Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2.

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

Sustainable production and pharmaceutical applications of ß-glucan from microbial sources.

ß-glucans are a large class of complex polysaccharides found in abundant sources. Our dietary sources of ß-glucans are cereals that include oats and barley, and non-cereal sources can consist of mushrooms, microalgae, bacteria, and seaweeds. There is substantial clinical interest in ß-glucans; as they can be used for a variety of diseases including cancer and cardiovascular conditions. Suitable sources of ß-glucans for biopharmaceutical applications include bacteria, microalgae, mycelium, and yeast. Environmental factors including culture medium can influence the biomass and ultimately ß-glucan content. Therefore, cultivation conditions for the above organisms can be controlled for sustainable enhanced production of ß-glucans. This review discusses the various sources of ß-glucans and their cultivation conditions that may be optimised to exploit sustainable production. Finally, this article discusses the immune-modulatory potential of ß-glucans from these sources.

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.

Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria, which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide-containing compound, USP30inh. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics inferred slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to this thumb-palm cleft, which guides the ubiquitin C terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

Polysaccharides-Naturally Occurring Immune Modulators.

The prevention of disease and infection requires immune systems that operate effectively. This is accomplished by the elimination of infections and abnormal cells. Immune or biological therapy treats disease by either stimulating or inhibiting the immune system, dependent upon the circumstances. In plants, animals, and microbes, polysaccharides are abundant biomacromolecules. Due to the intricacy of their structure, polysaccharides may interact with and impact the immune response; hence, they play a crucial role in the treatment of several human illnesses. There is an urgent need for the identification of natural biomolecules that may prevent infection and treat chronic disease. This article addresses some of the naturally occurring polysaccharides of known therapeutic potential that have already been identified. This article also discusses extraction methods and immunological modulatory capabilities.

Treatment with levosimendan in an experimental model of early ventilator-induced diaphragmatic dysfunction.

Introduction: Mechanical ventilation (MV) is a life-saving approach in critically ill patients. However, it may affect the diaphragmatic structure and function, beyond the lungs. Levosimendan is a calcium sensitizer widely used in clinics to improve cardiac contractility in acute heart failure patients. In vitro studies have demonstrated that levosimendan increased force-generating capacity of the diaphragm in chronic obstructive pulmonary disease patients. Thus the aim of this study was to evaluate the effects of levosimendan administration in an animal model of ventilator-induced diaphragmatic dysfunction (VIDD) on muscle contraction and diaphragm muscle cell viability. Methods: Sprague-Dawley rats underwent prolonged MV (5 hours). VIDD+Levo group received a starting bolus of levosimendan immediately after intratracheal intubation and then an intravenous infusion of levosimendan throughout the study. Diaphragms were collected for ex vivo contractility measurement (with electric stimulation), histological analysis and Western blot analysis. Healthy rats were used as the control. Results: Levosimendan treatment maintained an adequate mean arterial pressure during the entire experimental protocol, preserved levels of autophagy-related proteins (LC3BI and LC3BII) and the muscular cell diameter demonstrated by histological analysis. Levosimendan did not affect the diaphragmatic contraction or the levels of proteins involved in the protein degradation (atrogin). Conclusions: Our data suggest that levosimendan preserves muscular cell structure (cross-sectional area) and muscle autophagy after 5 hours of MV in a rat model of VIDD. However, levosimendan did not improve diaphragm contractile efficiency.

Hyaluronic Acid: A Review of the Drug Delivery Capabilities of This Naturally Occurring Polysaccharide.

The inclusion of physiologically active molecules into a naturally occurring polymer matrix can improve the degradation, absorption, and release profile of the drug, thus boosting the therapeutic impact and potentially even reducing the frequency of administration. The human body produces significant amounts of polysaccharide hyaluronic acid, which boasts exceptional biocompatibility, biodegradability, and one-of-a-kind physicochemical features. In this review, we will examine the clinical trials currently utilizing hyaluronic acid and address the bright future of this versatile polymer, as well as summarize the numerous applications of hyaluronic acid in drug delivery and immunomodulation.

Immunomodulatory activity of ß-glucan polysaccharides isolated from different species of mushroom - A potential treatment for inflammatory lung conditions.

