Demonstrating Ligandability of the LC3A and LC3B Adapter Interface

Abstract

Autophagy is the common name for a number of lysosome-based degradation pathways of cytosolic cargos. The key components of autophagy are members of Atg8 family proteins involved in almost all steps of the process, from autophagosome formation to their selective fusion with lysosomes. In this study, we show that the homologous members of the human Atg8 family proteins, LC3A and LC3B, are druggable by a small molecule inhibitor novobiocin. Structure–activity relationship (SAR) studies of the 4-hydroxy coumarin core scaffold were performed, supported by a crystal structure of the LC3A dihydronovobiocin complex. The study reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated degraders (AUTACs).

Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein

“…Thei nteraction between DC-LC3in and LC3B was also validated by a2 Dn uclear resonance experiment using an 1 H- 15 Nl abeled LC3B protein. As expected, anumber of HN cross-peaks in the HSQC spectrum of LC3B showed obvious chemical shift changes or resonance signal attenuations upon addition of the ligand (Figure 2B), results clearly demonstrating the binding of DC-LC3in with LC3B.S ubsequent chemical shift perturbation (CSP) analysis indicated that the affected residues,i ncluding Phe52, Leu53, Va l5 4, Va l58, Leu63, Ile66, and Ile67 (Figure 2C), were mainly hydro-phobic and were located around the L-site of LIR-LC3 interface,which indicates that DC-LC3in may block the LIR-LC3interaction.…”

“…Recently,i tw as reported that LC3A and LC3B LIRinteraction interface are druggable by as mall molecule inhibitor novobiocin though with weak potency. [15] Autophagy-tethering compounds (ATTECs) like HTT-LC3 or lipid droplets-LC3 linker compounds which could bind with LC3 were also reported but they showed no inhibition on autophagy. [16] We were thus prompted to conduct as creen exploiting the ATG8-LIR interaction in an effort to identify much more potent ATG8 inhibitors to modulate autophagy.…”

Coumarins as Fungal Metabolites with Potential Medicinal Properties

“…Furthermore, novobiocin participates in a number of lysosome-based degradation pathways of cytosolic cargos by means of interaction with the members of the Atg8 family proteins as the key components of autophagy, in particular, with the human Atg8 family proteins, LC3A and LC3B. Novobiocin was very recently reported to be among the first nonpeptide inhibitors for these protein interaction targets [ 132 ].…”

Closantel is an allosteric inhibitor of human Taspase1

“…The HTRF based Taspase1 activity assay utilizes fusion proteins with N-terminal SNAP followed by a Taspase1 recognition , 2006). Plasmids for expression of such assay constructs were cloned based on the recently described pET29b derivative pET29BH4 (Hartmann et al, 2021) that encodes an open reading frame (ORF) for Met-Gly-[His 10 -tag]-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a cleavage site for Tobacco Etch Virus protease [TEV site] and restriction sites for KpnI (in frame; first triplet is Gly codon of TEV site) and XhoI. The SNAP ORF (residues 2-177) was amplified by PCR using Q5 DNA polymerase (NEB), pSNAP-tag(T7)2 (NEB) as the template, and forward primer #495 in combination with either oligo #496 for attachment of CS2 or oligo #498 for attachment of CS1.…”