Enhanced expression and solubility of main protease (Mpro) of SARS-CoV-2 from E. coli.

The main protease (Mpro) of SARS-CoV-2 is a essential enzyme that facilitates viral transcription and replication. Furthermore, the conservation of Mpro across different variants and its non-overlapping nature with human proteases make it an appealing target for therapeutic interventions against SARS-CoV-2. Multiple inhibitors specifically target Mpro to mitigate the infection caused by SARS-CoV-2. In the current study, successful cloning and expression of SARS-CoV-2 Mpro were achieved using two E. coli hosts, namely BL21-DE3 and BL21-DE3-RIL. By optimizing the conditions for induction, the expression of Mpro in the soluble fraction of E. coli was improved. Subsequently, Mpro was purified using affinity chromatography, yielding significantly higher quantities from the BL21-DE3-RIL strain compared to the BL21-DE3 strain, with the former producing nearly twice as much as the latter. The purified Mpro was further characterized by mass spectrometry, fluorescence spectroscopy and circular dichroism (CD). Through fluorescence quenching studies, it was discovered that both GC376 and chitosan, which are inhibitors of Mpro, induced structural changes in the purified Mpro protein. This indicates that the protein retained its functional activity even after being expressed in a bacterial host. Further, FRET-based assay highlighted that the enzymatic activity of Mpro was significantly reduced in presence of both GC376 and chitosan. Consequently, the utilization of optimal conditions and the BL21-DE3-RIL bacterial host facilitates the cost-effective production of Mpro on a large scale, enabling high yields. This production approach can be applied for the screening of potent therapeutic drugs, making it a valuable resource for drug development endeavors.

Targeting DPP4-RBD interactions by sitagliptin and linagliptin delivers a potential host-directed therapy against pan-SARS-CoV-2 infections.

Highly mutated SARS-CoV-2 is known aetiological factor for COVID-19. Here, we have demonstrated that the receptor binding domain (RBD) of the spike protein can interact with human dipeptidyl peptidase 4 (DPP4) to facilitate virus entry, in addition to the usual route of ACE2-RBD binding. Significant number of residues of RBD makes hydrogen bonds and hydrophobic interactions with α/ß-hydrolase domain of DPP4. With this observation, we created a strategy to combat COVID-19 by circumventing the catalytic activity of DPP4 using its inhibitors. Sitagliptin, linagliptin or in combination disavowed RBD to establish a heterodimer complex with both DPP4 and ACE2 which is requisite strategy for virus entry into the cells. Both gliptins not only impede DPP4 activity, but also prevent ACE2-RBD interaction, crucial for virus growth. Sitagliptin, and linagliptin alone or in combination have avidity to impede the growth of pan-SARS-CoV-2 variants including original SARS-CoV-2, alpha, beta, delta, and kappa in a dose dependent manner. However, these drugs were unable to alter enzymatic activity of PLpro and Mpro. We conclude that viruses hijack DPP4 for cell invasion via RBD binding. Impeding RBD interaction with both DPP4 and ACE2 selectively by sitagliptin and linagliptin is an potential strategy for efficiently preventing viral replication.