The Effect of 4-Methylcatechol on Platelets in Familial Hypercholesterolemic Patients Treated with Lipid Apheresis and/or Proprotein Convertase Subtilisin Kexin 9 Monoclonal Antibodies.

Elevated low-density lipoprotein (LDL) cholesterol levels lead to atherosclerosis and platelet hyperaggregability, both of which are known culprits of arterial thrombosis. Normalization of LDL cholesterol in familial hypercholesterolemia (FH) is not an easy task and frequently requires specific treatment, such as regularly performed lipid apheresis and/or novel drugs such as proprotein convertase subtilisin kexin 9 monoclonal antibodies (PCSK9Ab). Moreover, a high resistance rate to the first-line antiplatelet drug acetylsalicylic acid (ASA) stimulated research of novel antiplatelet drugs. 4-methylcatechol (4-MC), a known metabolite of several dietary flavonoids, may be a suitable candidate. The aim of this study was to analyse the antiplatelet effect of 4-MC in FH patients and to compare its impact on two FH treatment modalities via whole-blood impedance aggregometry. When compared to age-matched, generally healthy controls, the antiplatelet effect of 4-MC against collagen-induced aggregation was higher in FH patients. Apheresis itself improved the effect of 4-MC on platelet aggregation and blood from patients treated with this procedure and pretreated with 4-MC had lower platelet aggregability when compared to those solely treated with PCKS9Ab. Although this study had some inherent limitations, e.g., a low number of patients and possible impact of administered drugs, it confirmed the suitability of 4-MC as a promising antiplatelet agent and also demonstrated the effect of 4-MC in patients with a genetic metabolic disease for the first time.

Proprotein convertases regulate trafficking and maturation of key proteins within the secretory pathway.

Proprotein Convertases (PCs) are serine endoproteases that regulate the homeostasis of protein substrates in the cell. The PCs family counts 9 members-PC1/3, PC2, PC4, PACE4, PC5/6, PC7, Furin, SKI-1/S1P, and PCSK9. The first seven PCs are known as Basic Proprotein Convertases due to their propensity to cleave after polybasic clusters. SKI-1/S1P requires the additional presence of hydrophobic residues for processing, whereas PCSK9 is catalytically dead after autoactivation and exerts its functions using mechanisms alternative to direct cleavage. All PCs traffic through the canonical secretory pathway, reaching different compartments where the various substrates reside. Despite PCs members do not share the same subcellular localization, most of the cellular organelles count one or more Proprotein Convertases, including ER, Golgi stack, endosomes, secretory granules, and plasma membranes. The widespread expression of these enzymes at the systemic level speaks for their importance in the homeostasis of a large number of biological functions. Among others, PCs cleave precursors of hormones and growth factors and activate receptors and transcription factors. Notably, dysregulation of the enzymatic activity of Proprotein Convertases is associated to major human pathologies, such as cardiovascular diseases, cancer, diabetes, infections, inflammation, autoimmunity diseases, and Parkinson. In the current COVID-19 pandemic, Furin has further attracted the attention as a key player for conferring high pathogenicity to SARS-CoV-2. Here, we review the Proprotein Convertases family and their most important substrates along the secretory pathway. Knowledge about the complex functions of PCs is important to identify potential drug strategies targeting this class of enzymes.

The emerging role of furin in neurodegenerative and neuropsychiatric diseases.

Furin is an important mammalian proprotein convertase that catalyzes the proteolytic maturation of a variety of prohormones and proproteins in the secretory pathway. In the brain, the substrates of furin include the proproteins of growth factors, receptors and enzymes. Emerging evidence, such as reduced FURIN mRNA expression in the brains of Alzheimer's disease patients or schizophrenia patients, has implicated a crucial role of furin in the pathophysiology of neurodegenerative and neuropsychiatric diseases. Currently, compared to cancer and infectious diseases, the aberrant expression of furin and its pharmaceutical potentials in neurological diseases remain poorly understood. In this article, we provide an overview on the physiological roles of furin and its substrates in the brain, summarize the deregulation of furin expression and its effects in neurodegenerative and neuropsychiatric disorders, and discuss the implications and current approaches that target furin for therapeutic interventions. This review may expedite future studies to clarify the molecular mechanisms of furin deregulation and involvement in the pathogenesis of neurodegenerative and neuropsychiatric diseases, and to develop new diagnosis and treatment strategies for these diseases.

