Toward Unveiling Putative Binding Sites of Interleukin-33: Insights from Mixed-Solvent Molecular Dynamics Simulations of the Interleukin-1 Family.

The interleukin (IL)-1 family is a major proinflammatory cytokine family, ranging from the well-studied IL-1s to the most recently discovered IL-33. As a new focus, IL-33 has attracted extensive research for its crucial immunoregulatory roles, leading to the development of notable monoclonal antibodies as clinical candidates. Efforts to develop small molecules disrupting IL-33/ST2 interaction remain highly desired but encounter challenges due to the shallow and featureless interfaces. The information from relative cytokines has shown that traditional binding site identification methods still struggle in mapping cryptic sites, necessitating dynamic approaches to uncover druggable pockets on IL-33. Here, we employed mixed-solvent molecular dynamics (MixMD) simulations with diverse-property probes to map the hotspots of IL-33 and identify potential binding sites. The protocol was first validated using the known binding sites of two IL-1 family members and then applied to the structure of IL-33. Our simulations revealed several binding sites and proposed side-chain rearrangements essential for the binding of a known inhibitor, aligning well with experimental NMR findings. Further microsecond-time scale simulations of this IL-33-protein complex unveiled distinct binding modes with varying occurrences. These results could facilitate future efforts in developing ligands to target challenging flexible pockets of IL-33 and IL-1 family cytokines in general.

Acute Kidney Injury After Percutaneous Coronary Intervention Guided by Intravascular Ultrasound.

Purpose We investigated the impact of intravascular ultrasound guidance on reducing the incidence of contrast-induced acute kidney injury (CI-AKI) in patients undergoing percutaneous coronary intervention (PCI). Methods Ninety-nine patients were enrolled in this prospective cohort who were not randomly assigned to angiography-guided percutaneous coronary intervention or intravascular ultrasound-guided percutaneous coronary intervention. The patients were hospitalized at the Vietnam National Heart Institute - Bach Mai Hospital between 2019 and 2020. Acute kidney injury incidence during hospitalization was the primary endpoint. Results A total of 99 patients were divided into two groups: the intravascular ultrasound-guided group (33 participants) and the angiography-guided group (66 participants). The mean ± SD contrast volume of each group was 95.2 ± 37.1 mL and 133.0 ± 36.0 mL for the ultrasound-guided and angiography-guided groups, with P < 0.0001. Intravascular imaging-guided percutaneous coronary intervention (IVUS-guided PCI) was associated with reduced acute kidney injury incidence during hospitalization: 0.0% vs. 12.12% and P = 0.049. Conclusions Intravascular ultrasound is a safe imaging tool that guides percutaneous coronary intervention and significantly reduces the rate of acute kidney injury compared to angiography alone. Patients who have a high chance of experiencing acute kidney injury benefit from using intravascular ultrasound.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center.

Nitrosuccinate is a biosynthetic building block in many microbial pathways. The metabolite is produced by dedicated L-aspartate hydroxylases that use NADPH and molecular oxygen as co-substrates. Here, we investigate the mechanism underlying the unusual ability of these enzymes to perform successive rounds of oxidative modifications. The crystal structure of Streptomyces sp. V2 L-aspartate N-hydroxylase outlines a characteristic helical domain wedged between two dinucleotide-binding domains. Together with NADPH and FAD, a cluster of conserved arginine residues forms the catalytic core at the domain interface. Aspartate is found to bind in an entry chamber that is close to but not in direct contact with the flavin. It is recognized by an extensive H-bond network that explains the enzyme's strict substrate-selectivity. A mutant designed to create steric and electrostatic hindrance to substrate binding disables hydroxylation without perturbing the NADPH oxidase side-activity. Critically, the distance between the FAD and the substrate is far too long to afford N-hydroxylation by the C4a-hydroperoxyflavin intermediate whose formation is confirmed by our work. We conclude that the enzyme functions through a catch-and-release mechanism. L-aspartate slides into the catalytic center only when the hydroxylating apparatus is formed. It is then re-captured by the entry chamber where it waits for the next round of hydroxylation. By iterating these steps, the enzyme minimizes the leakage of incompletely oxygenated products and ensures that the reaction carries on until nitrosuccinate is formed. This unstable product can then be engaged by a successive biosynthetic enzyme or undergoes spontaneous decarboxylation to produce 3-nitropropionate, a mycotoxin.

