A novel thiol-saccharide mucolytic for the treatment of muco-obstructive lung diseases.

BACKGROUND: Mucin disulfide cross-links mediate pathologic mucus formation in muco-obstructive lung diseases. MUC-031, a novel thiol-modified carbohydrate compound, cleaves disulfides to cause mucolysis. The aim of this study was to determine the mucolytic and therapeutic effects of MUC-031 in sputum from patients with cystic fibrosis (CF) and mice with muco-obstructive lung disease (ßENaC-Tg mice). METHODS: We compared the mucolytic efficacy of MUC-031 and existing mucolytics (N-acetylcysteine (NAC) and recombinant human deoxyribonuclease I (rhDNase)) using rheology to measure the elastic modulus (G') of CF sputum, and we tested effects of MUC-031 on airway mucus plugging, inflammation and survival in ßENaC-Tg mice to determine its mucolytic efficacy in vivo. RESULTS: In CF sputum, compared to the effects of rhDNase and NAC, MUC-031 caused a larger decrease in sputum G', was faster in decreasing sputum G' by 50% and caused mucolysis of a larger proportion of sputum samples within 15 min of drug addition. Compared to vehicle control, three treatments with MUC-031 in 1 day in adult ßENaC-Tg mice decreased airway mucus content (16.8±3.2 versus 7.5±1.2 nL·mm-2, p<0.01) and bronchoalveolar lavage cells (73 833±6930 versus 47 679±7736 cells·mL-1, p<0.05). Twice-daily treatment with MUC-031 for 2 weeks also caused decreases in these outcomes in adult and neonatal ßENaC-Tg mice and reduced mortality from 37% in vehicle-treated ßENaC-Tg neonates to 21% in those treated with MUC-031 (p<0.05). CONCLUSION: MUC-031 is a potent and fast-acting mucolytic that decreases airway mucus plugging, lessens airway inflammation and improves survival in ßENaC-Tg mice. These data provide rationale for human trials of MUC-031 in muco-obstructive lung diseases.

Exploring antiviral and anti-inflammatory effects of thiol drugs in COVID-19.

The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for the binding of SARS-2-S to angiotensin-converting enzyme 2 (ACE2), suggesting that drugs with a functional thiol group ("thiol drugs") may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore the antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection, but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury, and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.

Thiol drugs decrease SARS-CoV-2 lung injury in vivo and disrupt SARS-CoV-2 spike complex binding to ACE2 in vitro.

Neutrophil-induced oxidative stress is a mechanism of lung injury in COVID-19, and drugs with a functional thiol group ("thiol drugs"), especially cysteamine, have anti-oxidant and anti-inflammatory properties that could limit this injury. Thiol drugs may also alter the redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) and thereby disrupt ACE2 binding. Using ACE2 binding assay, reporter virus pseudotyped with SARS-CoV-2 spikes (ancestral and variants) and authentic SARS-CoV-2 (Wuhan-1), we find that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus entry into cells. Pseudoviruses carrying variant spikes were less efficiently inhibited as compared to pseudotypes bearing an ancestral spike, but the most potent drugs still inhibited the Delta variant in the low millimolar range. IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. In hamsters infected with SARS-CoV-2, intraperitoneal (IP) cysteamine decreased neutrophilic inflammation and alveolar hemorrhage in the lungs but did not decrease viral infection, most likely because IP delivery could not achieve millimolar concentrations in the airways. These data show that thiol drugs inhibit SARS-CoV-2 infection in vitro and reduce SARS-CoV-2-related lung injury in vivo and provide strong rationale for trials of systemically delivered thiol drugs as COVID-19 treatments. We propose that antiviral effects of thiol drugs in vivo will require delivery directly to the airways to ensure millimolar drug concentrations and that thiol drugs with lower thiol pKa values are most likely to be effective.

Using ion channel-forming peptides to quantify protein-ligand interactions.

This paper proposes a method for sensing affinity interactions by triggering disruption of self-assembly of ion channel-forming peptides in planar lipid bilayers. It shows that the binding of a derivative of alamethicin carrying a covalently attached sulfonamide ligand to carbonic anhydrase II (CA II) resulted in the inhibition of ion channel conductance through the bilayer. We propose that the binding of the bulky CA II protein (MW approximately 30 kD) to the ion channel-forming peptides (MW approximately 2.5 kD) either reduced the tendency of these peptides to self-assemble into a pore or extracted them from the bilayer altogether. In both outcomes, the interactions between the protein and the ligand lead to a disruption of self-assembled pores. Addition of a competitive inhibitor, 4-carboxybenzenesulfonamide, to the solution released CA II from the alamethicin-sulfonamide conjugate and restored the current flow across the bilayer by allowing reassembly of the ion channels in the bilayer. Time-averaged recordings of the current over discrete time intervals made it possible to quantify this monovalent ligand binding interaction. This method gave a dissociation constant of approximately 2 microM for the binding of CA II to alamethicin-sulfonamide in the bilayer recording chamber: this value is consistent with a value obtained independently with CA II and a related sulfonamide derivative by isothermal titration calorimetry.

Eliminating positively charged lysine epsilon-NH3+ groups on the surface of carbonic anhydrase has no significant influence on its folding from sodium dodecyl sulfate.

This study compares the folding of two polypeptides--bovine carbonic anhydrase (BCA) and peracetylated BCA (BCA-Ac(18))--having the same sequence of amino acids but differing by 18 formal units of charge, from a solution containing denaturing concentrations of sodium dodecyl sulfate (SDS). Acetylation of BCA with acetic anhydride converts all 18 lysine-epsilon-NH(3)(+) groups to lysine-epsilon-NHCOCH(3) groups and generates BCA-Ac(18). Both BCA and BCA-Ac(18) are catalytically active, and circular dichroism spectroscopy (CD) suggests that they have similar secondary and tertiary structures. SDS at concentrations above approximately 10 mM denatured both proteins. When the SDS was removed by dialysis, both proteins were regenerated in native form. This study suggests that large differences in the net charge of the polypeptide have no significant influence on the structure, the ability to refold, or the rate of refolding of this protein from solutions containing SDS. This study reinforces the idea that charged residues on the surface of BCA do not guide protein folding and raises the broader question of why proteins have charged residues on their surface, outside of the region of the active site.