A Structural Investigation of the Interaction between a GC-376-Based Peptidomimetic PROTAC and Its Precursor with the Viral Main Protease of Coxsackievirus B3.

The conservation of the main protease in viral genomes, combined with the absence of a homologous protease in humans, makes this enzyme family an ideal target for developing broad-spectrum antiviral drugs with minimized host toxicity. GC-376, a peptidomimetic 3CL protease inhibitor, has shown significant efficacy against coronaviruses. Recently, a GC-376-based PROTAC was developed to target and induce the proteasome-mediated degradation of the dimeric SARS-CoV-2 3CLPro protein. Extending this approach, the current study investigates the application of the GC-376 PROTAC to the 3CPro protease of enteroviruses, specifically characterizing its interaction with CVB3 3CPro through X-ray crystallography, NMR (Nuclear Magnetic Resonance) and biochemical techniques. The crystal structure of CVB3 3CPro bound to the GC-376 PROTAC precursor was obtained at 1.9 Å resolution. The crystallographic data show that there are some changes between the binding of CVB3 3CPro and SARS-CoV-2 3CLPro, but the overall similarity is strong (RMSD on C-alpha 0.3 Å). The most notable variation is the orientation of the benzyloxycarbonyl group of GC-376 with the S4 subsite of the proteases. NMR backbone assignment of CVB3 3CPro bound and unbound to the GC-376 PROTAC precursor (80% and 97%, respectively) was obtained. This information complemented the investigation, by NMR, of the interaction of CVB3 3CPro with the GC-376 PROTAC, and its precursor allows us to define that the GC-376 PROTAC binds to CVB3 3CPro in a mode very similar to that of the precursor. The NMR relaxation data indicate that a quench of dynamics of a large part of the protein backbone involving the substrate-binding site and surrounding regions occurs upon GC-376 PROTAC precursor binding. This suggests that the substrate cavity, by sampling different backbone conformations in the absence of the substrate, is able to select the suitable one necessary to covalently bind the substrate, this being the latter reaction, which is the fundamental step required to functionally activate the enzymatic reaction. The inhibition activity assay showed inhibition potency in the micromolar range for GC-376 PROTAC and its precursor. Overall, we can conclude that the GC-376 PROTAC fits well within the binding sites of both proteases, demonstrating its potential as a broad-spectrum antiviral agent.

Development of a GC-376 Based Peptidomimetic PROTAC as a Degrader of 3-Chymotrypsin-like Protease of SARS-CoV-2.

We have applied a proteolysis targeting chimera (PROTAC) technology to obtain a peptidomimetic molecule able to trigger the degradation of SARS-CoV-2 3-chymotrypsin-like protease (3CLPro). The PROTAC molecule was designed by conjugating a GC-376 based dipeptidyl 3CLPro ligand to a pomalidomide moiety through a piperazine-piperidine linker. NMR and crystallographic data complemented with enzymatic and cellular studies showed that (i) the dipeptidyl moiety of PROTAC binds to the active site of the dimeric state of SARS-CoV-2 3CLPro forming a reversible covalent bond with the sulfur atom of catalytic Cys145, (ii) the linker and the pomalidomide cereblon-ligand of PROTAC protrude from the protein, displaying a high degree of flexibility and no interactions with other regions of the protein, and (iii) PROTAC reduces the protein levels of SARS-CoV-2 3CLPro in cultured cells. This study paves the way for the future applicability of peptidomimetic PROTACs to tackle 3CLPro-dependent viral infections.

Gold-Based Metal Drugs as Inhibitors of Coronavirus Proteins: The Inhibition of SARS-CoV-2 Main Protease by Auranofin and Its Analogs.

Gold compounds have a long tradition in medicine and offer many opportunities for new therapeutic applications. Herein, we evaluated the lead compound Auranofin and five related gold(I) complexes as possible inhibitors of SARS-CoV-2 Main Protease (SARS-CoV-2 Mpro), a validated drug target for the COVID-19 disease. The investigational panel of gold compounds included Auranofin; three halido analogues, i.e., Au(PEt3)Cl, Au(PEt3)Br, and Au(PEt3)I; and two gold carbene complexes, i.e., Au(NHC)Cl and [Au(NHC)2]PF6. Notably, all these gold compounds, with the only exception of [Au(NHC)2]PF6, turned out to be potent inhibitors of the catalytic activity of SARS-CoV-2 Mpro: the measured Ki values were in the range 2.1-0.4 µM. The reactions of the various gold compounds with SARS-CoV-2 Mpro were subsequently investigated through electrospray ionization (ESI) mass spectrometry (MS) upon a careful optimization of the experimental conditions; the ESI MS spectra provided clear evidence for the formation of tight metallodrug-protein adducts and for the coordination of well defined gold-containing fragments to the SARS-CoV-2 Mpro, again with the only exception of [Au(NHC)2]PF6, The metal-protein stoichiometry was unambiguously determined for the resulting species. The crystal structures of the metallodrug- Mpro adducts were solved in the case of Au(PEt3)Br and Au(NHC)Cl. These crystal structures show that gold coordination occurs at the level of catalytic Cys 145 in the case of Au(NHC)Cl and at the level of both Cys 145 and Cys 156 for Au(PEt3)Br. Tight coordination of gold atoms to functionally relevant cysteine residues is believed to represent the true molecular basis of strong enzyme inhibition.