Opportunistic retrospective assessment of HUAC, bone density, SFT and breast density on CT images and relationship with of COVID-19

Aim. To evaluate mean Hounsfield unit calculation (HUAC), bone density, subcutaneous fat thickness (SFT), breast density (constitutional imaging biomarkers) and age in symptomatic patients with COVID-19, to assess their correlation with pneumonia severity. Materials and Methods. Between 11 March and 30 May 2020, 272 consecutive symptomatic female patients with COVID-19 who underwent chest CT imaging at baseline were reviewed. HUAC, bone density, SFT and breast density were evaluated retrospectively and statistically compared in cases with negative/positive PCR test results, with/without pneumonia and with mild/moderate-severe pneumonia. Univariate/multivariate logistic regression analyses were applied for estimation of moderate/severe pneumonia. Results. The parameters of age, HUAC, bone density, SFT and breast density were significantly different between patients with/without pneumonia. Additionally, the patients with moderate-severe pneumonia were older, had lower bone density, lower HUAC values, greater SFT and mostly fatty breast density. ROC analysis showed the highest AUC values of 0.763 and 0.744 for age and HUAC, respectively. A combination of HUAC and age was the most accurate model for estimation of moderate/severe pneumonia on logistic regression. Good intraobserver and interobserver reliabilities were detected. Conclusions. The severity of COVID-19 pneumonia among adult females was associated with older age, lower bone density, a lower HUAC value, greater SFT and fatty breast parenchyma. All these factors can be responsible for 21.9% of the development of moderate/severe pneumonia.

A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals.

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an α-ketoamide warhead, a P1 ß-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and 293T cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and cytotoxicity and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a ß-(S-2-oxopyrrolidin-3-yl)-alanyl (Opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 Opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency to inhibit ectopically expressed MPro in human 293T cells. In general, inhibitors with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to cytotoxicity tests on 293T cells and antiviral potency tests on three SARS-CoV-2 variants. They all have relatively low cytotoxicity and high antiviral potency with EC50 values around 1 µM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

Do the rapid influenza diagnostic tests results reflect the truth?

Objectives: Influenza (flu) causes seasonal epidemics and has led to numerous pandemics worldwide. Its rapid diagnosis and treatment are critical. This study evaluated the correlation between rapid influenza diagnostic test (RIDT) results and clinical reflection. Materials and Methods: A total of 795 patients who were performed the RIDT during the 2018-2019 influenza season were included. According to the test results, the patients were then divided into two groups. The Flu+ group was composed of patients with positive diagnostic test results, while the Flu- group was those with negative test results.

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals.

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 positions and the N-terminal protection group, we synthesized 18 tripeptidyl MPro inhibitors that contained also an aldehyde warhead and ß-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with MPro, their inhibition of MPro transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors. These inhibitors have a large variation of determined in vitro enzymatic inhibition IC50 values that range from 4.8 to 650 nM. The determined in vitro enzymatic inhibition IC50 values reveal that relatively small side chains at both P2 and P3 positions are favorable for achieving high in vitro MPro inhibition potency, the P3 position is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. In all structures, the MPro active site cysteine interacts covalently with the aldehyde warhead of the bound inhibitor to form a hemithioacetal that takes an S configuration. For all inhibitors, election density around the N-terminal protection group is weak indicating possible flexible binding of this group to MPro. In MPro, large structural variations were observed on residues N142 and Q189. Unlike their high in vitro enzymatic inhibition potency, most inhibitors showed low potency to inhibit MPro that was transiently expressed in 293T cells. Inhibitors that showed high potency to inhibit MPro transiently expressed in 293T cells all contain O-tert-butyl-threonine at the P3 position. These inhibitors also exhibited relatively low cytotoxicity and high antiviral potency. Overall, our current and previous studies indicate that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for tripeptidyl aldehyde inhibitors of MPro.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 µM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 μM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests. A cell sorting-based assay was developed to study about 30 SARS-CoV-2 main protease inhibitors, revealing MPI8 as the most potent one and others with likely different mechanisms.

Front Cover: MPI8 is Potent against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L (ChemMedChem 1/2022)

The Front Cover shows that MPI8, a SARS-CoV-2 main protease inhibitor, dually inhibits human cathepsin?L to prevent the SARS-CoV-2 RNA genome from leaving the endosome. MPI8 is a reversible covalent inhibitor of the SARS-CoV-2 main protease that displays high potency in inhibiting the virus in vitro. It also selectively inhibits human cathepsin?L over other tested human cathepsins. Cover design by Dr. Xinyu Ma. More information can be found in the Full Paper by Xinyu?R. Ma, Shiqing Xu, Wenshe?Ray Liu et al.

The P3 O-Tert-Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors (preprint)

As an essential enzyme to SARS-CoV-2, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of MPro inhibitors that contain -(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC50 values that range from 4.8 to 650 nM. The determined IC50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro MPro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the -carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

The N-Terminal Carbamate is Key to High Cellular and Antiviral Potency for Boceprevir-Based SARS-CoV-2 Main Protease Inhibitors (preprint)

Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an -ketoamide warhead, a P1 {beta}-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and human host cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a {beta}-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the war-head with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 opal residue, P2 dime-thylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed MPro in human 293T cells. All inhibitors including PF-07321332 with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC50 values around 1 M. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

MPI8 is Potent against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L.

A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro ) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10-3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.

Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics (preprint)

As an essential enzyme of SARS-CoV-2, the pathogen of COVID-19, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor with cellular potency. By coupling this toxicity alleviation with the expression of an MPro-eGFP fusion protein in a human cell host for straightforward characterization with fluorescent flow cytometry, we developed an effective method that allows bulk analysis of cellular potency of MPro inhibitors. In comparison to an antiviral assay in which MPro inhibitors may target host proteases or other processes in the SARS-CoV-2 life cycle to convene strong antiviral effects, this novel assay is more advantageous in providing precise cellular MPro inhibition information for assessment and optimization of MPro inhibitors. We used this assay to analyze 30 literature reported MPro inhibitors including MPI1-9 that were newly developed aldehyde-based reversible covalent inhibitors of MPro, GC376 and 11a that are two investigational drugs undergoing clinical trials for the treatment of COVID-19 patients in United States, boceprevir, calpain inhibitor II, calpain inhibitor XII, ebselen, bepridil that is an antianginal drug with potent anti-SARS-CoV-2 activity, and chloroquine and hydroxychloroquine that were previously shown to inhibit MPro. Our results showed that most inhibitors displayed cellular potency much weaker than their potency in direct inhibition of the enzyme. Many inhibitors exhibited weak or undetectable cellular potency up to 10 M. On contrary to their strong antiviral effects, 11a, calpain inhibitor II, calpain XII, ebselen, and bepridil showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle to convene potent antiviral effects. characterization of these molecules on their antiviral mechanisms will likely reveal novel drug targets for COVID-19. Chloroquine and hydroxychloroquine showed close to undetectable cellular potency to inhibit MPro. Kinetic recharacterization of these two compounds rules out their possibility as MPro inhibitors. Our results also revealed that MPI5, 6, 7, and 8 have high cellular and antiviral potency with both IC50 and EC50 values respectively below 1 M. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Given its strong cellular and antiviral potency, we cautiously suggest that MPI8 is ready for preclinical and clinical investigations for the treatment of COVID-19.

Bepridil is potent against SARS-CoV-2 in vitro.

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC50) value below 100 µM in inhibiting Mpro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting Mpro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors*.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.