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A Genome-wide CRISPR Screen Identifies ZCCHC14 as a Host Factor Required for Hepatitis B Surface Antigen Production
No full textA hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.
Hyrina et al. employ a non-biased functional CRISPR screening approach to identify host factors regulating HBsAg expression as well as those targeted by RG7834, a HBsAg inhibitor. The screen highlighted over 60 genes and identified a mechanism by which ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing
Structural basis of species-selective antagonist binding to the succinate receptor
No full textThe tricarboxylic acid cycle intermediate succinate is involved in metabolic processes
and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species.
The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a
member of the G-protein-coupled-receptor family and links succinate signalling to
renin-induced hypertension, retinal angiogenesis and inflammation. Because
SUCNR1 senses succinate as an immunological danger signal—which has relevance for
diseases including ulcerative colitis, liver fibrosis, diabetes and rheumatoid
arthritis—it is of interest as a therapeutic target. Here we report the high-resolution
crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in
the inactive conformation. Structure-based mutagenesis and radioligand-binding
studies, in conjunction with molecular modelling, identified key residues for speciesselective
antagonist binding and enabled the determination of the high-resolution
crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, humanselective
antagonist denoted NF-56-EJ40. We anticipate that these structural insights
into the architecture of the succinate receptor and its antagonist selectivity will enable
structure-based drug discovery and will further help to elucidate the function of
SUCNR1 in vitro and in vivo
Investigation of drug product and container-closure interactions: A case study of diluent containing prefilled syringe.
No full textPrefilled syringes (PFS) constitute a widely used medical device for drug delivery particularly for the drugs of biological origin. Interactions between the product contents and the components of the PFS play a critical role in determining the suitability of selected PFS. A diluent (with benzyl alcohol/BzOH as a preservative) containing PFS used for reconstitution of the lyophilized product revealed a systematic decrease in the BzOH content during accelerated and stress stability program. Investigation was carried out to understand and identify the underlying causes of this phenomenon. BzOH has a varying propensity to bind to the rubber components (stopper and tip-cap) of the PFS. Vapor permeation behavior across the tip-cap of the PFS was studied via headspace-gas chromatography-mass spectroscopy (HS-GC-MS) enabled analysis. Depending on the properties of the rubber components, BzOH can not only bind but also traverse across them, resulting in a systematic loss during the course of the stability. PFS can allow not only water vapor permeation across the tip-cap as shown in previous studies, but also molecules like benzyl alcohol. This phenomenon stresses the need for careful selection of the components of the primary packaging and also provides an opportunity to deploy novel tools like HS-GC-MS in the early selection of the optimal primary packaging configuration
Previously Uncharacterized Vacuolar-type ATPase Binding Site Discovered from Structurally Similar Compounds with Distinct Mechanisms of Action
No full textUsing a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex
COPDGene 2019: Redefining the Diagnosis of Chronic Obstructive Pulmonary Disease
No full textAbstract
Background
Chronic Obstructive Pulmonary Disease (COPD) remains a major cause of morbidity
and mortality. Present-day diagnostic criteria are largely based solely on spirometric
criteria. Accumulating evidence has identified a substantial number of individuals
without spirometric evidence of COPD who suffer from respiratory symptoms and/or
increased morbidity and mortality. There is a clear need for an expanded definition of
COPD that is linked to physiologic, structural (CT) and clinical evidence of disease.
Using data from COPDGene, we hypothesized that an integrated approach that
includes environmental exposure, clinical symptoms, chest CT imaging and spirometry
better defines disease and captures the likelihood of progression of respiratory
obstruction and mortality.
Methods
Four key disease characteristics – environmental exposure (cigarette smoking), clinical
symptoms (dyspnea and/or chronic bronchitis), chest CT imaging abnormalities
(emphysema, gas trapping and/or airway wall thickening), and abnormal spirometry –
were evaluated in a group of 8,784 current and former smokers who were participants in
COPDGene Phase 1. Using these four disease characteristics, eight categories of
subjects were identified and evaluated for odds of spirometric disease progression
(FEV1 > 350 ml loss over 5 years), and the hazard ratio for all-cause mortality was
examined.
Results
Using smokers without symptoms, CT imaging abnormalities or airflow obstruction as
the reference population, individuals were classified as Possible COPD, Probable
COPD and Definite COPD. Current GOLD criteria would diagnose 4,062 (46%) of the
8,784 study subjects with COPD. The proposed COPDGene 2019 diagnostic criteria
would add an additional 3,144 subjects. Under the new criteria, 82% of the 8,784 study
subjects would be diagnosed with Possible, Probable or Definite COPD. These COPD
groups showed increased risk of disease progression and mortality. Mortality increased
in patients as the number of their COPD characteristics increased, with a maximum
hazard ratio for all cause-mortality of 5.18 (95% CI: 4.15-6.48) in those with all four
disease characteristics.
