Correlation of Experimental and Calculated Inhibition Constants of Protease Inhibitor Complexes.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.

Structure and biochemical characterization of l-2-hydroxyglutarate dehydrogenase and its role in the pathogenesis of l-2-hydroxyglutaric aciduria.

l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.

Lactate dehydrogenase D is a general dehydrogenase for D-2-hydroxyacids and is associated with D-lactic acidosis.

Mammalian lactate dehydrogenase D (LDHD) catalyzes the oxidation of D-lactate to pyruvate. LDHD mutations identified in patients with D-lactic acidosis lead to deficient LDHD activity. Here, we perform a systematic biochemical study of mouse LDHD (mLDHD) and determine the crystal structures of mLDHD in FAD-bound form and in complexes with FAD, Mn2+ and a series of substrates or products. We demonstrate that mLDHD is an Mn2+-dependent general dehydrogenase which exhibits catalytic activity for D-lactate and other D-2-hydroxyacids containing hydrophobic moieties, but no activity for their L-isomers or D-2-hydroxyacids containing hydrophilic moieties. The substrate-binding site contains a positively charged pocket to bind the common glycolate moiety and a hydrophobic pocket with some elasticity to bind the varied hydrophobic moieties of substrates. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of LDHD, and the functional roles of mutations in the pathogenesis of D-lactic acidosis.

Molecular insights into the catalysis and regulation of mammalian NAD-dependent isocitrate dehydrogenases.

Eukaryotic NAD-dependent isocitrate dehydrogenases (NAD-IDHs) are mitochondria-localized enzymes which catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate using NAD as a cofactor. In mammals, NAD-IDHs (or IDH3) consist of three types of subunits (α, ß, and γ), and exist as (α2ßγ)2 heterooctamer. Mammalian NAD-IDHs are regulated allosterically and/or competitively by a diversity of metabolites including citrate, ADP, ATP, NADH, and NADPH, which are associated with cellular metabolite flux, energy demands, and redox status. Proper assembly of the component subunits is essential for the catalysis and regulation of the enzymes. Recently, crystal structures of human IDH3 have been solved in apo form and in complex with various ligands, revealing the molecular mechanisms for the assembly, catalysis, and regulation of the enzyme.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Digital Therapeutics in Migraine Management: A Novel Treatment Option in the COVID-19 Era.

Migraine is a chronic and often lifelong disease that directly affects over one billion people globally. Because access to migraine medical services is limited, only a minority of migraine patients are treated adequately. This situation worsened during the COVID-19 pandemic. Digital therapeutics (DTx) is an emerging therapeutic approach that opens up many new possibilities for remote migraine management. For instance, migraine management tools, online migraine diagnosis, guideline-based treatment options, digitally networked patients, and collecting anonymized information about migraine attacks and course parameters for scientific evaluation. Various applications of DTx in migraine management have been studied in recent years, such as the usefulness of digital migraine self-management tools in diagnosing and tracking migraine attacks, and the efficacy and safety of digital cognitive behavioural therapy. However, the development of DTx is still in its infancy and still faces many obstacles. The primary goal of this study is to review the latest research on DTx in migraine management, identify challenges, and outline future trends.

Structures of a constitutively active mutant of human IDH3 reveal new insights into the mechanisms of allosteric activation and the catalytic reaction.