Targeting Olokizumab-Interleukin 6 interaction interface to discover novel IL-6 inhibitors.

The IL-6/IL-6R or IL-6/GP130 protein-protein interactions play a significant role in controlling the development of chronic inflammatory diseases, such as rheumatoid arthritis, Castleman disease, psoriasis, and, most recently, COVID-19. Modulating or antagonizing protein-protein interactions of IL6 binding to its receptors by oral drugs promises similar efficacy to biological therapy in patients, namely monoclonal antibodies. In this study, we used a crystal structure of the Fab part of olokizumab in a complex with IL-6 (PDB ID: 4CNI) to uncover starting points for small molecule IL-6 antagonist discovery. Firstly, a structure­based pharmacophore model of the protein active site cavity was generated to identify possible candidates, followed by virtual screening with a significant database Drugbank. After the docking protocol validation, a virtual screening by molecular docking was carried out and a total of 11 top hits were reported. Detailed analysis of the best scoring molecules was performed with ADME/T analysis and molecular dynamics simulation. Furthermore, the Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) technique has been utilized to evaluate the free binding energy. Based on the finding, one newly obtained compound in this study, namely DB15187, may serve as a lead compound for the discovery of IL-6 inhibitors.Communicated by Ramaswamy H. Sarma.

In silico approach to identify novel allosteric intracellular antagonist for blocking the interleukin-8/CXCR2 receptor signaling pathway.

The role of interleukin-8 (IL-8) and its receptor CXCR2 in inflammatory responses and tumor development and progression has been well documented. Our study aims to discover novel compounds as CXCR2 antagonists to block the IL-8 signaling pathway using an in silico drug design. Herein, a structure-based pharmacophore model was developed based on the crystal structure of inactive CXCR2 in a complex with an allosteric inhibitor. This model was validated and refined, followed by virtual screening with the ZINC15 database. Subsequent molecular docking allows for predicting the best pose of a ligand inside a receptor binding site. We found that the 35 top-ranked hits exhibited docking scores from -30.81 to -25.28 kJ/mol and better interaction potential comparing the reference inhibitor. Analysis of ADME and toxicity properties revealed the efficacy and safety of the selected seven compounds. To validate the stability of the protein-ligand complex structure MD simulations approach has also been performed and confirmed via the critical parameters. The MD results explained that the CXCR2 receptor bound with two best-proposed molecules, including ZINC77105530 and ZINC93176465, was quite stable states as observed from low RMSD, RMSF, Rg, SASA values, and high occupancy of the interaction types. Finally, our data identified that these compounds play as potential inhibitors of IL-8 signaling pathways with the MM/GBSA binding free energies of -41.77 ± 6.45 kcal/mol and -38.84 ± 6.16 kcal/mol, respectively.Communicated by Ramaswamy H. Sarma.

Identification of small molecules as potential inhibitors of interleukin 6: a multi-computational investigation.

IL(interleukin)-6 is a multifunctional cytokine crucial for immunological, hematopoiesis, inflammation, and bone metabolism. Strikingly, IL-6 has been shown to significantly contribute to the initiation of cytokine storm-an acute systemic inflammatory syndrome in Covid-19 patients. Recent study has showed that blocking the IL-6 signaling pathway with an anti-IL-6 receptor monoclonal antibody (mAb) can reduce the severity of COVID-19 symptoms and enhance patient survival. However, the mAb has several drawbacks, such as high cost, potential immunogenicity, and invasive administration due to the large-molecule protein product. Instead, these issues could be mitigated using small molecule IL-6 inhibitors, but none are currently available. This study aimed to discover IL-6 inhibitors based on the PPI with a novel camelid Fab fragment, namely 68F2, in a crystal protein complex structure (PDB ID: 4ZS7). The pharmacophore models and molecular docking were used to screen compounds from DrugBank databases. The oral bioavailability of the top 24 ligands from the screening was predicted by the SwissAMDE tool. Subsequently, the selected molecules from docking and MD simulation illustrated a promising binding affinity in the formation of stable complexes at the active binding pocket of IL-6. Binding energies using the MM-PBSA technique were applied to the top 4 hit compounds. The result indicated that DB08402 and DB12903 could form strong interactions and build stable protein-ligand complexes with IL-6. These potential compounds may serve as a basis for further developing small molecule IL-6 inhibitors in the future.