Conclusions
A substantial portion of smokers with respiratory symptoms and imaging abnormalities
do not manifest spirometric obstruction as defined by population normals. These
individuals are at significant risk of death and spirometric disease progression. We
propose to redefine the diagnosis of COPD through an integrated approach using
environmental exposure, clinical symptoms, CT imaging and spirometric criteria. These
expanded criteria offer the potential to stimulate both current and future interventions
that could slow or halt disease progression in patients before disability or irreversible
lung structural changes develop
Contribution of the WNK1 kinase to corneal wound healing using the tissue-engineered human cornea as an in vitro model
No full textDamage to the corneal epithelium triggers important changes in the composition of the extracellular matrix (ECM) to which the basal human corneal epithelial cells (hCECs) attach. These changes are perceived by integrins, a family of trans-membrane receptors that activate different intracellular signaling pathways, ultimately leading to re-epithelialization of the injured epithelium. In this study, we investigated the impact of the pharmacological inhibition of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas (hTECs) as in vitro models. Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays. The impact of WNK1 inhibition was evaluated on both the wounded hTECs as well as on hCECs monolayers using a scratch wound assay. Gene profiling analyses and protein kinases arrays revealed that expression and activity of several mediators from the integrin-dependent signaling pathways were altered in response to the ECM changes taking place during corneal wound healing. Phosphorylation of the WNK1 kinase turned out to be the most striking activation event occurring during wound healing. The pharmacological inhibition of WNK1 by WNK463 reduced the rate of corneal wound closure in both the hTEC and hCECs grown in monolayer compared to their respective negative controls. WNK463 also considerably reduced phosphorylation of the WNK1 downstream targets SPAK/OSR1 in wounded hTECs. These results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing. Most of all, they identified the WNK1 kinase as an important player in ensuring proper wound healing of the cornea since its inhibition considerably impedes that process
Fragment-Based Drug Discovery: Advancing Fragments in the Absence of Crystal Structures
No full textFragment-based drug discovery typically requires an interplay between screening methods, structural methods, and medicinal chemistry. X-ray crystallography is generally the method of choice to obtain three-dimensional structures of the bound ligand/protein complex, but this can sometimes be difficult, particularly for early, low-affinity fragment hits. In this Perspective, we discuss strategies to advance and evolve fragments in the absence of crystal structures of protein-fragment complexes, although the structure of the unliganded protein may be available. The strategies can involve other structural techniques, such as NMR spectroscopy, molecular modeling, or a variety of chemical approaches. Often, these strategies are aimed at guiding evolution of initial fragment hits to a stage where crystal structures can be obtained for further structure-based optimization.
Fragment-based drug discovery (FBDD) generally relies on crystal structures of fragments bound to the target protein. Erlanson, Davis, and Jahnke review strategies and approaches for successful FBDD in cases when these crystal structures are not available
Metascape provides a biologist-oriented resource for the analysis of systems-level datasets
No full textA critical component in the interpretation of systems-level studies is the inference of enriched biological pathways and protein complexes contained within OMICs datasets. Successful analysis requires the integration of a broad set of current biological databases and the application of a robust analytical pipeline to produce readily interpretable results. Metascape is a web-based portal designed to provide a comprehensive gene list annotation and analysis resource for experimental biologists. In terms of design features, Metascape combines functional enrichment, interactome analysis, gene annotation, and membership search to leverage over 40 independent knowledgebases within one integrated portal. Additionally, it facilitates comparative analyses of datasets across multiple independent and orthogonal experiments. Metascape provides a significantly simplified user experience through a one-click Express Analysis interface to generate interpretable outputs. Taken together, Metascape is an effective and efficient tool for experimental biologists to comprehensively analyze and interpret OMICs-based studies in the big data era
Identification of a Xist silencing domain 1 by Tiling CRISPR
No full textDespite the essential roles of long noncoding RNAs (lncRNAs) in development and disease, methods determine lncRNA cis-elements are lacking. Here, we developed a screening method named “Tiling CRISPR” to identify lncRNA functional domains. Using this approach, we identified Xist A-Repeats as the silencing domain, an observation in agreement with published work. Mechanistic analysis suggested a novel function for Xist A-repeats in promoting Xist transcription
The next-generation sphingosine 1 receptor modulator BAF312 (siponimod) improves cortical network functionality in autoimmune neurodegeneration
No full textBackground. Autoimmune diseases of the central nervous system (CNS) like multiple sclerosis (MS) are characterized by inflammation and demyelinated lesions in white and grey matter regions. While inflammation is present at all stages of MS, it is more pronounced in the relapsing forms of the disease (RMS), whereas progressive MS (PMS) shows significant neuroaxonal damage and grey and white matter atrophy. Hence, disease-modifying treatments beneficial in patients with RMS have limited success in PMS. BAF312 (siponimod) is a novel sphingosine 1-phosphate (S1P) receptor modulator recently shown to delay progression in PMS. It sequesters lymphocytes in the lymphoid tissues, thereby reducing CNS inflammation. Unlike most other immune-modulatory agents, BAF312 crosses the blood-brain barrier (BBB) and binds to S1P receptors on brain-resident cells like neurons, astrocytes and oligodendrocytes.
Methods. To evaluate direct neuroprotective effects of BAF312 on neuroaxonal damage, BAF312 was locally administered in the CNS of experimental autoimmune encephalomyelitis (EAE) mice with distinct grey and white matter lesions (focal EAE) using an osmotic mini-pump. Focal lesions were induced by stereotactical injection of pro-inflammatory cytokines in pre-immunized mice. The effects of intracerebral versus systemic BAF312 treatment were compared. Ex vivo flow cytometric assays were performed to investigate the effects on local inflammatory cells. Voltage-sensitive dye imaging was used to investigate the spatio-temporal patterns of neuronal network activity in cortical grey matter.
Results. Immune cell infiltration of animals with both grey and white matter lesions was lowered upon systemic administration of BAF312, while intracerebral treatment did not shift the immune phenotype. Examination of neuronal circuits with focal inflammatory lesions revealed an altered activity pattern of activation compared to controls within the cortical layers in response to electrical stimulation of white matter fiber tracts entering the cortex. BAF312 partially restored cortical neuronal circuit function.
Conclusion. Intracerebral BAF312 treatment did not influence 1 EAE disease course at the tested doses, irrespectively of the location of focal EAE lesions. However, administration of BAF312 in acute brain slices from focal EAE mice showed improvement of neuronal network functionality. The data suggest that BAF312 exerts a neuroprotective effect after crossing the BBB independently of peripheral effects on immune cells