Inhibitor Development against p7 Channel in Hepatitis C Virus.

Hepatitis C Virus (HCV) is the key cause of chronic and severe liver diseases. The recent direct-acting antiviral agents have shown the clinical success on HCV-related diseases, but the rapid HCV mutations of the virus highlight the sustaining necessity to develop new drugs. p7, the viroporin protein from HCV, has been sought after as a potential anti-HCV drug target. Several classes of compounds, such as amantadine and rimantadine have been testified for p7 inhibition. However, the efficacies of these compounds are not high. Here, we screened some novel p7 inhibitors with amantadine scaffold for the inhibitor development. The dissociation constant (Kd) of 42 ARD-series compounds were determined by nuclear magnetic resonance (NMR) titrations. The efficacies of the two best inhibitors, ARD87 and ARD112, were further confirmed using viral production assay. The binding mode analysis and binding stability for the strongest inhibitor were deciphered by molecular dynamics (MD) simulation. These ARD-series compounds together with 49 previously published compounds were further analyzed by molecular docking. Key pharmacophores were identified among the structure-similar compounds. Our studies suggest that different functional groups are highly correlated with the efficacy for inhibiting p7 of HCV, in which hydrophobic interactions are the dominant forces for the inhibition potency. Our findings provide guiding principles for designing higher affinity inhibitors of p7 as potential anti-HCV drug candidates.

Deciphering Cholesterol's Role in PD-L2 Stability: A Distinct Regulatory Mechanism From PD-L1.

Programmed cell death 1 ligand 2 (PD-L2), a member of the B7 immune checkpoint protein family, emerges as a crucial player in immune modulation. Despite its functional overlap with programmed cell death 1 ligand 1 (PD-L1) in binding to the programmed cell death protein 1 (PD-1) on T cells, PD-L2 exhibits a divergent expression pattern and a higher affinity for PD-1. However, the regulatory mechanisms of PD-L2 remain under-explored. Here, our investigations illustrate the pivotal role of cholesterol in modulating PD-L2 stability. Using advanced nuclear magnetic resonance (NMR) and biochemical analyses, we demonstrate a direct and specific binding between cholesterol and PD-L2, mediated by an F-xxx-V-xx-LR motif in its transmembrane domain, distinct from that in PD-L1. This interaction stabilizes PD-L2 and prevents its downstream degradation. Disruption of this binding motif compromises PD-L2's cellular stability, underscoring its potential significance in cancer biology. These findings not only deepen our understanding of PD-L2 regulation in the context of tumors, but also open avenues for potential therapeutic interventions.

Identification of a novel class of cyclic penta-peptides against hepatitis C virus as p7 channel blockers.

The hepatitis C virus (HCV) p7 viroporin protein is essential for viral assembly and release, suggesting its unrealised potential as a target for HCV interventions. Several classes of small molecules that can inhibit p7 through allosteric mechanisms have shown low efficacy. Here, we used a high throughput virtual screen to design a panel of eight novel cyclic penta-peptides (CPs) that target the p7 channel with high binding affinity. Further examination of the effects of these CPs in viral production assays indicated that CP7 exhibits the highest potency against HCV among them. Moreover, the IC50 efficacy of CP7 in tests of strain Jc1-S282T suggested that this cyclopeptide could also effectively inhibit a drug-resistant HCV strain. A combination of nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations revealed that CP7 blocking activity relies on direct binding to the p7 channel lumen at the N-terminal bottleneck region. These findings thus present a promising anti-HCV cyclic penta-peptide targeting p7 viroporin, while also describing an alternative strategy for designing a new class of p7 channel blockers for strains resistant to direct-acting antiviral agents (DAA).

NMR Detection and Structural Modeling of the Ethylene Receptor LeETR2 from Tomato.

The gaseous plant hormone ethylene influences many physiological processes in plant growth and development. Plant ethylene responses are mediated by a family of ethylene receptors, in which the N-terminal transmembrane domains are responsible for ethylene binding and membrane localization. Until now, little structural information was available on the molecular mechanism of ethylene responses by the transmembrane binding domain of ethylene receptors. Here, we screened different constructs, fusion tags, detergents, and purification methods of the transmembrane sensor domain of ethylene receptors. However, due to their highly hydrophobic transmembrane domain (TMD), only a KSI-fused LeETR21-131 from tomato yielded a good-quality nuclear magnetic resonance (NMR) spectrum in the organic solvent. Interestingly, a dimer model of LeETR21-131 built by the AlphaFold2 algorithm showed greatly converged structures. The interaction analysis of ethylene and LeETR21-131 using molecular docking and molecular dynamics (MD) simulations demonstrated the potential binding sites of ethylene in LeETR21-131. Our exploration provides valuable knowledge for further understanding of the ethylene-perception process in ethylene receptors.

Regulation of PD-L1 through direct binding of cholesterol to CRAC motifs.

Cholesterol, an essential molecule for cell structure, function, and viability, plays crucial roles in the development, progression, and survival of cancer cells. Earlier studies have shown that cholesterol-lowering drugs can inhibit the high expression of programmed-death ligand 1 (PD-L1) that contributes to immunoevasion in cancer cells. However, the regulatory mechanism of cell surface PD-L1 abundance by cholesterol is still controversial. Here, using nuclear magnetic resonance and biochemical techniques, we demonstrated that cholesterol can directly bind to the transmembrane domain of PD-L1 through two cholesterol-recognition amino acid consensus (CRAC) motifs, forming a sandwich-like architecture and stabilizing PD-L1 to prevent downstream degradation. Mutations at key binding residues prohibit PD-L1-cholesterol interactions, decreasing the cellular abundance of PD-L1. Our results reveal a unique regulatory mechanism that controls the stability of PD-L1 in cancer cells, providing an alternative method to overcome PD-L1-mediated immunoevasion in cancers.

Cholesterol Binds in a Reversed Orientation to TCRß-TM in Which Its OH Group is Localized to the Center of the Lipid Bilayer.

T cell receptor (TCR) signaling in response to antigen recognition is essential for the adaptive immune response. Cholesterol keeps TCRs in the resting conformation and mediates TCR clustering by directly binding to the transmembrane domain of the TCRß subunit (TCRß-TM), while cholesterol sulfate (CS) displaces cholesterol from TCRß. However, the atomic interaction of cholesterol or CS with TCRß remains elusive. Here, we determined the cholesterol and CS binding site of TCRß-TM in phospholipid bilayers using solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation. Cholesterol binds to the transmembrane residues within a CARC-like cholesterol recognition motif. Surprisingly, the polar OH group of cholesterol is placed in the hydrophobic center of the lipid bilayer stabilized by its polar interaction with K154 of TCRß-TM. An aromatic interaction with Y158 and hydrophobic interactions with V160 and L161 stabilize this reverse orientation. CS binds to the same site, explaining how it competes with cholesterol. Site-directed mutagenesis of the CARC-like motif disrupted the cholesterol/CS binding to TCRß-TM, validating the NMR and MD results.