Structure-based 3D-Pharmacophore modeling to discover novel interleukin 6 inhibitors: An in silico screening, molecular dynamics simulations and binding free energy calculations.

Interleukin 6 (IL-6) is a cytokine with various biological functions in immune regulation, hematopoiesis, and inflammation. Elevated IL-6 levels have been identified in several severe disorders such as sepsis, acute respiratory distress syndrome (ARDS), and most recently, COVID-19. The biological activity of IL-6 relies on interactions with its specific receptor, IL-6Rα, including the membrane-bound IL-6 receptor (mIL-6R) and the soluble IL-6 receptor (sIL-6R). Thus, inhibition of the interaction between these two proteins would be a potential treatment for IL-6 related diseases. To date, no orally available small-molecule drug has been approved. This study focuses on finding potential small molecules that can inhibit protein-protein interactions between IL-6 and its receptor IL-6Rα using its crystal structure (PDB ID: 5FUC). First, two pharmacophore models were constructed based on the interactions between key residues of IL-6 (Phe74, Phe78, Leu178, Arg179, Arg182) and IL-6Rα (Phe229, Tyr230, Glu277, Glu278, Phe279). A database of approximately 22 million compounds was screened using 3D-pharmacophore models, molecular docking models, and ADMET properties. By analyzing the interactive capability of successfully docked compounds with important amino acids, 12 potential ligands were selected for further analysis via molecular dynamics simulations. Based on the stability of the complexes, the high interactions rate of each ligand with the key residues of IL-6/IL-6Rα, and the low binding free energy calculation, two compounds ZINC83804241 and ZINC02997430, were identified as the most potential IL-6 inhibitor candidates. These results will pave the way for the design and optimization of more specific compounds to combat cytokine storm in severe coronavirus patients.

Identification of potential interleukin-8 inhibitors acting on the interactive site between chemokine and CXCR2 receptor: A computational approach.

Interactions between interleukin (IL)-8 and its receptors, CXCR1, and CXCR2, serve crucial roles in inflammatory conditions and various types of cancers. Inhibition of this signaling pathway has been exploited as a promising strategy in treating these diseases. However, most studies only focused on the design of allosteric antagonists-bound receptors on the intracellular side of IL-8 receptors. Recently, the first cryo-EM structures of IL-8-CXCR2-Gi complexes have been solved, revealing the unique binding and activation modes of the endogenous chemokine IL-8. Hence, we set to identify small molecule inhibitors for IL-8 using critical protein-protein interaction between IL-8 and CXCR2 at the orthosteric binding site. The pharmacophore models and molecular docking screened compounds from DrugBank and NCI databases. The oral bioavailability of the top 23 ligands from the screening was then predicted by the SwissAMDE tool. Molecular dynamics simulation and free binding energy calculation were performed for the best compounds. The result indicated that DB14770, DB12121, and DB03916 could form strong interactions and stable protein-ligand complexes with IL-8. These three candidates are potential IL-8 inhibitors that can be further evaluated by in vitro experiments in the next stage.

Discovery of small molecular inhibitors for interleukin-33/ST2 protein-protein interaction: a virtual screening, molecular dynamics simulations and binding free energy calculations.

