Binding Subunit Research Articles

Recombinant Ricin Toxin Binding Subunit B (RTB) Stimulates Production of TNF-α by Mouse Macrophages Through Activation of TLR4 Signaling Pathway.

Ricin toxin binding subunit B (RTB) is a galactose-binding lectin protein derived from the beans of the castor oil plant (Ricinus communis). Our previous studies have reported a direct immunomodulatory effect of recombinant RTB, which stimulates RAW264.7 cells to produce cytokines including TNF-α. However, the role of RTB in innate immune response and its specific mechanism have not been reported in detail. In this work, the results showed that RTB treatment of macrophages significantly increased TLR4 protein levels. RTB also activated TLR4 downstream events, including MyD88, IRAK, and TRAF6, resulting in macrophage activation and TNF-α production. This process is reflected in the increase of IκB phosphorylation. TLR4 knockdown macrophages treated with RTB exhibited greatly reduced IκB phosphorylation and TNF-α secretion. Moreover, treatment with MyD88 inhibitor also suppressed TNF-α production. The docking of RT and TLR4 was simulated by computer, and the contact residues were concentrated on RTB. Our results suggest that recombinant RTB can activate mouse macrophages to secrete TNF-α through activation of NF-κB via the TLR4 signaling pathways.

Computer simulations of the interaction between SARS-CoV-2 spike glycoprotein and different surfaces

A prominent feature of coronaviruses is the presence of a large glycoprotein spike protruding from a lipidic membrane. This glycoprotein spike determines the interaction of coronaviruses with the environment and the host. In this paper, we perform all atomic molecular dynamics simulations of the interaction between the SARS-CoV-2 trimeric glycoprotein spike and surfaces of materials. We considered a material with high hydrogen bonding capacity (cellulose) and a material capable of strong hydrophobic interactions (graphite). Initially, the spike adsorbs to both surfaces through essentially the same residues belonging to the receptor binding subunit of its three monomers. Adsorption onto cellulose stabilizes in this configuration, with the help of a large number of hydrogen bonds developed between cellulose and the three receptor-binding domains of the glycoprotein spike. In the case of adsorption onto graphite, the initial adsorption configuration is not stable and the surface induces a substantial deformation of the glycoprotein spike with a large number of adsorbed residues not pertaining to the binding subunits of the spike monomers.

Analysis of COVID-19 convalescent plasma for SARS-CoV-2 IgG using two commercial immunoassays

Coronavirus Disease 2019 (COVID-19) convalescent plasma (CCP) was approved by the FDA for use in severe cases of COVID-19 under an emergency Investigational New Drug (IND) protocol. Eligibility criteria for CCP donors includes documentation of evidence of COVID-19 either by viral RNA detection at the time of illness or positive SARS-CoV-2 IgG after recovery if diagnostic testing for COVID-19 was not performed at the time of illness. In addition to analysis of CCP, analysis of SARS-CoV-2 IgG provides information for possible past exposure and may support diagnosis when SARS-CoV-2 PCR is negative and clinical suspicion for COVID-19 is high. Furthermore, assays with high sensitivity and specificity for SARS-CoV-2 IgG are critical for understanding community exposure rates to SARS-CoV-2. Currently, there are several assays that test for antibodies to SARS-CoV-2 using a variety of methods, including point-of-care lateral flow-based devices, high throughput immunoassay analyzers, and manual methods such as ELISA. These assays target a number of SARS-CoV-2 antigens, including the nucleocapsid protein (N), full length spike protein (S), S1 subunit, or receptor binding domain (RBD) of the S protein. Given the heterogeneity among methods for, and antigenic targets used in SARS-CoV-2 antibody assays, it is necessary for careful evaluation of these assays prior to implementation for clinical use. We compared two assays that had received the CE mark of regulatory approval and that used either the N antigen or S1-RBD antigen as the target for analysis of a large set of CCP samples. Our data indicates that sensitivity and specificity vary between these assays and that more than one antigenic target may be required to improve the sensitivity and specificity of IgG detection to SARS-CoV-2.

A Collision Coupling Model Governs the Activation of Neuronal GIRK1/2 Channels by Muscarinic-2 Receptors.

