Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Substrate specificity of MtSerB2.

<p>Relative change in the hydrolysis of different substrates. l- PS depicts l-phosphoserine, l- PT depicts l-phosphothreonine, l- PTy depicts l-phosphotyrosine, G-6-P depicts Glucose-6-phosphate and ATP is adenosine triphosphate. The experiment was performed in triplicate and the values represent the average. Inset shows a close-up of the active site and the docked moiety is indicated in stick representation. l-phosphotyrosine is occluded from the active site due to steric hindrance while l-phosphoserine fits well in the active site.</p

Characterization of <i>M. tuberculosis</i> SerB2, an Essential HAD-Family Phosphatase, Reveals Novel Properties

<div><p><i>M. tuberculosis</i> harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.</p></div

Molecular Docking and Isothermal calorimetry experiments.

<p>(<b>A</b>) <b>Docking modes of l-serine with MtSerB2</b>. l-serine was docked against the binding sites predicted in the ACTI and ACTII domains respectively. Interactions of l-serine with ACTI (<i>top</i>) and ACTII (<i>bottom</i>) domains are shown here. Key interacting residues are labeled in <i>black</i> and shown in stick representation while the rest of the site is shown in cartoon representation. l-serine is depicted in ball- and-stick representation. (<b>B</b>) <b>ITC experiments involving interactions of l-serine with MtSerB2</b>. Titration of l-serine (300 µM) into MtSerB2 solution (30 µM). The experiments were performed in 50 mM sodium phosphate buffer, pH 7.4, and 50 mM NaCl and 2 mM β mercaptoethanol at 25°C. The cell volume was 1.43 ml while the injection volume was 6 µl.</p

Determination of K_m and K_cat values for l-phosphoserine.

<p>Michaelis-Menten plots calculated using l-phosphoserine as the substrate, for (<b>A</b>) MtSerB2 and (<b>B</b>) PSPD respectively. Inset shows double-reciprocal plots of the initial velocities (1/Vo) against the reciprocal of l-phosphoserine phosphate. The experiment was performed in triplicate and the values represent their average.</p

Effect of various factors on functional properties of MtSerB2 and its phosphatase domain (PSPD) respectively.

<p>(<b>A</b>) Relative change in hydrolysis of l-phosphoserine with increasing pH. Hydrolysis at pH 7.5 was taken as 100%. (<b>B</b>) Changes in the enzyme activity of MtSerB2 on increasing the temperature. Data are shown in percentages with enzyme activity observed for MtSerB2 at 37°C taken as 100%. (<b>C</b>) Effect of divalent cations on enzymatic hydrolysis of l-phosphoserine by MtSerB2. Relative activity was measured at concentrations of different divalent cations and that with Mg²⁺ was considered as 100%. Inset shows inhibition by Calcium Chloride. (<b>D</b>) Effect of varying the temperature on the phosphatase activity of PSPD. (<b>E</b>) Optimal pH for maximum substrate hydrolysis.</p

Fluorescent microscopy experiments involving THP-1 cells shows alteration in cell microtubules in the presence of MtSerB2.

<p>Exogenous addition of purified MtSerB2 protein to THP-1 cells induces microtubule rearrangements. Cells were stained for α-tubulin (<i>red</i>) and images were taken at 60× magnification. Arrows indicate the presence of enriched tubulin at the cell surface. Controls (vehicle) contained enzyme buffer only. Additional controls involve the D341N catalytic site mutant which induced very less/negligible tubulin rearrangement and this is attributed to the residual activity possessed by the mutant.</p

Piscidin-1-analogs with double L- and D-lysine residues exhibited different conformations in lipopolysaccharide but comparable anti-endotoxin activities

To become clinically effective, antimicrobial peptides (AMPs) should be non-cytotoxic to host cells. Piscidins are a group of fish-derived AMPs with potent antimicrobial and antiendotoxin activities but limited by extreme cytotoxicity. We conjectured that introduction of cationic residue(s) at the interface of polar and non-polar faces of piscidins may control their insertion into hydrophobic mammalian cell membrane and thereby reducing cytotoxicity. We have designed several novel analogs of piscidin-1 by substituting threonine residue(s) with L and D-lysine residue(s). L/D-lysine-substituted analogs showed significantly reduced cytotoxicity but exhibited either higher or comparable antibacterial activity akin to piscidin-1. Piscidin-1-analogs demonstrated higher efficacy than piscidin-1 in inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory responses in THP-1 cells. T15,21K-piscidin-1 (0.5 mg/Kg) and T15,21dK-piscidin-1 (1.0 mg/Kg) demonstrated 100% survival of LPS (12.0 mg/Kg)-administered mice. High resolution NMR studies revealed that both piscidin-1 and T15,21K-piscidin-1 adopted helical structures, with latter showing a shorter helix, higher amphipathicity and cationic residues placed at optimal distances to form ionic/hydrogen bond with lipid A of LPS. Remarkably, T15,21dK-piscidin-1 showed a helix-loop-helix structure in LPS and its interactions with LPS could be sustained by the distance of separation of side chains of R7 and D-Lys-15 which is close to the inter-phosphate distance of lipid A.MOE (Min. of Education, S’pore)Published versio

Size exclusion chromatography experiments involving MtSerB2, its mutants and l-serine.

<p>(<b>A</b>) <b>Wildtype SerB2</b> (<b>B</b>) <b>D341N</b> (<b>C</b>) <b>G18A</b> (D) <b>G108A</b>. Chromatogram in the absence of l-serine is in <i>black</i>, whereas the chromatogram in the presence of of l-serine, and MtSerB2 and its mutants, are shown in grey. Wild-type MtSerB2 and D341N show a shift to the tetrameric/higher order oligomeric forms in the presence of ∼0.8 molar ratio of l-serine.</p