats. e–g Binding mode analysis between PF-07321332 (e), GA (f) or AA (g) and SARS-CoV-2 3CLpro using HPLC-Q-TOF MS. According to the published protocol [10], purified SARS-CoV-2 3CLpro (5 μM) was incubated with or without PF-07321332, GA or AA (500 μM) in TBS (10 mM Tris, 50 mM NaCl pH 8.0) at RT for 30 min. The desalted samples were analyzed by the quadrupole time-of-flight (Q-TOF) mass spectrum (Agilent, USA) for detecting the molecular weight of intact 3CLpro. Mass spectrum were deconvoluted using Mass Hunter software (Agilent), and maximum entropy was performed for deconvolute algorithm. h, i Evaluation of binding activity of GA (h) and AA (i) to SARS-CoV-2 3CLpro using SPR. The affinity of GA and AA (25, 50, 125, 250, and 500 μM) to SARS-CoV-2 3CLpro was examined separately by real-time SPR spectroscopy, with 20 μL SARS-CoV-2 3CLpro (1.6 mg/mL) in 10 mM NaAc buffer (pH5.5) immobilized on the flow cell of the sensor chip CM5. The kinetics parameters (ka, kd and KD) were calculated using the analyte binding kinetic curve. j, l The Lineweaver–Burk plots for analysis the inhibition mechanisms of GA (j) and AA (l) against 3CLpro using the FRET assay. k, m The secondary plots for the inhibitory constant (Ki) values of GA (k) and AA (m) in the FRET substrate