Human NAD-dependent isocitrate dehydrogenase or IDH3 (HsIDH3) catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the tricarboxylic acid cycle. It consists of three types of subunits (α, ß, and γ) and exists and functions as the (αßαγ)2 heterooctamer. HsIDH3 is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. Our previous studies have revealed the molecular basis for the activity and regulation of the αß and αγ heterodimers. However, the molecular mechanism for the allosteric activation of the HsIDH3 holoenzyme remains elusive. In this work, we report the crystal structures of the αß and αγ heterodimers and the (αßαγ)2 heterooctamer containing an α-Q139A mutation in the clasp domain, which renders all the heterodimers and the heterooctamer constitutively active in the absence of activators. Our structural analysis shows that the α-Q139A mutation alters the hydrogen-bonding network at the heterodimer-heterodimer interface in a manner similar to that in the activator-bound αγ heterodimer. This alteration not only stabilizes the active sites of both αQ139Aß and αQ139Aγ heterodimers in active conformations but also induces conformational changes of the pseudo-allosteric site of the αQ139Aß heterodimer enabling it to bind activators. In addition, the αQ139AICT+Ca+NADßNAD structure presents the first pseudo-Michaelis complex of HsIDH3, which allows us to identify the key residues involved in the binding of cofactor, substrate, and metal ion. Our structural and biochemical data together reveal new insights into the molecular mechanisms for allosteric regulation and the catalytic reaction of HsIDH3.

Neutralization mechanism of a human antibody with pan-coronavirus reactivity including SARS-CoV-2.

Frequent outbreaks of coronaviruses underscore the need for antivirals and vaccines that can counter a broad range of coronavirus types. We isolated a human antibody named 76E1 from a COVID-19 convalescent patient, and report that it has broad-range neutralizing activity against multiple α- and ß-coronaviruses, including the SARS-CoV-2 variants. 76E1 also binds its epitope in peptides from γ- and δ-coronaviruses. 76E1 cross-protects against SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and therapeutic murine animal models. Structural and functional studies revealed that 76E1 targets a unique epitope within the spike protein that comprises the highly conserved S2' site and the fusion peptide. The epitope that 76E1 binds is partially buried in the structure of the SARS-CoV-2 spike trimer in the prefusion state, but is exposed when the spike protein binds to ACE2. This observation suggests that 76E1 binds to the epitope at an intermediate state of the spike trimer during the transition from the prefusion to the postfusion state, thereby blocking membrane fusion and viral entry. We hope that the identification of this crucial epitope, which can be recognized by 76E1, will guide epitope-based design of next-generation pan-coronavirus vaccines and antivirals.

Imaging moiré deformation and dynamics in twisted bilayer graphene.

In 'magic angle' twisted bilayer graphene (TBG) a flat band forms, yielding correlated insulator behavior and superconductivity. In general, the moiré structure in TBG varies spatially, influencing the overall conductance properties of devices. Hence, to understand the wide variety of phase diagrams observed, a detailed understanding of local variations is needed. Here, we study spatial and temporal variations of the moiré pattern in TBG using aberration-corrected Low Energy Electron Microscopy (AC-LEEM). We find a smaller spatial variation than reported previously. Furthermore, we observe thermal fluctuations corresponding to collective atomic displacements over 70 pm on a timescale of seconds. Remarkably, no untwisting is found up to 600 ∘C. We conclude that thermal annealing can be used to decrease local disorder. Finally, we observe edge dislocations in the underlying atomic lattice, the moiré structure acting as a magnifying glass. These topological defects are anticipated to exhibit unique local electronic properties.

Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase.

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Amino Acid Scanning at P5' within the Bowman-Birk Inhibitory Loop Reveals Specificity Trends for Diverse Serine Proteases.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-amino acid cyclic peptide that shares an inhibitory loop with a sequence and structure similar to a larger family of serine protease inhibitors, the Bowman-Birk inhibitors. Here, we focus on the P5' residue in the Bowman-Birk inhibitory loop and produce a library of SFTI variants to characterize the P5' specificity of 11 different proteases. We identify seven amino acids that are generally preferred by these enzymes and also correlate with P5' sequence diversity in naturally occurring Bowman-Birk inhibitors. Additionally, we show that several enzymes have divergent specificities that can be harnessed in engineering studies. By optimizing the P5' residue, we improve the potency or selectivity of existing inhibitors for kallikrein-related peptidase 5 and show that a variant with substitutions at 7 of the scaffold's 14 residues retains a similar structure to SFTI-1. These findings provide new insights into P5' specificity requirements for the Bowman-Birk inhibitory loop.

