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). 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 βENaC-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 βENaC-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 βENaC-Tg mice and reduced mortality from 37% in vehicle-treated βENaC-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 βENaC-Tg mice. These data provide rationale for human trials of MUC-031 in muco-obstructive lung diseases. MUC-031, a novel thiol-saccharide mucolytic drug, is potent and fast acting in rheology-based sputum assays and improves mucus obstruction, airway inflammation and survival in a mouse model of muco-obstructive lung disease http://bit.ly/3Z2UIVQ

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.

Higher than normal secretion of mucin, a molecular component of mucus, is a feature of many lung diseases. The development of a peptide that blocks mucin secretion in airway epithelial cells might lead to therapies.

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.e. L-leucine and L-isoleucine) in enhancing aerosolization performance and in mitigating moisture-induced changes in spray dried trehalose formulations. Trehalose was spray dried with 20-60% w/w of amino acid (i.e. L-leucine or L-isoleucine). The spray dried formulations were stored at 25 degrees C/50% RH for 28 days. Solid state characterization and in vitro aerosolization performance studies were performed on the spray dried formulations before and after storage. The addition of 20-60% w/w of amino acid (i.e. L-leucine or L-isoleucine) improved the emitted fractions of spray dried trehalose formulations from a dry powder inhaler. However, >= 40% w/w of L-leucine/L-isoleucine was needed to prevent recrystallization of trehalose in the formulations when exposed to 25 degrees C/50% RH for 28 days. X-ray photoelectron spectroscopy (XPS) demonstrated that samples with 40-60% w/w L-isoleucine had more amino acid on the surfaces of the particles compared to their L-leucine counterparts. This may explain the greater ability of the L-isoleucine (40-60% w/w) samples to cope with elevated humidity compared to L-leucine samples of the same concentrations, as observed in the dynamic vapour sorption (DVS) studies. In conclusion, this study demonstrated that both L-leucine and L-isoleucine were effective in enhancing aerosolization performance and mitigating moisture-induced reduction in aerosolization performance in spray dried trehalose formulations. L-isoleucine proved to be superior to L-leucine in terms of its moisture protectant effect when incorporated at the same concentration in the formulations.

The University of California San Francisco (UCSF) Catalyst program is an innovative technology translation mechanism, which supports the development of therapeutics, devices, diagnostics and digital health applications from academic research. The development of therapeutics is considered to be the most challenging area with highest risk of failure, costs and longest timelines. It is widely recognized that there is often a significant knowledge, expertise and capability gap between bench research in an academic environment and the requirements for successful development of therapeutics to achieve licensing or further investment. This includes the development of robust and appropriate target validation datasets, product profiles, hit discovery, compound optimization (i.e. medicinal chemistry, pharmacokinetics, pharmacology, formulation), candidate profile, regulatory documentation & development/regulatory and commercial strategy etc. The majority of academic centers do not have access to in‐house expertise and capabilities in these areas. The UCSF Catalyst program identifies high‐potential projects, links them to the key expertise and capabilities (industry expert advisors, CROs, collaborators) required by the project and funds key activities, with the goal to securing the next stage of investment. The UCSF Catalyst has assembled more than 200 Catalyst Advisors, who are experts from all sectors of the biomedical industry who review projects and and provide consultation and mentorship for projects with the highest product and commercialization potential based on assessment criteria that prioritize unmet need, innovation, develop‐ability, commercial potential and intellectual property. The Catalyst advisors serve as mentors and technical consultants to innovators whose projects are selected to receive funding, which ensures efficient and effective use of limited capital and increases the likelihood that the projects deliver viable and commercializable solutions. The catalyst program also supports the development of academic‐industry collaborations. In 11 cycles, over 5 years, to date Catalyst has supported over 40 therapeutics projects (~50:50 Small molecule: Biological). The majority of projects are in the early target validation through to hit discovery and initial optimization phases. The most frequently required support is with regard to expertise in target validation requirements, support for in vitro or in vivo proof of principle studies, identification and supply of suitable tool compounds, hit identification, competitive landscape and development strategy. In later stage projects, Catalyst advisors have been actively involved in regulatory discussions and the preparation of INDs. Since its initiation 6 years ago, projects supported by the Catalyst program (all tracks) have resulted in more than ten new start‐up companies and many disclosures or patents. Catalyst's return on investment has been greater than ten ‐fold. Many of the therapeutics projects have received significant follow‐on funding and have made significant progress towards clinical assessment. The Catalyst mechanism is a successful approach to enabling the translation of novel therapeutics from the academic bench to the clinic and product development. Support or Funding Information The UCSF Clinical and Translational Science Institute (CTSI) is part of the Clinical and Translational Science Award (CTSA) program funded by the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH) (Grant Number UL1 TR000004)