The G protein-activated Inwardly Rectifying K+-channel (GIRK) modulates heart rate and neuronal excitability. Following G-Protein Coupled Receptor (GPCR)-mediated activation of heterotrimeric G proteins (Gαβγ), opening of the channel is obtained by direct binding of Gβγ subunits. Interestingly, GIRKs are solely activated by Gβγ subunits released from Gαi/o-coupled GPCRs, despite the fact that all receptor types, for instance Gαq-coupled, are also able to provide Gβγ subunits. It is proposed that this specificity and fast kinetics of activation stem from pre-coupling (or pre-assembly) of proteins within this signaling cascade. However, many studies, including our own, point towards a diffusion-limited mechanism, namely collision coupling. Here, we set out to address this long-standing question by combining electrophysiology, imaging, and mathematical modeling. Muscarinic-2 receptors (M2R) and neuronal GIRK1/2 channels were coexpressed in Xenopus laevis oocytes, where we monitored protein surface expression, current amplitude, and activation kinetics. Densities of expressed M2R were assessed using a fluorescently labeled GIRK channel as a molecular ruler. We then incorporated our results, along with available kinetic data reported for the G-protein cycle and for GIRK1/2 activation, to generate a comprehensive mathematical model for the M2R-G-protein-GIRK1/2 signaling cascade. We find that, without assuming any irreversible interactions, our collision coupling kinetic model faithfully reproduces the rate of channel activation, the changes in agonist-evoked currents and the acceleration of channel activation by increased receptor densities.

Structural impact due to PPQEE deletion in multiple cancer associated protein - Integrin αV: An In silico exploration

A heterodimeric receptor subunit, Integrin αV, often complexed with Integrin β3 plays a vital role in cell signaling to regulate angiogenesis and promote cancer progression. The paramount β-turn formed from pentapeptide residues (PPQEE) in the cytoplasmic domain of Integrin αV was previously reported as crucial for cell signaling and its deletion was proved deleterious for protein's cell membrane adhesion and ligand binding properties. This study revealed conformational changes in the Integrin αV subunit upon deletion of PPQEE residues through in silico structural modelling approach followed by analysis of alteration of binding sites. Human Protein Atlas database helped to identify the association of Integrin αV to the unfavourable prognosis of three gastrointestinal cancers: stomach, liver and pancreatic cancers. Molecular modelling and docking techniques were carried out for the necessary complex formations (wild-type and mutant-type). Further comparison was performed for the complexes. The changes in protein's conformation and stability due to PPQEE deletion were observed in both independent subunit and heterodimer. The most noteworthy conformational shift was the disruption of a transmembrane helix into coil, which accounted for protein's impaired cell membrane adhesion, increased solvent accessibility and decreased stability. The deletion also caused a reduction of beta-turn regions, which disrupted ligand binding in the cytoplasmic domain of Integrin αV subunit. This study emphasized on structural basis of how the deletion of PPQEE residues alters stability, ligand binding and signaling activity of Integrin αV subunit highlighting the importance of these residues in maintenance of protein's native structure.

EDF1 coordinates cellular responses to ribosome collisions.

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation.

Allosteric pluripotency as revealed by protein kinase A.

The functional response of a signaling system to an allosteric stimulus often depends on subcellular conditions, a phenomenon known as pluripotent allostery. For example, a single allosteric modulator, Rp-cAMPS, of the prototypical protein kinase A (PKA) switches from antagonist to agonist depending on MgATP levels. However, the mechanism underlying such pluripotent allostery has remained elusive for decades. Using nuclear magnetic resonance spectroscopy, ensemble models, kinase assays, and molecular dynamics simulations, we show that allosteric pluripotency arises from surprisingly divergent responses of highly homologous tandem domains. The differential responses perturb domain-domain interactions and remodel the free-energy landscape of inhibitory excited states sampled by the regulatory subunit of PKA. The resulting activation threshold values are comparable to the effective free energy of regulatory and catalytic subunit binding, which depends on metabolites, substrates, and mutations, explaining pluripotent allostery and warranting a general redefinition of allosteric targets to include specific subcellular environments.

Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site.

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct four amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability, and transmission.

Potential pathogenesis of severe acute respiratory syndrome coronavirus 2.

The emergence of novel coronavirus pneumonia which was named as coronavirus disease 2019 (COVID-19) by the World Health Organization, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to public health. Notably, COVID-19 has rapidly spread around the world and large amount of people have been infected. There is imminent need to investigate the pathogenesis of SARS-CoV-2 and develop effective therapeutic strategies to contain the epidemic. The spike (S) protein of SARS-CoV-2 mediates viral entry into target cells, with S1 subunit binding to a cellular receptor and S2 subunit fusing viral and host membranes. Angiotensin-converting enzyme 2 (ACE2), previously known as a cell receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), is putatively responsible for mediating COVID-19. In this review, we detail our current understanding of the interaction between S protein and ACE2 in the process of virus infection and the potential pathogenesis of SARS-CoV-2, which has critical implications for exploring the potential therapeutic strategies for COVID-19.