Acute respiratory distress syndrome (ARDS) is the most common form of acute severe hypoxemic respiratory failure in the critically ill with a hospital mortality of 40%. Alveolar inflammation is one of the hallmarks for this disease. ß-Glucans are polysaccharides isolated from a variety of natural sources including mushrooms, with documented immune modulating properties. To investigate the immunomodulatory activity of ß-glucans and their potential as a treatment for ARDS, we isolated and measured glucan-rich polysaccharides from seven species of mushrooms. We used three models of in-vitro injury in THP-1 macrophages, Peripheral blood mononuclear cells (CD14+) (PMBCs) isolated from healthy volunteers and lung epithelial cell lines. We observed variance between ß-glucan content in extracts isolated from seven mushroom species. The extracts with the highest ß-glucan content found was Lentinus edodes which contained 70% w/w and Hypsizygus tessellatus which contained 80% w/w with low levels of α-glucan. The extracts had the ability to induce secretion of up to 4000 pg/mL of the inflammatory cytokine IL-6, and up to 5000 pg/mL and 500 pg/mL of the anti-inflammatory cytokines IL-22 and IL-10, respectively, at a concentration of 1 mg/mL in THP-1 macrophages. In the presence of cytokine injury, IL-8 was reduced from 15,000 pg/mL to as low as 10,000 pg/mL in THP-1 macrophages. After insult with LPS, phagocytosis dropped from 70-90% to as low 10% in CD14+ PBMCs. After LPS insult CCL8 relative gene expression was reduced, and IL-10 relative gene expression increased from 50 to 250-fold in THP-1 macrophages. In lung epithelial cells, both A549 and BEAS-2B after IL-1ß insult, IL-8 levels dropped from 10,000 pg/mL to as low as 6000 pg/mL. TNF-α levels dropped 10-fold from 100 pg/mL to just below 10 pg/mL. These results demonstrate the therapeutic potential of ß-glucans in inflammatory lung conditions. Findings also advance bio-based research that connects green innovation with One Health applications for the betterment of society.

ß-Glucan Metabolic and Immunomodulatory Properties and Potential for Clinical Application.

ß-glucans are complex polysaccharides that are found in several plants and foods, including mushrooms. ß-glucans display an array of potentially therapeutic properties. ß-glucans have metabolic and gastro-intestinal effects, modulating the gut microbiome, altering lipid and glucose metabolism, reducing cholesterol, leading to their investigation as potential therapies for metabolic syndrome, obesity and diet regulation, gastrointestinal conditions such as irritable bowel, and to reduce cardiovascular and diabetes risk. ß-glucans also have immune-modulating effects, leading to their investigation as adjuvant agents for cancers (solid and haematological malignancies), for immune-mediated conditions (e.g., allergic rhinitis, respiratory infections), and to enhance wound healing. The therapeutic potential of ß-glucans is evidenced by the fact that two glucan isolates were licensed as drugs in Japan as immune-adjuvant therapy for cancer in 1980. Significant challenges exist to further clinical testing and translation of ß-glucans. The diverse range of conditions for which ß-glucans are in clinical testing underlines the incomplete understanding of the diverse mechanisms of action of ß-glucans, a key knowledge gap. Furthermore, important differences appear to exist in the effects of apparently similar ß-glucan preparations, which may be due to differences in sources and extraction procedures, another poorly understood issue. This review will describe the biology, potential mechanisms of action and key therapeutic targets being investigated in clinical trials of ß-glucans and identify and discuss the key challenges to successful translation of this intriguing potential therapeutic.

A Bilayer Vaginal Tablet for the Localized Delivery of Disulfiram and 5-Fluorouracil to the Cervix.

This study was performed to develop an adjuvant therapy in the form of a self-administered vaginal tablet regimen for the localized delivery of chemotherapeutic drugs. This therapy will help to reduce relapse by eradicating cancerous cells in the margin of cervical tumors. The vaginal tablet is a very common formulation that is easy to manufacture, easy to place in the vagina, and has a low cost of manufacture, making them ideal for use in developing countries. A combination of disulfiram and 5-fluorouracil, which are both off-patent drugs and provide different modes of action, were evaluated. The tablets developed were evaluated for weight variation, thickness, hardness, friability, swelling index, differential scanning calorimetry (DSC), particle morphology, in vitro drug release, and cytotoxicity on Ca-Ski cells. Both layers were designed to release both drugs concurrently for a synergistic effect. The polymer-polymer interaction between the layers was able to reduce the loss of formulation due to chitosan. While the bilayer tablet had satisfactory performance in the physicochemical tests, in vitro cell culture with Ca-Ski also showed a synergistic effect using a combination of drugs at a low dose. However, the formulation only had 24-h dose release before degradation. Further drug combinations should be evaluated in subsequent studies.

