Elettra | Structural Biology, XRD2 During the COVID-19 pandemic, numerous studies were conducted on the virus responsible- SARS-CoV-2- with the aim of thoroughly understanding its life cycle in order to identify effective therapeutic targets. Like all viruses, SARS-CoV-2 is a complex and well-structured assembly of biological macromolecules- i.e. proteins, lipids, polysaccharides, and nucleic acids (RNA or DNA as genome)- which must rely on the functional macromolecular machinery of living cells to survive and replicate. SARS-CoV-2 is an RNA virus whose genome encodes for non-structural proteins (NSPs), accessory proteins, and structural proteins. Once internalized, SARS-CoV-2 exploits the host's biological processes to translate its genome, producing a single, inactive polypeptide chain composed of various protein elements- commonly referred to as viral polyprotein. To become functional, these elements must be cleaved from the parent protein by one of the most important SARS-CoV-2 proteins: the protease 3CLpro. The therapeutic significance of 3CLpro lies in the fact that its inhibition prevents the maturation of the viral polyprotein, thereby blocking all the vital functions SARS-CoV-2 requires to survive, reproduce, and spread. It is no coincidence that since the beginning of the pandemic, more than 1,000 scientific articles have focused on this particular protein. 3CLpro is an enzyme: it acts as a biological catalyst, accelerating specific biochemical reactions. In particular, on the basis of the specific reaction that 3CLpro promotes—the breaking of chemical bonds that characterize proteins (called peptide bonds)—this enzyme is included in the family of proteases. More precisely, it is classified as a cysteine protease, as a cysteine, occurring at position 145 in its amino acid sequence (Cys145), is involved in the catalytic process. The active form of this enzyme is a dimer composed of two copies of the same protein (called subunits) that must interact in a specific way to adopt the correct (native) conformation required for catalytic function. Alternative modes of interaction—despite the presence of the catalytically essential residue Cys145— are often less efficient or completely inactive. Even the mere misalignment or incorrect orientation of the amino acid chains can impair catalytic efficiency. Therefore, strategies to inhibit this 3CLpro are aimed either at specifically blocking Cys145, to counteract/destabilize the dimer in its native conformation, or at achieving both with a synergistic effect. Numerous drugs and molecules have been studied for their ability to inhibit 3CLpro by blocking the catalytic cysteine in the active site. However, active site stability depends on the dimerization of 3CLpro and the correct orientation of its subdomains. In this wide and intricate field, our research was focused to the identification and description of the mode of action of a drug able to inhibit 3CLpro and, therefore, to contrast SARS-CoV-2 viral infection. Exscalate4CoV (“EXaSCale smart pLatform Against paThogEns for CoronaVirus” or E4C; www.exscalate4cov. eu) an EU H2020 supported project (grant agreement 101003551) exploiting the most powerful and costefficient supercomputing platform and experimental screening facilities, performed an initial in silico screening of thousands of drugs already employed in preclinical and clinical studies for antiviral treatments. We predicted 62 potential inhibitors of 3CLpro, identifying halicin among them. Halicin covalently binds the catalytic Cys145, as well as other reactive cysteines present in the 3CLpro, leading to its enzymatic inhibition. Although blocking the catalytic residue causes loss of function of the SARS-CoV-2 protease, the other 11 cysteine residues might influence the protease's catalytic activity through different mechanisms. In this study, we characterized the 3CLpro-halicin complex by identifying and quantifying all the reactive cysteine residues through integrated molecular dynamics, macromolecular crystallography, mass spectrometry and biophysical approaches. Based on previous studies and our investigations, Cys145 was found the most targeted residue by halicin. By solving the 1,93Å co-crystal structure (crystals and data generated at the crystallization facility and XRD2 beam line of Elettra, PDB ID 7NBY) we found that Cys44 was covalently modified, along with Cys85 and Cys300 at a lesser extent. These results were confirmed by mass spectrometry. To further understand the role of Cys44, we expressed and purified a 3CLpro protein mutant (Cys44Ala), replacing the Cys44 with alanine (Ala), an amino acid lacking of the specific cysteine features, and assessed its activity. Surprisingly, we observed a 14-fold loss of potency of the mutated form (Cys44Ala) in comparison to the wild type (wt) protein in a well-established enzymatic assay, corroborating the theory that Cys44 plays a key role in halicin's inhibitory mechanism, beyond the effect due to the catalytic Cys145 modification. Moreover, we also evaluated the 3CLPro wt the stability through Differential Scanning Fluorimetry (DSF) and the monomerdimer equilibrium (oligomerisation state) in native conditions, in comparison with the mutated Cys44Ala protein. The mutant showed a significant lowering in melting temperature, estimated as - 9 °C in comparison to the wt, suggesting that also in absence of any drugs, the Cys44Ala mutant is less stable than the 3CLpro wt. Finally, by comparing the oligomerisation state of the mutated vs the wt protein, we found a prevalence of the monomer in the protein lacking Cys in position 44, confirming crucial role of Cys44 on 3CLpro monomer-dimer equilibrium, and therefore, in stabilising active conformation. In conclusion, this study demonstrates for the first time that, despite halicin predominantly targets Cys145, Cys44 supports the catalytic activity of 3CLpro, through a stabilizing effect of monomer and therefore, on the consequent active dimer formation. The substitution of Cys44 with an alanine residue induces a local alteration which also has a longrange impact on protein conformation and on monomer-dimer equilibrium. Characterisation of covalent inhibition of 3CLpro from SARS-CoV-2 by halicin Elettra Highlights 2024–2025 58
RkJQdWJsaXNoZXIy MjQ4NzI=