19b

Amino Acid Sequence

3D Structure (/structures/struct-20261026-145126-490070.cif)

Function

Plays an essential role in viral RNA transcription and replication by forming the heterotrimeric polymerase complex together with PB1 and PB2 subunits. The complex transcribes viral mRNAs by using a unique mechanism called cap-snatching. It consists in the hijacking and cleavage of host capped pre-mRNAs. These short capped RNAs are then used as primers for viral mRNAs. The PB2 subunit is responsible for the binding of the 5' cap of cellular pre-mRNAs which are subsequently cleaved after 10-13 nucleotides by the PA subunit that carries the endonuclease activity.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

The degradation of PA was prevented by the addition of 50 μM dipeptide RA (Figure 5A). Therefore, we hypothesized that the degradation induced by Boc2-Lys is linked to ubiquitination. We found that the PA turnover triggered by 19b was sensitive to the 10 μM proteasome inhibitor MG-132 and bortezomib. (Figure 5B,C). 3-MA and CQ substantially inhibited the degradation function of 19b, confirming that degradation was partially mediated by autophagy (Figure 5D,E). the proteasome pathway is one of the manners of PA protein degradation mediated by Boc2−Lys, perhaps owing to the inhibition of both A2 and B1, excessive accumulation of the ubiquitinated complex containing PA protein, and repositioning to autophagy for degradation (path C). CQ and subcytotoxic concentrations of 3-MA slightly inhibit PA protein degradation, verifying that autophagy was involved in the degradation process. MG-132 or bortezomib treatment resulted in the inhibition of the 20S proteasome and 26S proteasome, but some PA was still degraded by the autophagy pathway.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

Therefore, it is reasonable to hypothesize the simultaneous existence of the ubiquitin-dependent proteasome pathway (path A) and the ubiquitin-independent proteasome pathway (path B). Similarly, we observed that MG132 and bortezomib were able to substantially rebound PA protein levels affected by 19b with inhibited functions of the 20S proteasome and the 26S proteasome. This suggested that the proteasome pathway is one of the manners of PA protein degradation mediated by Boc2−Lys. RA could effectively block the consequent degradation of PA proteins at 50 μM, implying that the degradation process involved ubiquitin ligase Ubr1 (A1 stage). We suggested that PA degradation induced by 19b showed a significant correlation with both autophagy and proteasome. MG-132 or bortezomib treatment resulted in the inhibition of the 20S proteasome and 26S proteasome, but some PA was still degraded by the autophagy pathway.

Description

Briefly, all of the synthesized compounds exhibited moderate to high anti-H1N1 efficacy, and most of them were better than ligand 11, AMD, and T-705 but were weaker than baloxavir. 19b (EC50 = 0.015 ± 0.001 μM, SI = 6666.7), 19c (EC50 =0.021 ± 0.002 μM, SI > 4761.9), and 19d (EC50 = 0.045 ±0.014 μM, SI > 2222.2) exhibited strong inhibitory activity with safety index (SI) superior to that of ligand 11 (EC50 =0.332 ± 0.017, SI = 217.6). HEK293T cells were infected with A/WSN/33/H1N1 at a multiplicity of infection (MOI) of 0.1 and were treated with different concentrations of compounds for 48 h, and then cell viability was evaluated using the CCK-8 reagent (n = 3). Amantadine (AMD), favipiravir (T-705), and baloxavir acid (BXA) served as positive controls. Data were acquired from at least three independent experiments (mean ± SD). EC50: effective concentration protecting 50% of cells from virus infection.

Description

The most potent HyT degrader 19b exhibited great anti-influenza efficacy (EC50 = 0.015 ± 0.001 μM) and a strong capacity for degrading the PA of A/WSN/33/H1N1 via proteasomal and autophagy−lysosomal pathways (DC50 = 0.06 μM). Furthermore, it exhibited lower cytotoxicity (CC50 = 97.58 μM) than the original ligand 11 (CC50 = 72.26 μM). HEK293T cells were infected with A/WSN/33/H1N1 at a multiplicity of infection (MOI) of 0.1 and were treated with different concentrations of compounds for 48 h, and then cell viability was evaluated using the CCK-8 reagent (n = 3). CC50: minimum concentration inducing 50% death of normal cells.

Description

We chose 19b and 19c for further investigation, as they exhibited no significant difference in A/WSN/33/H1N1 inhibition activity (Table 1). Western blot analysis revealed that the PA protein degradation ability of 19b (Figure 4A,D, DC50 = 0.06 μM) was excellent, concentration-dependent, and more potent than that of 19c (Figure 4B,D, DC50 = 0.65 μM).