ß-Glucan extracts from the same edible shiitake mushroom Lentinus edodes produce differential in-vitro immunomodulatory and pulmonary cytoprotective effects - Implications for coronavirus disease (COVID-19) immunotherapies.

Coronavirus pneumonia is accompanied by rapid virus replication, where a large number of inflammatory cell infiltration and cytokine storm may lead to acute lung injury, acute respiratory distress syndrome (ARDS) and death. The uncontrolled release of pro-inflammatory cytokines, including interleukin (IL)-1ß and IL-6, is associated with ARDS. This constituted the first study to report on the variability in physicochemical properties of ß-glucans extracts from the same edible mushroom Lentinus edodes on the reduction of these pro-inflammatory cytokines and oxidative stress. Specifically, the impact on the immunomodulatory and cytoprotective properties of our novel in 'house' (IH-Lentinan, IHL) and a commercial (Carbosynth-Lentinan, CL) Lentinan extract were investigated using in vitro models of lung injury and macrophage phagocytosis. CL comprised higher amounts of α-glucans and correspondingly less ß-glucans. The two lentinan extracts demonstrated varying immunomodulatory activities. Both Lentinan extracts reduced cytokine-induced NF-κB activation in human alveolar epithelial A549 cells, with the IHL extract proving more effective at lower doses. In contrast, in activated THP-1 derived macrophages, the CL extract more effectively attenuated pro-inflammatory cytokine production (TNF-α, IL-8, IL-2, IL-6, IL-22) as well as TGF-ß and IL-10. The CL extract attenuated oxidative stress-induced early apoptosis, while the IHL extract attenuated late apoptosis. Our findings demonstrate significant physicochemical differences between Lentinan extracts, which produce differential in vitro immunomodulatory and pulmonary cytoprotective effects that may also have positive relevance to candidate COVID-19 therapeutics targeting cytokine storm.

Aedes aegypti Odorant Binding Protein 22 selectively binds fatty acids through a conformational change in its C-terminal tail.

Aedes aegypti is the primary vector for transmission of Dengue, Zika and chikungunya viruses. Previously it was shown that Dengue virus infection of the mosquito led to an in increased expression of the odorant binding protein 22 (AeOBP22) within the mosquito salivary gland and that siRNA mediated knockdown of AeOBP22 led to reduced mosquito feeding behaviors. Insect OBPs are implicated in the perception, storage and transport of chemosensory signaling molecules including air-borne odorants and pheromones. AeOBP22 is unusual as it is additionally expressed in multiple tissues, including the antenna, the male reproductive glands and is transferred to females during reproduction, indicating multiple roles in the mosquito life cycle. However, it is unclear what role it plays in these tissues and what ligands it interacts with. Here we present solution and X-ray crystallographic studies that indicate a potential role of AeOBP22 binding to fatty acids, and that the specificity for longer chain fatty acids is regulated by a conformational change in the C-terminal tail that leads to creation of an enlarged binding cavity that enhances binding affinity. This study sheds light onto the native ligands for AeOBP22 and provides insight into its potential functions in different tissues.

Correction to: Complete NMR chemical shift assignments of odorant binding protein 22 from the yellow fever mosquito, Aedes aegypti, bound to arachidonic acid.

The article listed above was initially published with incorrect copyright information. Upon publication of this Correction, the copyright of the article is changed to "The Author(s)". The original article has been corrected.

Complete NMR chemical shift assignments of odorant binding protein 22 from the yellow fever mosquito, Aedes aegypti, bound to arachidonic acid.

Aedes aegypti mosquitoes are the vector for transmission of Dengue, Zika and chikungunya viruses. These mosquitos feed exclusively on human hosts for a blood meal. Previous studies have established that Dengue virus infection of the mosquito results in increased expression of the odorant binding proteins 22 and 10 within the mosquito salivary gland and silencing of these genes dramatically reduces blood-feeding behaviors. Odorant binding proteins are implicated in modulating the chemosensory perception of external stimuli that regulate behaviors such as host location, feeding and reproduction. However, the role that AeOBP22 plays in the salivary gland is unclear. Here, as a first step to a more complete understanding of the function of AeOBP22, we present the complete backbone and side chain chemical shift assignments of the protein in the complex it forms with arachidonic acid. These assignments reveal that the protein consists of seven α-helices, and that the arachidonic acid is bound tightly to the protein. Comparison with the chemical shift assignments of the apo-form of the protein reveals that binding of the fatty acid is accompanied by a large conformational change in the C-terminal helix, which appears disordered in the absence of lipid. This NMR data provides the basis for determining the structure of AeOBP22 and understanding the nature of the conformational changes that occur upon ligand binding. This information will provide a path to discover novel compounds that can interfere with AeOBP22 function and impact blood feeding by this mosquito.