The interleukin-1 receptor like ST2 has emerged as a potential drug discovery target since it was identified as the receptor of the novel cytokine IL-33, which is involved in many inflammatory and autoimmune diseases. For the treatment of such IL-33-related disorders, efforts have been made to discover molecules that can inhibit the protein-protein interactions (PPIs) between IL-33 and ST2, but to date no drug has been approved. Although several anti-ST2 antibodies have entered clinical trials, the exploration of small molecular inhibitors is highly sought-after because of its advantages in terms of oral bioavailability and manufacturing cost. The aim of this study was to discover ST2 receptor inhibitors based on its PPIs with IL-33 in crystal structure (PDB ID: 4KC3) using virtual screening tools with pharmacophore modeling and molecular docking. From an enormous chemical space ZINC, a potential series of compounds has been discovered with stronger binding affinities than the control compound from a previous study. Among them, four compounds strongly interacted with the key residues of the receptor and had a binding free energy < - 20 kcal/mol. By intensive calculations using data from molecular dynamics simulations, ZINC59514725 was identified as the most potential candidate for ST2 receptor inhibitor in this study.

p-Coumaric Acid Enhances Hypothalamic Leptin Signaling and Glucose Homeostasis in Mice via Differential Effects on AMPK Activation.

AMP-activated protein kinase (AMPK) plays a crucial role in the regulation of energy homeostasis in both peripheral metabolic organs and the central nervous system. Recent studies indicated that p-Coumaric acid (CA), a hydroxycinnamic phenolic acid, potentially activated the peripheral AMPK pathway to exert beneficial effects on glucose metabolism in vitro. However, CA's actions on central AMPK activity and whole-body glucose homeostasis have not yet been investigated. Here, we reported that CA exhibited different effects on peripheral and central AMPK activation both in vitro and in vivo. Specifically, while CA treatment promoted hepatic AMPK activation, it showed an inhibitory effect on hypothalamic AMPK activity possibly by activating the S6 kinase. Furthermore, CA treatment enhanced hypothalamic leptin sensitivity, resulting in increased proopiomelanocortin (POMC) expression, decreased agouti-related peptide (AgRP) expression, and reduced daily food intake. Overall, CA treatment improved blood glucose control, glucose tolerance, and insulin sensitivity. Together, these results suggested that CA treatment enhanced hypothalamic leptin signaling and whole-body glucose homeostasis, possibly via its differential effects on AMPK activation.

Reconstructing the evolutionary history of F420-dependent dehydrogenases.

During the last decade the number of characterized F420-dependent enzymes has significantly increased. Many of these deazaflavoproteins share a TIM-barrel fold and are structurally related to FMN-dependent luciferases and monooxygenases. In this work, we traced the origin and evolutionary history of the F420-dependent enzymes within the luciferase-like superfamily. By a thorough phylogenetic analysis we inferred that the F420-dependent enzymes emerged from a FMN-dependent common ancestor. Furthermore, the data show that during evolution, the family of deazaflavoproteins split into two well-defined groups of enzymes: the F420-dependent dehydrogenases and the F420-dependent reductases. By such event, the dehydrogenases specialized in generating the reduced deazaflavin cofactor, while the reductases employ the reduced F420 for catalysis. Particularly, we focused on investigating the dehydrogenase subfamily and demonstrated that this group diversified into three types of dehydrogenases: the already known F420-dependent glucose-6-phosphate dehydrogenases, the F420-dependent alcohol dehydrogenases, and the sugar-6-phosphate dehydrogenases that were identified in this study. By reconstructing and experimentally characterizing ancestral and extant representatives of F420-dependent dehydrogenases, their biochemical properties were investigated and compared. We propose an evolutionary path for the emergence and diversification of the TIM-barrel fold F420-dependent dehydrogenases subfamily.

Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol.

Glycerol is a major byproduct of biodiesel production, and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here, we describe the comprehensive structural and biochemical characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active toward methanol, ethanol, and 1-propanol ( kcat = 18, 19, and 11 s-1, respectively), but showed very limited activity toward glycerol ( kobs = 0.2 s-1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was determined at a resolution of 2.6 Å. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the observed preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity toward glycerol. Among them, the F101S variant had a high kcat value of 3 s-1, retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.

Enantio- and regioselective ene-reductions using F420H2-dependent enzymes.