Potent, multi-target serine protease inhibition achieved by a simplified ß-sheet motif.

Engagement of an extended ß-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like ß-sheet to enzyme inhibition. Here we report the crystal structure of an simplified ß-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by ß-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.

Exposure to Nitrogen Oxide in the First Trimester and Risk of Cardiovascular-Related Malformations: A Dose-Response Meta-Analysis of Observational Studies.

Nitrogen oxide (NO x ) is produced during combustion at high temperature, which is a major constituent of air pollutants. Recent studies suggested inconsistent results on the association between NO x exposure and cardiovascular-related malformations. We aimed to assess aforementioned association in pregnant women in the first trimester and cardiovascular-related malformations of infants. A systematic literature review identified studies for observational studies about NO x exposure and cardiovascular-related malformation in PubMed. Random-effect models were used to estimate summary odds ratio (SOR) and 95% confidence intervals (CIs) for aforementioned association. Finally, nine studies met the inclusion criteria. Overall, the SOR of cardiovascular-related malformation per 10 ppb increment in NO x and NO2 concentration was 1.01 (95% CI: 0.98-1.04; I2 = 38.6%, P = 0.09) and 0.99 (95% CI: 0.95-1.04; I2 = 37.8%, P = 0.13), respectively. Stratifying by study design, geographic locations, and confounded adjustments, the majority of strata showed negative results, which were consistent with the main findings. However, we found that exposure to NO x and NO2 in the first trimester increased the risk of coarctation of the aorta (COA) malformation by 13% and 19%, respectively. Our study provided limited evidence regarding the association between NO x exposure in the first trimester and cardiovascular-related malformations in infants.

EGF stimulates glioblastoma metastasis by induction of matrix metalloproteinase-9 in an EGFR-dependent mechanism.

Epidermal growth factor (EGF) and EGF receptor (EGFR) play prominent roles in the metastasis of glioblastoma (GBM). However, the molecular mechanisms for the function of EGF and EGFR in GBM metastasis have not been elucidated. Herein, we demonstrate that coactivation of EGF and EGFR drives tumor metastasis in a matrix metalloproteinase-9 (MMP-9)-dependent manner. Expression levels of EGF, EGFR, and MMP-9 were substantially upregulated in the GBM and edema zones of patients, compared with those of paired unaffected participants. Secretion of EGF and MMP-9 was reduced in the cerebrospinal fluid (CSF) after removing GBM for 2 weeks by operation. To the mechanism, MMP-9 was upregulated by activating EGF and EGFR via PI3K/AKT- and ERK1/2-dependent pathways. Moreover, signal transducer and activator of transcription (STAT) 3 and STAT5 mediated the activation of NF-κB by PI3K/AKT and ERK1/2 pathways. This resulted in transactivation of MMP-9 in GBM. Finally, MMP-9 induction facilitated abnormal proliferation, migration, and invasion of cells, which contributed to GBM metastasis.

Developing an institute of medicine-aligned framework for categorizing primary care indicators for quality assessment.

The Institute of Medicine (IOM) framework has been used frequently to assess and monitor quality in secondary and tertiary care, but not in primary care. This article describes and proposes a conceptual framework for categorizing primary care indicators that align with the IOM's six aims for quality in healthcare performance (Safe, Effective, Patient-Centred, Timely, Efficient and Equitable.) Using an iterative process, the authors developed and compared a primary care framework for categorizing indicators in the Quality in Family Practice Book of Tools (QBT) with the IOM aims and other local healthcare systems frameworks (Integrated and Continuous, Appropriate Practice Resources). They also compared, cross-matched and analyzed their QBT categories and indicators with other international primary care assessment tools. And they compared the QBT titles and descriptions of groups of indicators with those published in the international tools.