Peptidomimetic Small-Molecule Inhibitors of 3CLPro Activity and Spike-ACE2 Interaction: Toward Dual-Action Molecules against Coronavirus Infections.

The development of molecules able to target protein-protein interactions (PPIs) is of interest for the development of novel therapeutic agents. Since a high percentage of PPIs are mediated by α-helical structure at the interacting surface, peptidomimetics that reproduce the essential conformational components of helices are useful templates for the development of PPIs inhibitors. In this work, the synthesis of a constrained dipeptide isostere and insertion in the short peptide epitope EDLFYQ of the angiotensin-converting enzyme 2 (ACE2) α1 helix domain resulted in the identification of a molecule capable of inhibiting the SARS-CoV-2 ACE2/spike interaction in the micromolar range. Moreover, inhibition of SARS-CoV-2 3CLPro main protease activity was assessed as an additional inhibitory property of the synthesized peptidomimetics, taking advantage of the C-terminal Q amino acid present in both the ACE2 epitope and the Mpro recognizing motif (APSTVxLQ), thus paving the way to the development of multitarget therapeutics toward coronavirus infections.

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.