In the past decade it has become clear that many microbes harbor enzymes that employ an unusual flavin cofactor, the F420 deazaflavin cofactor. Herein we show that F420-dependent reductases (FDRs) can successfully perform enantio-, regio- and chemoselective ene-reductions. For the first time, we have demonstrated that F420H2-driven reductases can be used as biocatalysts for the reduction of α,ß-unsaturated ketones and aldehydes with good conversions (>99%) and excellent regioselectivities and enantiomeric excesses (>99% ee). Noteworthily, FDRs typically display an opposite enantioselectivity when compared to the well established FMN-dependent Old Yellow Enzymes (OYEs).

Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase.

A number of oxidoreductases from the VAO/para-cresol methylhydroxylase flavoprotein family catalyze the oxidation of para-substituted phenols. One of the best-studied is vanillyl-alcohol oxidase (VAO) from the fungus Penicillium simplicissimum For oxidation of phenols by VAO to occur, they must first be bound in the active site of the enzyme in their phenolate anion form. The crystal structure of VAO reveals that two tyrosine residues, Tyr-108 and Tyr-503, are positioned to facilitate this deprotonation. To investigate their role in catalysis, we created three VAO variants, Y108F, Y503F, and Y108F/Y503F, and studied their biochemical properties. Steady-state kinetics indicated that the presence of at least one of the tyrosine residues is essential for efficient catalysis by VAO. Stopped-flow kinetics revealed that the reduction of VAO by chavicol or vanillyl alcohol occurs at two different rates: kobs1, which corresponds to its reaction with the deprotonated form of the substrate, and kobs2, which corresponds to its reaction with the protonated form of the substrate. In Y108F, Y503F, and Y108F/Y503F, the relative contribution of kobs2 to the reduction is larger than in wild-type VAO, suggesting deprotonation is impaired in these variants. Binding studies disclosed that the competitive inhibitor isoeugenol is predominantly in its deprotonated form when bound to wild-type VAO, but predominantly in its protonated form when bound to the variants. These results indicate that Tyr-108 and Tyr-503 are responsible for the activation of substrates in VAO, providing new insights into the catalytic mechanism of VAO and related enzymes that oxidize para-substituted phenols.

Isolation and characterization of a thermostable F420:NADPH oxidoreductase from Thermobifida fusca.

F420H2-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed reduction, and their potential for applications in biocatalysis has attracted increasing attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F420: NADPH oxidoreductase (Tfu-FNO) from T. fusca, the first F420-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg of recombinant enzyme per liter of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F420 reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus We obtained the crystal structure of FNO in complex with NADP+ at 1.8 Å resolution, providing the first bacterial FNO structure. The overall architecture and NADP+-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2'-phosphate of NADP+ were probed by targeted mutagenesis, indicating that Thr-28, Ser-50, Arg-51, and Arg-55 are important for discriminating between NADP+ and NAD+ Interestingly, a T28A mutant increased the kinetic efficiency >3-fold as compared with the wild-type enzyme when NADH is the substrate. The biochemical and structural data presented here provide crucial insights into the molecular recognition of the two cofactors, F420 and NAD(P)H by FNO.

Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1.

Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.

Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst.

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol-4-(1-hydroxyethyl)-2-methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent-inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p-substituents while tolerating o-methoxy substituents.

Metabolomics methods for the synthetic biology of secondary metabolism.

Many microbial secondary metabolites are of high biotechnological value for medicine, agriculture, and the food industry. Bacterial genome mining has revealed numerous novel secondary metabolite biosynthetic gene clusters, which encode the potential to synthesize a large diversity of compounds that have never been observed before. The stimulation or "awakening" of this cryptic microbial secondary metabolism has naturally attracted the attention of synthetic microbiologists, who exploit recent advances in DNA sequencing and synthesis to achieve unprecedented control over metabolic pathways. One of the indispensable tools in the synthetic biology toolbox is metabolomics, the global quantification of small biomolecules. This review illustrates the pivotal role of metabolomics for the synthetic microbiology of secondary metabolism, including its crucial role in novel compound discovery in microbes, the examination of side products of engineered metabolic pathways, as well as the identification of major bottlenecks for the overproduction of compounds of interest, especially in combination with metabolic modeling. We conclude by highlighting remaining challenges and recent technological advances that will drive metabolomics towards fulfilling its potential as a cornerstone technology of synthetic microbiology.