Invalidation of geraniin as a potential inhibitor against SARS-CoV-2 main protease

Abstract Recently, geraniin has been identified as a potent antiviral agent targeting SARS-CoV-2 main protease (Mpro). Considering the potential of geraniin in COVID-19 treatment, a stringent validation for its Mpro inhibition is necessary. Herein, we rigorously evaluated the in vitro inhibitory effect of geraniin on Mpro using the fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and dimerization-dependent red fluorescent protein (ddRFP) assays. Our data indicate that geraniin is not a potential inhibitor against Mpro based on the results from a set of in vitro assays. These results suggest a stringent in vitro validation with diverse biochemical assays is essential for the discovery of Mpro inhibitors, and the fluorescence quenching effect caused by natural products should be considered when evaluating Mpro inhibitors. GRAPHICAL ABSTRACT


Introduction
SARS-CoV-2 is highly transmissible and has caused COVID-19 pandemic.However, therapeutic options for this disease are still limited.An attractive therapeutic target for COVID-19 is the main protease (Mpro), as this highly conserved enzyme plays a key role in virus replication and host immune evasion.At present, docking-based virtual screening approaches have been widely used for rapid discovery of Mpro inhibitors (Arokiyaraj et al. 2020;Li et al. 2020;Guo et al. 2021;Yu et al. 2022).This computer-aided technique reduces the time and costs for drug discovery, but it remains challenging to efficiently remove the false positive hits (Ma and Wang 2021).Recently, geraniin has been identified as a potent Mpro inhibitor using a virtual screening method, and its inhibitory effect on Mpro is examined by the fluorescence resonance energy transfer (FRET) assay with an IC 50 value of 15.85 μg/mL (Yu et al. 2022).Considering the potential of geraniin in COVID-19 treatment, a rigorous validation for its Mpro inhibition is necessary.
We have developed a combined high-throughput screening (HTS) platform for rapid discovery and assessment of Mpro inhibitors, including FRET, fluorescence polarization (FP), and dimerization-dependent red fluorescent protein (ddRFP) assays (Yan et al. 2021;Zhang et al. 2022;Yan et al. 2023).Herein, we rigorously evaluated Mpro inhibition by geraniin in vitro using this robust HTS pipeline (Figure S1).To ensure the reliability of these assays, nirmatrelvir (PF-07321332, PF-332) was reevaluated with these assays.

Results and discussion
using FRET assay, our results showed that geraniin exhibits obvious inhibition of Mpro with an IC 50 value of 13.74 μM (Figures 1A and S2), but the presence of geraniin at the testing concentrations was able to quench the fluorescence signal of MCA-AVLQ peptide, which is generated by the cleaved FRET substrate (Figure S3A).Importantly, this quenching effect fully contributed to the observed Mpro inhibition of geraniin in the FRET assay, suggesting this inhibition is false positive (Figure 1B and S3B).We speculate that the overlap region between the absorption spectrum of geraniin and an emission wavelength of MCA-AVLQ peptide may cause this fluorescence quenching effect.We further evaluated Mpro inhibition by geraniin using FP assay.As expected, geraniin did not show dose-dependent inhibition of Mpro in the FP assay (IC 50 > 100 μM) (Figure 1C).As a control, PF-332 showed obvious inhibition of Mpro using FRET and FP assays with an equal IC 50 value of 0.13 μM (Figure S4), and this value was consistent with the previous study (Sacco et al. 2022).Moreover, a versatile ddRFP biosensor has been used to assess Mpro inhibitors by monitoring the change of relative fluorescence unit (RFu) value as well as the cleavage of ddRFP biosensor using SDS-PAGE analysis.Notably, PF-332 also exhibited significant inhibition of Mpro using ddRFP assay with an IC 50 value of 0.15 μM (Figure S5A), and the similar Mpro inhibition by PF-332 could be detected using SDS-PAGE analysis (Figure S5B).unlike PF-332, geraniin failed to inhibit the cleavage of ddRFP biosensor catalyzed by Mpro, and the IC 50 value was more than 100 μM (Figure 1D).In addition, geraniin did not show detectable inhibition of Mpro using SDS-PAGE analysis because of the cleavage of ddRFP biosensor in the gels (Figure 1E).Therefore, our results suggest that the in vitro inhibition of Mpro by geraniin is false positive, while PF-332 is a potent inhibitor against Mpro.
Geraniin exhibits wide pharmacological properties, such as antioxidant, antimicrobial, antidiabetic, and anti-inflammation (Zhang et al. 2022).Recent studies reveal that geraniin is likely to be a promising antiviral agent for COVID-19 treatment by blocking virus entry into human host cells (Arokiyaraj et al. 2020;Kim et al. 2021).
Considering the limitations of this study, we understand that more in vivo experiments for its antiviral target engagement and mechanism of false inhibition should be explored in the future.

Experimental
See Supplemental material section.

Conclusions
In summary, our data indicate that geraniin is not a potential inhibitor against Mpro based on the results from a set of in vitro assays.These results suggest a stringent in vitro validation with diverse biochemical assays is essential for the discovery of Mpro inhibitors, and the fluorescence quenching effect caused by natural products should be considered when evaluating Mpro inhibitors.

Figure 1 .
Figure 1.Inhibitory effect of geraniin on sars-coV-2 Mpro in vitro.(a) Inhibition of Mpro by geraniin using Fret assay.(B) the comparison between Mpro inhibition and fluorescence quenching effect of geraniin in the Fret assay.*p<0.05;n.s, not statiatically significant.(c) Inhibition of Mpro by geraniin using FP assay.In these assays, Gc-376 (1 μM) and dMso served as the positive and negative controls, respectively.the Ic 50 value of geraniin was shown.(d) Inhibition of Mpro by geraniin using ddrFP assay.the Ic 50 value of geraniin was shown.(e) Gel-based assay of ddrFP biosensor cleavage by geraniin in vitro.In sds-PaGe analysis, the ddrFP biosensor (55 kda) can be cleaved by Mpro (34 kda) to generate rFP-a 1 (top band, 29 kda) and rFP-B 1 fragments (bottom band, 26 kda).two rFP fragments were enclosed in a box with dashed red line in the gels.the testing concentration of geraniin was 1, 10, or 100 μM.Gc-376 (10 μM) and dMso served as the positive and negative controls, respectively.