Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Auto-amplification and spatial propagation of neutrophil extracellular traps.

The release of cellular DNA as neutrophil extracellular traps (NETs) plays a pivotal role in the immune response to pathogens by physically entrapping and killing microbes. NET release occurs at a greater frequency within neutrophil clusters and swarms, indicating a potential for collective behavior. However, little is known about how dense clustering of cells influences the frequency of NET release. Using an image-based assay for NETosis in nanowells, we show that the frequency of NETosis increases with cell density. We then co-incubate NETotic neutrophils with naïve neutrophils and find that NETotic neutrophils can induce secondary NETosis in naïve neutrophils in a cell density-dependent manner. Further mechanistic studies show that secondary NETosis is caused by a combination of DNA and protein factors. Finally, we immobilize NETotic neutrophils in a plaque, and then place the plaque near naïve neutrophils to characterize the spatial propagation of secondary NETosis. We find that secondary NETosis from naïve neutrophils increases over time, but remains spatially restricted to the periphery of the plaque. Together, we show that NETosis is an auto-amplified process, but that the spatial propagation of NET release is strictly regulated.

Lactobacillus for preventing recurrent urinary tract infections in women: meta-analysis.

INTRODUCTION: Urinary tract infections (UTIs) are the most common infections affecting women, and often recur. Lactobacillus probiotics could potentially replace low dose, long term antibiotics as a safer prophylactic for recurrent UTI (rUTI). This systematic review and meta-analysis was performed to compile the results of existing randomized clinical trials (RCTs) to determine the efficacy of probiotic Lactobacillus species in preventing rUTI. MATERIALS AND METHODS: MEDLINE and EMBASE were searched from inception to July 2012 for RCTs using a Lactobacillus prophylactic against rUTI in premenopausal adult women. A random-effects model meta-analysis was performed using a pooled risk ratio, comparing incidence of rUTI in patients receiving Lactobacillus to control. RESULTS: Data from 294 patients across five studies were included. There was no statistically significant difference in the risk for rUTI in patients receiving Lactobacillus versus controls, as indicated by the pooled risk ratio of 0.85 (95% confidence interval of 0.58-1.25, p = 0.41). A sensitivity analysis was performed, excluding studies using ineffective strains and studies testing for safety. Data from 127 patients in two studies were included. A statistically significant decrease in rUTI was found in patients given Lactobacillus, denoted by the pooled risk ratio of 0.51 (95% confidence interval 0.26-0.99, p = 0.05) with no statistical heterogeneity (I2 = 0%). CONCLUSION: Probiotic strains of Lactobacillus are safe and effective in preventing rUTI in adult women. However, more RCTs are required before a definitive recommendation can be made since the patient population contributing data to this meta-analysis was small.

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome.

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

Hydroxamic Acid Inhibitors Provide Cross-Species Inhibition of Plasmodium M1 and M17 Aminopeptidases.

There is an urgent clinical need for antimalarial compounds that target malaria caused by both Plasmodium falciparum and Plasmodium vivax. The M1 and M17 metalloexopeptidases play key roles in Plasmodium hemoglobin digestion and are validated drug targets. We used a multitarget strategy to rationally design inhibitors capable of potent inhibition of the M1 and M17 aminopeptidases from both P. falciparum ( Pf-M1 and Pf-M17) and P. vivax ( Pv-M1 and Pv-M17). The novel chemical series contains a hydroxamic acid zinc binding group to coordinate catalytic zinc ion/s, and a variety of hydrophobic groups to probe the S1' pockets of the four target enzymes. Structural characterization by cocrystallization showed that selected compounds utilize new and unexpected binding modes; most notably, compounds substituted with bulky hydrophobic substituents displace the Pf-M17 catalytic zinc ion. Excitingly, key compounds of the series potently inhibit all four molecular targets and show antimalarial activity comparable to current clinical candidates.

Novel Human Aminopeptidase N Inhibitors: Discovery and Optimization of Subsite Binding Interactions.

Aminopeptidase N (APN/CD13) is a zinc-dependent M1 aminopeptidase that contributes to cancer progression by promoting angiogenesis, metastasis, and tumor invasion. We have previously identified hydroxamic acid-containing analogues that are potent inhibitors of the APN homologue from the malarial parasite Plasmodium falciparum M1 aminopeptidase (PfA-M1). Herein, we describe the rationale that underpins the repurposing of PfA-M1 inhibitors as novel APN inhibitors. A series of novel hydroxamic acid analogues were developed using a structure-based design approach and evaluated their inhibition activities against APN. N-(2-(Hydroxyamino)-2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide (6ad) proved to be an extremely potent inhibitor of APN activity in vitro, selective against other zinc-dependent enzymes such as matrix metalloproteases, and possessed limited cytotoxicity against Ad293 cells and favorable physicochemical and metabolic stability properties. The combined results indicate that compound 6ad may be a useful lead for the development of anticancer agents.

Low-density lipoprotein (LDL)-dependent uptake of Gram-positive lipoteichoic acid and Gram-negative lipopolysaccharide occurs through LDL receptor.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Body temperature and mouse scoring systems as surrogate markers of death in cecal ligation and puncture sepsis.