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.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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+)). These ionotropic hydrogels experience a force in a magnetic field gradient that correlates with the concentration of the paramagnetic cations crosslinking the polymer. In an externally applied magnetic field, the paramagnetic hydrogel spheres assemble into ordered arrays or confined geometrical structures in the regions of highest magnetic field. These spheres can be separated from heterogeneous mixtures of diamagnetic materials using a simple bar magnet. Two applications using these recoverable hydrogel spheres were demonstrated: i) When prepared with embedded indicator dyes bound to paper, the spheres were used as colorimetric sensors for pH. ii) When prepared with embedded activated carbon powder, they were used to remove organic materials from aqueous solutions.

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. This method involves chemical modification of those residues to generate a large number of derivatives of the protein that differ in charge. CE separates those derivatives into groups with the same number of modified charged groups. By studying the influence of charge on the properties of proteins using charge ladders, it is possible to estimate the net charge and hydrodynamic radius and to infer the role of charged residues in ligand binding and protein folding.

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. In urea, the large change in electrostatic interactions not only modifies the free energy of denaturation, but also introduces a stable intermediate into the unfolding pathway. This work shows that modifications of charges on the surfaces of proteins can have a large effect—positive or negative, depending on the denaturant—on the stability of the proteins despite the exposure of these charges to high dielectric solvent and buffer ions.

AbstractFast alle Proteine enthalten geladene Aminosäuren. Während für einzelne Ladungen im aktiven Zentrum die Funktion bei der Katalyse oder der Bindung häufig aufgeklärt werden kann, ist es weniger ersichtlich, welche Funktion den Ladungen außerhalb dieser Region zuzuordnen ist. Sind sie für die Löslichkeit notwendig? Oder haben sie andere Aufgaben? Lassen sich durch Verändern dieser Ladungen die Proteineigenschaften variieren? Eine Kombination aus Kapillarelektrophorese (CE) und “Proteinladungsleitern” ermöglicht es, die Funktionen geladener Reste auf der Proteinoberfläche außerhalb des aktiven Zentrums zu studieren. Dazu werden diese Reste durch Reaktionen modifiziert, die eine große Zahl unterschiedlich geladener Derivate des Proteins liefern. CE trennt diese Derivate in Fraktionen mit der gleichen Zahl modifizierter geladener Gruppen. Die Untersuchung des Ladungseinflusses auf die Proteineigenschaften unter Verwendung von Ladungsleitern ermöglicht es, die Nettoladung und den hydrodynamischen Radius der Proteine zu bestimmen und auf die Rolle geladener Reste bei Ligandbindung und Proteinfaltung zu schließen.

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. Based on a survey of 18 different proteins, we demonstrate that proteins differ in the concentrations of SDS at which they denature, in the rates of unfolding in SDS, and in the profiles of the denaturation pathways. We also demonstrate that these differences can be exploited in the analysis of mixtures.

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. The rates of renaturation of BCA-Ac18 are greater than those of BCA, when these proteins are allowed to refold from a denatured state ([SDS]=10 mM) to a folded state ([SDS]=0.1 to 1.5 mM). On renaturation, the yields of the correctly folded protein (either BCA or BCA-Ac18) decrease with increasing concentration of SDS. At intermediate concentrations of SDS (from 0.7 to 2 mM for BCA, and from 1.5 to 2 mM for BCA-Ac18), both unfolding and refolding of the proteins are too slow to be observed; an alternative process-probably aggregation-competes with refolding of the denatured proteins at those intermediate concentrations. Because it is experimentally impractical to prove equilibrium, it is not possible to establish whether there is a difference in the thermodynamics of unfolding/refolding between BCA and BCA-Ac18.

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 ε−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. The proteins with an intermediate number of modifications are the most stable to denaturation by SDS. There are four competing interactions—two resulting from the change in electrostatics and two resulting from the change in exposed hydrophobic surface area—that determine how a modification affects the stability of a rung of a charge ladder of BCA to denaturation with SDS. A model based on assumptions about how these interactions affect the folded and transition states has been developed and fits the experimental results. Modeling indicates that for each additional acylation, the magnitude of the change in the activation energy of denaturation (ΔΔG‡) due to changes in the electrostatics is much larger than the change in ΔΔG‡ due to changes in the hydrophobicity, but the intermolecular and intramolecular electrostatic effects are opposite in sign. At the high numbers of acylations, hydrophobic interactions cause the hexanoyl-modified BCA to denature nearly three orders of magnitude more rapidly than the acetyl-modified BCA.