Furin cleavage is required for swine acute diarrhea syndrome coronavirus spike protein-mediated cell - cell fusion.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was reported in China in 2017 and is a causative agent of porcine enteric disease. Recent studies indicate that cells from various hosts are susceptible to SADS-CoV, suggesting the zoonotic potential of this virus. However, little is known about the mechanisms through which this virus enters cells. In this study, we investigated the role of furin in SADS-CoV spike (S)-mediated cell - cell fusion and entry. We found that the SADS-CoV S protein induced the fusion of various cells. Cell - cell fusion was inhibited by the proprotein convertase inhibitor dec-RVKR-cmk, and between cells transfected with mutant S proteins resistant to furin cleavage. These findings revealed that furin-induced cleavage of the SADS-CoV S protein is required for cell - cell fusion. Using mutagenesis analysis, we demonstrated that furin cleaves the SADS-CoV S protein near the S1/S2 cleavage site, 446RYVR449 and 543AVRR546. We used pseudotyped viruses to determine whether furin-induced S cleavage is also required for viral entry. Pseudotyped viruses expressing S proteins with a mutated furin cleavage site could be transduced into target cells, indicating that furin-induced cleavage is not required for pseudotyped virus entry. Our data indicate that S cleavage is critical for SADS-CoV S-mediated cell - cell fusion and suggest that furin might be a host target for SADS-CoV antivirals.

Dichlorophenylpyridine-Based Molecules Inhibit Furin through an Induced-Fit Mechanism.

Inhibitors of the proprotein convertase furin might serve as broad-spectrum antiviral therapeutics. High cellular potency and antiviral activity against acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we characterized the binding mechanism of this inhibitor class using structural, biophysical, and biochemical methods. We established a MALDI-TOF-MS-based furin activity assay, determined IC50 values, and solved X-ray structures of (3,5-dichlorophenyl)pyridine-derived compounds in complex with furin. The inhibitors induced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. These changes formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into a newly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strong structural stabilization in surface plasmon resonance and differential scanning fluorimetry experiments, respectively. The discovered furin conformation offers new opportunities for structure-based drug discovery.

A novel antibody against the furin cleavage site of SARS-CoV-2 spike protein: Effects on proteolytic cleavage and ACE2 binding.

SARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, including with a P681R mutation, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.

How Do Enveloped Viruses Exploit the Secretory Proprotein Convertases to Regulate Infectivity and Spread?

Inhibition of the binding of enveloped viruses surface glycoproteins to host cell receptor(s) is a major target of vaccines and constitutes an efficient strategy to block viral entry and infection of various host cells and tissues. Cellular entry usually requires the fusion of the viral envelope with host plasma membranes. Such entry mechanism is often preceded by "priming" and/or "activation" steps requiring limited proteolysis of the viral surface glycoprotein to expose a fusogenic domain for efficient membrane juxtapositions. The 9-membered family of Proprotein Convertases related to Subtilisin/Kexin (PCSK) serine proteases (PC1, PC2, Furin, PC4, PC5, PACE4, PC7, SKI-1/S1P, and PCSK9) participate in post-translational cleavages and/or regulation of multiple secretory proteins. The type-I membrane-bound Furin and SKI-1/S1P are the major convertases responsible for the processing of surface glycoproteins of enveloped viruses. Stefan Kunz has considerably contributed to define the role of SKI-1/S1P in the activation of arenaviruses causing hemorrhagic fever. Furin was recently implicated in the activation of the spike S-protein of SARS-CoV-2 and Furin-inhibitors are being tested as antivirals in COVID-19. Other members of the PCSK-family are also implicated in some viral infections, such as PCSK9 in Dengue. Herein, we summarize the various functions of the PCSKs and present arguments whereby their inhibition could represent a powerful arsenal to limit viral infections causing the present and future pandemics.

Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry.

Antibody-dependent enhancement (ADE) of viral entry has been a major concern for epidemiology, vaccine development, and antibody-based drug therapy. However, the molecular mechanism behind ADE is still elusive. Coronavirus spike protein mediates viral entry into cells by first binding to a receptor on the host cell surface and then fusing viral and host membranes. In this study, we investigated how a neutralizing monoclonal antibody (MAb), which targets the receptor-binding domain (RBD) of Middle East respiratory syndrome (MERS) coronavirus spike, mediates viral entry using pseudovirus entry and biochemical assays. Our results showed that MAb binds to the virus surface spike, allowing it to undergo conformational changes and become prone to proteolytic activation. Meanwhile, MAb binds to cell surface IgG Fc receptor, guiding viral entry through canonical viral-receptor-dependent pathways. Our data suggest that the antibody/Fc-receptor complex functionally mimics viral receptor in mediating viral entry. Moreover, we characterized MAb dosages in viral-receptor-dependent, Fc-receptor-dependent, and both-receptors-dependent viral entry pathways, delineating guidelines on MAb usages in treating viral infections. Our study reveals a novel molecular mechanism for antibody-enhanced viral entry and can guide future vaccination and antiviral strategies.IMPORTANCE Antibody-dependent enhancement (ADE) of viral entry has been observed for many viruses. It was shown that antibodies target one serotype of viruses but only subneutralize another, leading to ADE of the latter viruses. Here we identify a novel mechanism for ADE: a neutralizing antibody binds to the surface spike protein of coronaviruses like a viral receptor, triggers a conformational change of the spike, and mediates viral entry into IgG Fc receptor-expressing cells through canonical viral-receptor-dependent pathways. We further evaluated how antibody dosages impacted viral entry into cells expressing viral receptor, Fc receptor, or both receptors. This study reveals complex roles of antibodies in viral entry and can guide future vaccine design and antibody-based drug therapy.