A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6.

INTRODUCTION: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding. METHODS: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System. RESULTS: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a KD in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC50. DISCUSSION: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.

Discovery of a PCAF Bromodomain Chemical Probe.

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(-)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Heme enzymes. Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase.

Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O-O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

A novel mechanism of ligand binding and release in the odorant binding protein 20 from the malaria mosquito Anopheles gambiae.

Anopheles gambiae mosquitoes that transmit malaria are attracted to humans by the odor molecules that emanate from skin and sweat. Odorant binding proteins (OBPs) are the first component of the olfactory apparatus to interact with odorant molecules, and so present potential targets for preventing transmission of malaria by disrupting the normal olfactory responses of the insect. AgamOBP20 is one of a limited subset of OBPs that it is preferentially expressed in female mosquitoes and its expression is regulated by blood feeding and by the day/night light cycles that correlate with blood-feeding behavior. Analysis of AgamOBP20 in solution reveals that the apo-protein exhibits significant conformational heterogeneity but the binding of odorant molecules results in a significant conformational change, which is accompanied by a reduction in the conformational flexibility present in the protein. Crystal structures of the free and bound states reveal a novel pathway for entrance and exit of odorant molecules into the central-binding pocket, and that the conformational changes associated with ligand binding are a result of rigid body domain motions in α-helices 1, 4, and 5, which act as lids to the binding pocket. These structures provide new insights into the specific residues involved in the conformational adaptation to different odorants and have important implications in the selection and development of reagents targeted at disrupting normal OBP function.

Interactions of Anopheles gambiae odorant-binding proteins with a human-derived repellent: implications for the mode of action of n,n-diethyl-3-methylbenzamide (DEET).

The Anopheles gambiae mosquito, which is the vector for Plasmodium falciparum malaria, uses a series of olfactory cues emanating from human sweat to select humans as their source for a blood meal. Perception of these odors within the mosquito olfactory system involves the interplay of odorant-binding proteins (OBPs) and odorant receptors and disrupting the normal responses to those odorants that guide mosquito-human interactions represents an attractive approach to prevent the transmission of malaria. Previously, it has been shown that DEET targets multiple components of the olfactory system, including OBPs and odorant receptors. Here, we present the crystal structure of A. gambiae OBP1 (OBP1) in the complex it forms with a natural repellent 6-methyl-5-heptene-2-one (6-MH). We find that 6-MH binds to OBP1 at exactly the same site as DEET. However, key interactions with a highly conserved water molecule that are proposed to be important for DEET binding are not involved in binding of 6-MH. We show that 6-MH and DEET can compete for the binding of attractive odorants and in doing so disrupt the interaction that OBP1 makes with OBP4. We further show that 6-MH and DEET can bind simultaneously to OBPs with other ligands. These results suggest that the successful discovery of novel reagents targeting OBP function requires knowledge about the specific mechanism of binding to the OBP rather than their binding affinity.

Crystal structure of guaiacol and phenol bound to a heme peroxidase.

Guaiacol is a universal substrate for all peroxidases, and its use in a simple colorimetric assay has wide applications. However, its exact binding location has never been defined. Here we report the crystal structures of guaiacol bound to cytochrome c peroxidase (CcP). A related structure with phenol bound is also presented. The CcP-guaiacol and CcP-phenol crystal structures show that both guaiacol and phenol bind at sites distinct from the cytochrome c binding site and from the δ-heme edge, which is known to be the binding site for other substrates. Although neither guaiacol nor phenol is seen bound at the δ-heme edge in the crystal structures, inhibition data and mutagenesis strongly suggest that the catalytic binding site for aromatic compounds is the δ-heme edge in CcP. The functional implications of these observations are discussed in terms of our existing understanding of substrate binding in peroxidases [Gumiero A et al. (2010) Arch Biochem Biophys 500, 13-20].