Although much progress has been made in the surveillance and control of coronavirus disease 2019 (COVID-19) pandemic around the world since its outbreak in 2019, the pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been undergoing mutations continuously, raising the concerns that the SARS-CoV-2 variants may gain resistance to the current SARS-CoV-2 neutralizing antibodies, vaccines, RNA-dependent RNA polymerase (RdRp) inhibitors and 3-chymotrypsin-like protease or main protease (3CLpro or Mpro) inhibitors [1,2,3,4]. If nothing else, SARS-CoV-2 has taught us that widespread proliferation, low fidelity genome synthesis, and selective pressure will quickly produce drug resistant phenotypes [3]. Therefore, there is still an urgent need to develop safe, effective, and affordable prevention/treatment agents for SARS-CoV-2 infection and future drug-resistance.
Several viral proteins, including 3CLpro, Papain-like protease (PLpro) and RdRp have been prioritized as promising anti-COVID-19 drug targets. Among the three viral proteases, 3CLpro appears to be a high-profile drug target for the development of broad-spectrum antivirals: first, 3CLpro plays an essential role in coronavirus replication by cleaving the viral polyproteins at more than 11 sites; second, 3CLpros have relatively high sequence similarity within each CoV group; moreover, 3CLpro has an unique substrate preference for glutamine at the P1 site (Leu-Gln↓(Ser,Ala,Gly)), a feature that is absent in closely related host proteases; last but not least, up to now, none of the 25 most common 3CLpro mutants involve residues in the active site or at the dimerization interface [3]. Even with several exceptions, including P132H, the resulting amino acid is often similar in size and physicochemical properties, such as K → R, which does not compromise small-molecule drug inhibition [3]. Therefore, it is feasible to design 3CLpro inhibitors with high selectivity and high potential to monitor drug-resistance. Our primary goal was to identify lead compounds targeting SARS-CoV-2 3CLpro using our newly established sandwich-like FP screening assay [5]. Among 3000 tested compounds, ginkgolic acid (GA) and anacardic acid (AA) displayed the most potent inhibitory effects on the hydrolytic activity of SARS-CoV-2 3CLpro. As illustrated in Fig. 1a, SARS-CoV-2 3CLpro was inhibited by GA in a dose-dependent manner, with half maximal inhibitory concentration (IC50) value of 11.29 ± 0.48 μM. The IC50 value of AA against SARS-CoV-2 3CLpro was 12.19 ± 0.50 μM (Fig. 1b), showing similar inhibitory efficiency as GA. Subsequently, we confirmed the proteolytic inhibition of GA and AA against 3CLpro using fluorescence resonance energy transfer (FRET) assay. The IC50 values of GA and AA towards 3CLpro were 4.89 ± 0.30 μM (Fig. 1c) and 7.60 ± 0.30 μM (Fig. 1d), respectively, which were similar to the previously published results [6, 7].
Covalent inhibitors usually use electrophilic moieties, prominently nitrile, disulfide or cyanoacrylate to react with a corresponding site in its target [8]. The majority of current reported SARS-CoV-2 3CLpro inhibitors are peptidomimetic covalent inhibitors with a reactive warhead such as ketone, aldehyde or ketoamide [9]. A highlighting milestone is Nirmatrelvir (PF-07321332), a covalent inhibitor carrying a nitrile warhead that targets SARS-CoV-2 3CLpro, which plus Ritonavir has received its first conditional authorization on 31 December 2021 for the treatment of COVID-19 in the United Kingdom [10]. Nirmatrelvir has also been authorized for emergency use in the USA (December 2021), and more recently received a conditional authorization in the EU (January 2022). Inspired by this fact, we used PF-07321332 as a positive control to demonstrate that the covalent conjugate can be detected by our developed mass spectrum assay. Notably, the presence of a mass shift of 500 Da after treatment with PF-07321332 indicated a covalent adduct formation (Fig. 1e), in agreement with the results presented in the previous study [10]. Considering the lack of reactive group in GA and AA, covalent inhibition against 3CLpro is usually unlikely. To test this idea, we compared the molecular weights of 3CLpro before and after its incubation with GA or AA using HPLC-Q-TOF–MS. A MS peak with a mass value of 34,863.01 (Fig. 1f)/34,862.24 Da (Fig. 1g) was detected for SARS-CoV-2 3CLpro incubated with GA or AA, respectively. These numbers were equal to the molecular weight of SARS-CoV-2 3CLpro alone (34,864.15 Da) (Fig. 1f, g), indicating non-covalent conjugation with either GA or AA. In conclusion, our results suggested that neither GA nor AA covalently reacted with SARS-CoV-2 3CLpro.
To further understand the nature of non-covalent interaction between SARS-CoV-2 3CLpro and GA or AA at the molecular level, surface plasmon resonance (SPR)-based binding assay was used to measure the affinity constant and characterize these interactions. Although GA could increase the real refractive index unit (RIU) response of SARS-CoV-2 3CLpro in a dose-dependent manner, the KD was 4.77 × 10–3 M (Fig. 1h), indicating poor affinity and reversible binding mode between GA and SARS-CoV-2 3CLpro. Very similar results were obtained regarding the binding of AA to SARS-CoV-2 3CLpro with KD of 6.21 × 10–3 M (Fig. 1i). Meanwhile, both GA and AA exhibited fast association rate (ka) and fast dissociation rate (kd) values in binding kinetics curves (Fig. 1h, i).
It is well documented that reversible inhibitors bind to enzymes by non-covalent bonds. The non-covalent adduct formation between SARS-CoV-2 3CLpro and GA or AA indicated that GA and AA are likely to exert inhibitory effects via reversible inhibition. Therefore, kinetic analysis was performed to investigate the inhibitory mechanisms of GA and AA using FRET assay. Based on enzyme kinetics analysis, the slopes and intercepts of the reciprocal Lineweaver–Burk plots elevated with the increase in inhibitor concentration, which is inconsistent with the three main types of inhibition, competitive, noncompetitive, or uncompetitive (Fig. 1j, l). The intersection of each trend line in the second quadrant suggested a mixed-type inhibitory mode, implying that these agents may bind this target enzyme at both catalytic active site and non-catalytic site. The inhibitory constant (Ki) values of GA and AA were 1.69 and 1.87 μM, respectively (Fig. 1k, m). Overall, these results suggested that GA and AA act as reversible and mixed-type inhibitors against SARS-CoV-2 3CLpro, which is supported by the previous finding [6].
In summary, our data provide insights into the binding modes between SARS-CoV-2 3CLpro and GA or AA, which is non-covalent, reversible and mixed-type of inhibition. Our results are inconsistent with a previously report about the covalent binding between SARS-CoV-2 3CLpro and GA or AA [7]. Regardless, these compounds are promising candidates worthy of structure modification for the treatment of COVID-19.
