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 (βENaC-Tg mice).

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.

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

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.

The use of hydrophobic amino acids in protecting spray dried trehalose formulations against moisture-induced changes.

Trehalose is commonly used as a protein stabilizer in spray dried protein formulations delivered via the pulmonary route. Spray dried trehalose formulations are highly hygroscopic, which makes them prone to deliquescence and recrystallization when exposed to moisture, leading to impairment in aerosolization performance. The main aim of this study was to investigate and compare the effect of hydrophobic amino acids (i.

Fabrication and Manipulation of Ionotropic Hydrogels Crosslinked by Paramagnetic Ions.

This manuscript describes the fabrication and manipulation of millimeter-scale spheres fabricated from ionotropic hydrogels that are crosslinked with paramagnetic metal ions (e.g., Ho(3+)).

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.

Effects of surface charge on denaturation of bovine carbonic anhydrase.

This work compares the denaturation of two proteins-bovine carbonic anhydrase II (BCA) and its derivative with all lysine groups acetylated (BCA-Ac(18))-by urea, guanidinium chloride (GuHCl), heat, and sodium dodecyl sulfate (SDS). It demonstrates that increasing the net negative charge of the protein by acetylation of lysines reduces its stability to urea, GuHCl, and heat, but increases its kinetic stability (its thermodynamic stability cannot be measured) towards denaturation by SDS. Increasing the ionic strength of the buffer improves the stability of BCA-Ac(18) to urea and heat, but still leaves it less stable than unacetylated BCA to those denaturants.

Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS.

This paper shows that proteins display an unexpectedly wide range of behaviors in buffers containing moderate (0.1-10 mM) concentrations of SDS (complete unfolding, formation of stable intermediate states, specific association with SDS, and various kinetic phenomena); capillary electrophoresis provides a convenient method of examining these behaviors. Examination of the dynamics of the response of proteins to SDS offers a way to differentiate and characterize proteins.

Why are proteins charged? Networks of charge-charge interactions in proteins measured by charge ladders and capillary electrophoresis.

Almost all proteins contain charged amino acids. While the function in catalysis or binding of individual charges in the active site can often be identified, it is less clear how to assign function to charges beyond this region. Are they necessary for solubility? For reasons other than solubility? Can manipulating these charges change the properties of proteins? A combination of capillary electrophoresis (CE) and protein charge ladders makes it possible to study the roles of charged residues on the surface of proteins outside the active site.

Increasing the net charge and decreasing the hydrophobicity of bovine carbonic anhydrase decreases the rate of denaturation with sodium dodecyl sulfate.

This study compares the rate of denaturation with sodium dodecyl sulfate (SDS) of the individual rungs of protein charge ladders generated by acylation of the lysine epsilon-NH3+ groups of bovine carbonic anhydrase II (BCA). Each acylation decreases the number of positively charged groups, increases the net negative charge, and increases the hydrophobic surface area of BCA. This study reports the kinetics of denaturation in solutions containing SDS of the protein charge ladders generated with acetic and hexanoic anhydrides; plotting these rates of denaturation as a function of the number of modifications yields a U-shaped curve.

Peracetylated bovine carbonic anhydrase (BCA-Ac18) is kinetically more stable than native BCA to sodium dodecyl sulfate.

Bovine carbonic anhydrase (BCA) and its derivative with all lysine groups acetylated (BCA-Ac18) have different stabilities toward denaturation by sodium dodecyl sulfate (SDS). This difference is kinetic: BCA-Ac18 denatures more slowly than BCA by several orders of magnitude over concentrations of SDS ranging from 2.5 to 10 mM.

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.

Influence of the Zn(II) cofactor on the refolding of bovine carbonic anhydrase after denaturation with sodium dodecyl sulfate.

This paper uses capillary electrophoresis to follow a globular metalloprotein--bovine carbonic anhydrase II (BCA, EC 4.2.1.

Micropatterned agarose gels for stamping arrays of proteins and gradients of proteins.

We describe a method for repetitive and rapid formation of planar microarrays and gradients of proteins using patterned agarose stamps. It demonstrates: (i) micropatterning of agarose gels with feature sizes as small as 2 microm; (ii) inking of posts (diameter 50-1000 microm) on patterned agarose stamps with one or multiple (here, eight) proteins and repetitive stamping of patterns (>100 times in the case of one protein) and arrays (20 times in the case of eight proteins) without the need for intermediate re-inking; (iii) transferring spots of proteins with good homogeneity in surface coverage to glass slides; (iv) applying this technique to surface-based immunoassays; (v) stamping that requires only sub-nanomolar amounts of protein (typically approximately 3 microg in approximately 0.6 microL of solution); (vi) stamping without the need for drying of the proteins, as opposed to stamping with stamps made of poly(dimethylsiloxane); and (vii) patterning gradients of proteins by allowing two proteins to diffuse toward each other in an agarose stamp, followed by printing the protein gradients onto a surface.

Synthesis of monodisperse polymers from proteins.

Proteins are functional biopolymers; viewed as molecules, they are also monodisperse polyamides with chemically reactive side chains. This paper describes the use of proteins as starting materials for the synthesis of monodisperse polymers with nonbiological functionalities attached to the side chains. It demonstrates the complete derivatization of amine groups (lysine side chains and N-termini) on three different proteins by addition of activated carboxylate reagents in aqueous solutions containing sodium dedecyl sulfate (SDS), under denaturing conditions.

Analysis by capillary electrophoresis of the kinetics of charge ladder formation for bovine carbonic anhydrase.

A series of charge ladders of bovine carbonic anhydrase II were synthesized and the relative abundances of the rungs analyzed by capillary electrophoresis as a function of the quantity of acylating agent used. A simulation that models the kinetics of formation of the members of the charge ladders is described. The observed rate constants decreased as the extent of acylation increased.