BACKGROUND: Despite increasing ethical standards for conducting animal research, death is still often used as an endpoint in mouse sepsis studies. Recently, the Murine Sepsis Score (MSS), Mouse Clinical Assessment Score for Sepsis (M-CASS), and Mouse Grimace Scale (MGS) were developed as surrogate endpoint scoring systems for assessing pain and disease severity in mice. The objective of our study was to compare the effectiveness of these scoring systems and monitoring of body temperature for predicting disease progression and death in the cecal ligation and puncture (CLP) sepsis model, in order to better inform selection of surrogate endpoints for death in experimental sepsis. METHODS: C57Bl/6J mice were subjected to control sham surgery, or moderate or severe CLP sepsis. All mice were monitored every 4 h for surrogate markers of death using modified versions of the MSS, M-CASS, and MGS scoring systems until 24 h post-operatively, or until endpoint (inability to ambulate) and consequent euthanasia. RESULTS: Thirty percent of mice subjected to moderate severity CLP reached endpoint by 24 h post-CLP, whereas 100% undergoing severe CLP reached endpoint within 20 h. Modified MSS, M-CASS, and MGS scores all increased, while body temperature decreased, in a time-dependent and sepsis severity-dependent manner, although modified M-CASS scores showed substantial variability. Receiver operating characteristic curves demonstrate that the last recorded body temperature (AUC = 0.88; 95% CI 0.77-0.99), change in body temperature (AUC = 0.89; 95% CI 0.78-0.99), modified M-CASS (AUC = 0.93; 95% CI 0.85-1.00), and modified MSS (AUC = 0.95; 95% CI 0.88-1.01) scores are all robust for predicting death in CLP sepsis, whereas modified MGS (AUC = 0.78; 95% CI 0.63-0.92) is less robust. CONCLUSIONS: The modified MSS and body temperature are effective markers for assessing disease severity and predicting death in the CLP model, and should thus be considered as valid surrogate markers to replace death as an endpoint in mouse CLP sepsis studies.

Resuscitation fluid composition affects hepatic inflammation in a murine model of early sepsis.

BACKGROUND: Fluid resuscitation is a crucial therapy for sepsis, and the use of balanced fluids and/or isotonic albumin may improve patient survival. We have previously demonstrated that resuscitation with normal saline results in increased hepatic leukocyte recruitment in a murine model of sepsis. Given that clinical formulations of albumin are in saline, our objectives were to develop a novel balanced electrolyte solution specifically for sepsis and to determine if supplementing this solution with albumin would improve the inflammatory response in sepsis. METHODS: We developed two novel buffered electrolyte solutions that contain different concentrations of acetate and gluconate, named Seplyte L and Seplyte H, and administered these solutions with or without 5% albumin. Normal saline with or without albumin and Ringer's lactate served as controls. Sepsis was induced by cecal ligation and puncture (CLP), and the liver microvasculature was imaged in vivo at 6 h after CLP to quantify leukocyte recruitment. Hepatic cytokine expression and plasma cell-free DNA (cfDNA) concentrations were also measured. RESULTS: Septic mice receiving either Seplyte fluid showed significant reductions in hepatic post-sinusoidal leukocyte rolling and adhesion compared to normal saline. Hepatic cytokine concentrations varied in response to different concentrations of acetate and gluconate in the novel resuscitation fluids but were unaffected by albumin. All Seplyte fluids significantly increased hepatic TNF-α levels at 6 h compared to control fluids. However, Seplyte H exhibited a similar cytokine profile to the control fluids for all other cytokines, whereas mice given Seplyte L had significantly elevated IL-6, IL-10, KC (CXCL1), and MCP-1 (CCL2). Plasma cfDNA was generally increased during sepsis, but resuscitation fluid composition did not significantly affect cfDNA concentrations. CONCLUSIONS: Electrolyte concentrations and buffer constituents of resuscitation fluids can modulate hepatic cytokine production and leukocyte recruitment in septic mice, while the effects of albumin are modest during early sepsis. Therefore, crystalloid fluid choice should be an important consideration for resuscitation in sepsis, and the effects of fluid composition on inflammation in other organ systems should be studied to better understand the physiological impact of this vital sepsis therapy.

Differential Expression of PCSK9 Modulates Infection, Inflammation, and Coagulation in a Murine Model of Sepsis.

INTRODUCTION: Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets lipoprotein receptors for degradation, thereby reducing hepatic lipid clearance. PCSK9 inhibition reduces mortality in septic mice, presumably through increased hepatic clearance of pathogen lipids due to increased lipoprotein receptor concentrations. However, PCSK9 overexpression in vivo has not been studied in sepsis. Therefore, this study aimed to evaluate the effects of differential PCSK9 expression on systemic infection, inflammation, and coagulation in sepsis. METHODS: Wild-type, PCSK9 knockout (KO), and transgenic (Tg) mice that overexpress PCSK9 were subjected to sham surgery or cecal ligation and puncture (CLP). Bacterial loads were measured in lungs, peritoneal cavity fluid, and blood. Organ pathology was assessed in lungs, liver, and kidneys. Lung myeloperoxidase activity, and plasma concentrations of alanine aminotransferase (ALT), creatinine, cell-free DNA (cfDNA), protein C, thrombin-antithrombin (TAT) complexes, interleukin (IL)-6, and IL-10 were also measured 6 h postoperatively. Morbidity was assessed for 16 h following CLP. RESULTS: Overexpression of PCSK9 in mice increased liver and kidney pathology, plasma IL-6, ALT, and TAT concentrations during sepsis, whereas PCSK9 KO mice exhibited reduced bacterial loads, lung and liver pathology, myeloperoxidase activity, plasma IL-10, and cfDNA during CLP-induced sepsis. All septic mice had reduced plasma levels of protein C, but the protein C ratio relative to normal was significantly decreased in PCSK9 Tg mice. Dyspnea, cyanosis, and overall grimace scores were greatest in septic mice overexpressing PCSK9, whereas PCSK9 KO mice retained core body temperature during sepsis. CONCLUSION: These findings demonstrate that PCSK9 deficiency confers protection against systemic bacterial dissemination, organ pathology, and tissue inflammation, particularly in the lungs and liver, while PCSK9 overexpression exacerbates multi-organ pathology as well as the hypercoagulable and pro-inflammatory states in early sepsis.