ACS Bio & Med Chem Au最新文献

This study introduces novel cospray-dried (Co-SD) formulations of simvastatin, a Nrf2 activator ROCK inhibitor, with l-carnitine as molecular mixtures in various molar ratios for targeted pulmonary inhalation aerosol delivery in pulmonary hypertension, optimized for excipient-free dry powder inhalers (DPIs). The two components were spray-dried at various molar ratios by using different starting feed solution concentrations and process parameters. In addition to comprehensive physicochemical characterization, in vitro aerosol dispersion performance as DPIs using two FDA-approved DPI devices with different shear stress properties, in vitro viability as a function of dose on 2D human pulmonary cellular monolayers and on 3D small airway epithelia human primary cultures at the air–liquid interface (ALI), and in vitro transepithelial electrical resistance (TEER) at the ALI were conducted. Solid-state physicochemical characterization confirmed homogeneous molecular mixtures and the crystalline nature of the Co-SD formulations. In vitro aerosolization dispersion performance demonstrated that all Co-SD dual combination molecular mixtures aerosolized successfully with both human FDA-approved DPI devices, had ∼100% emitted dose, and good fine particle fraction values. The in vitro viability and TEER assays demonstrated that all formulations were safe to the human pulmonary cell as 2D and 3D cultures as a function of dose.

This study introduces novel cospray-dried (Co-SD) formulations of simvastatin, a Nrf2 activator ROCK inhibitor, with l-carnitine as molecular mixtures in various molar ratios for targeted pulmonary inhalation aerosol delivery in pulmonary hypertension, optimized for excipient-free dry powder inhalers (DPIs). The two components were spray-dried at various molar ratios by using different starting feed solution concentrations and process parameters. In addition to comprehensive physicochemical characterization, in vitro aerosol dispersion performance as DPIs using two FDA-approved DPI devices with different shear stress properties, in vitro viability as a function of dose on 2D human pulmonary cellular monolayers and on 3D small airway epithelia human primary cultures at the air-liquid interface (ALI), and in vitro transepithelial electrical resistance (TEER) at the ALI were conducted. Solid-state physicochemical characterization confirmed homogeneous molecular mixtures and the crystalline nature of the Co-SD formulations. In vitro aerosolization dispersion performance demonstrated that all Co-SD dual combination molecular mixtures aerosolized successfully with both human FDA-approved DPI devices, had ∼100% emitted dose, and good fine particle fraction values. The in vitro viability and TEER assays demonstrated that all formulations were safe to the human pulmonary cell as 2D and 3D cultures as a function of dose.

Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes constitute a large subclass of radical SAM (RS) enzymes that use Cbl to catalyze various types of reactions, the most common of which are methylations. Most Cbl-dependent RS enzymes contain an N-terminal Rossmann fold that aids Cbl binding. Recently, it has been demonstrated that the methanogenesis marker protein 10 (Mmp10) requires Cbl to methylate an arginine residue in the α-subunit of methyl coenzyme M reductase. However, Mmp10 contains a Cbl-binding domain in the C-terminal region of its primary structure that does not share significant sequence similarity with canonical RS Cbl-binding domains. Bioinformatic analysis of Mmp10 identified DUF512 (Domain of Unknown Function 512) as a potential Cbl-binding domain in RS enzymes. In this paper, four randomly selected DUF512-containing proteins from various organisms were overexpressed, purified, and shown to bind Cbl. X-ray crystal structures of DUF512-containing proteins from Clostridium sporogenes and Pyrococcus furiosus were determined, confirming their C-terminal Cbl-binding domains. The structure of the DUF512-containing protein from C. sporogenes is the first of an RS enzyme containing a PDZ domain. Its RS domain has an unprecedented β₃α₄ core, whereas most RS enzymes adopt a (βα)₆ core. The DUF512-containing protein from P. furiosus has no PDZ domain, but its RS domain also has an uncommon (βα)₅ core.

Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes constitute a large subclass of radical SAM (RS) enzymes that use Cbl to catalyze various types of reactions, the most common of which are methylations. Most Cbl-dependent RS enzymes contain an N-terminal Rossmann fold that aids Cbl binding. Recently, it has been demonstrated that the methanogenesis marker protein 10 (Mmp10) requires Cbl to methylate an arginine residue in the α-subunit of methyl coenzyme M reductase. However, Mmp10 contains a Cbl-binding domain in the C-terminal region of its primary structure that does not share significant sequence similarity with canonical RS Cbl-binding domains. Bioinformatic analysis of Mmp10 identified DUF512 (Domain of Unknown Function 512) as a potential Cbl-binding domain in RS enzymes. In this paper, four randomly selected DUF512-containing proteins from various organisms were overexpressed, purified, and shown to bind Cbl. X-ray crystal structures of DUF512-containing proteins from Clostridium sporogenes and Pyrococcus furiosus were determined, confirming their C-terminal Cbl-binding domains. The structure of the DUF512-containing protein from C. sporogenes is the first of an RS enzyme containing a PDZ domain. Its RS domain has an unprecedented β₃α₄ core, whereas most RS enzymes adopt a (βα)₆ core. The DUF512-containing protein from P. furiosus has no PDZ domain, but its RS domain also has an uncommon (βα)₅ core.

The high throughput YESS 2.0 platform was used to screen a large library of SARS-CoV-2 M^pro variants in the presence of nirmatrelvir. Of the 100 individual most prevalent mutations identified in the screen and reported here, the most common were E166V, L27V, N142S, A173V, and Y154N, along with their various combinations. In vitro analysis revealed that resistance to nirmatrelvir for these individual mutations, as well as all of the combinations we analyzed, was accompanied by decreased catalytic activity with the native substrate. Importantly, the mutations we identified have not appeared as significantly enriched in SARS-CoV-2 M^pro sequences isolated from COVID-19 patients following the introduction of nirmatrelvir. We also analyzed three of the most common SARS-CoV-2 M^pro mutations that have been seen in patients recently, and only a measured increase in nirmatrelvir resistance was seen when the more recently appearing A285V is added to both P132H and K90R. Taken together, our results predict that resistance to nirmatrelvir will be slower to develop than expected based on experience with other viral protease inhibitors, perhaps due in part to the close structural correspondence between nirmatrelvir and SARS-CoV-2 M^pro's preferred substrates.

The high throughput YESS 2.0 platform was used to screen a large library of SARS-CoV-2 M^pro variants in the presence of nirmatrelvir. Of the 100 individual most prevalent mutations identified in the screen and reported here, the most common were E166V, L27V, N142S, A173V, and Y154N, along with their various combinations. In vitro analysis revealed that resistance to nirmatrelvir for these individual mutations, as well as all of the combinations we analyzed, was accompanied by decreased catalytic activity with the native substrate. Importantly, the mutations we identified have not appeared as significantly enriched in SARS-CoV-2 M^pro sequences isolated from COVID-19 patients following the introduction of nirmatrelvir. We also analyzed three of the most common SARS-CoV-2 M^pro mutations that have been seen in patients recently, and only a measured increase in nirmatrelvir resistance was seen when the more recently appearing A285V is added to both P132H and K90R. Taken together, our results predict that resistance to nirmatrelvir will be slower to develop than expected based on experience with other viral protease inhibitors, perhaps due in part to the close structural correspondence between nirmatrelvir and SARS-CoV-2 M^pro’s preferred substrates.