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.

Structural Plasticity of NFU1 Upon Interaction with Binding Partners: Insights into the Mitochondrial [4Fe-4S] Cluster Pathway.

In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]2+ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]2+ clusters, two [2Fe-2S]2+ clusters are converted into a [4Fe-4S]2+ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]2+ cluster from ISCA1-ISCA2 complex. A structural view of the protein-protein recognition events occurring along the [4Fe-4S]2+ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]2+ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]2+ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]2+ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]2+ cluster transfer.