The Beginning of the End: Initial Steps in the Degradation of Plasma Membrane Proteins.

The plasma membrane (PM), as border between the inside and the outside of a cell, is densely packed with proteins involved in the sensing and transmission of internal and external stimuli, as well as transport processes and is therefore vital for plant development as well as quick and accurate responses to the environment. It is consequently not surprising that several regulatory pathways participate in the tight regulation of the spatiotemporal control of PM proteins. Ubiquitination of PM proteins plays a key role in directing their entry into the endo-lysosomal system, serving as a signal for triggering endocytosis and further sorting for degradation. Nevertheless, a uniting picture of the different roles of the respective types of ubiquitination in the consecutive steps of down-regulation of membrane proteins is still missing. The trans-Golgi network (TGN), which acts as an early endosome (EE) in plants receives the endocytosed cargo, and here the decision is made to either recycled back to the PM or further delivered to the vacuole for degradation. A multi-complex machinery, the endosomal sorting complex required for transport (ESCRT), concentrates ubiquitinated proteins and ushers them into the intraluminal vesicles of multi-vesicular bodies (MVBs). Several ESCRTs have ubiquitin binding subunits, which anchor and guide the cargos through the endocytic degradation route. Basic enzymes and the mode of action in the early degradation steps of PM proteins are conserved in eukaryotes, yet many plant unique components exist, which are often essential in this pathway. Thus, deciphering the initial steps in the degradation of ubiquitinated PM proteins, which is the major focus of this review, will greatly contribute to the larger question of how plants mange to fine-tune their responses to their environment.

IL-1β dysregulates cGMP signaling in the newborn lung.

Cyclic guanosine monophosphate (cGMP) signaling is an important regulator of newborn lung function and development. Although cGMP signaling is decreased in many models of newborn lung injury, the mechanisms are poorly understood. We determined how IL-1β regulates the expression of the α1-subunit of soluble guanylate cyclase (sGCα1), a prime effector of pulmonary cGMP signaling. Physiologic levels of IL-1β were discovered to rapidly decrease sGCα1 mRNA expression in a human fetal lung fibroblast cell line (IMR-90 cells) and protein levels in primary mouse pup lung fibroblasts. This sGCα1 expression inhibition appeared to be at a transcriptional level; IL-1β treatment did not alter sGCα1 mRNA stability, although it reduced sGCα1 promoter activity. Transforming growth factor-β (TGFβ)-activated kinase-1 (TAK1) was determined to be required for IL-1β's regulation of sGCα1 expression; TAK1 knockdown protected sGCα1 mRNA expression in IL-1β-treated IMR-90 cells. Moreover, heterologously expressed TAK1 was sufficient to decrease sGCα1 mRNA levels in those cells. Nuclear factor-κB (NF-κB) signaling played a critical role in the IL-1β-TAK1-sGCα1 regulatory pathway; chromatin immunoprecipitation studies demonstrated enhanced activated NF-κB subunit (RelA) binding to the sGCα1 promoter after IL-1β treatment unless treated with an IκB kinase-2 inhibitor. Also, this NF-κB signaling inhibition protected sGCα1 expression in IL-1β-treated fibroblasts. Lastly, using transgenic mice in which active IL-1β was conditionally expressed in lung epithelial cells, we established that IL-1β expression is sufficient to stimulate TAK1 and decrease sGCα1 protein expression in the newborn lung. Together these results detail the role and mechanisms by which IL-1β inhibits cGMP signaling in the newborn lung.

Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency.

Cytokine signaling is challenging to study and therapeutically exploit as the effects of these proteins are often pleiotropic. A subset of cytokines can, however, achieve signal specificity via association with latency-inducing proteins, which cage the cytokine until disrupted by discreet biological stimuli. Inspired by this precision, here, we describe a strategy for synthetic induction of cytokine latency via modification with photolabile polymers that mimic latency while attached then restore protein activity in response to light, thus controlling the magnitude, duration, and location of cytokine signals. We characterize the high dynamic range of cytokine activity modulation and find that polymer-induced latency, alone, can prolong in vivo circulation and bias receptor subunit binding. We further show that protein derepression can be achieved with a near single-cell resolution and demonstrate the feasibility of transcutaneous photoactivation. Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and more precise immunotherapies.

Abstract B38: Ras clipping by bacterial toxin RRSP reduces viability and proliferation of Ras-dependent cancer cell lines in 2D and 3D in vitro models

Abstract Ras GTPases are critical molecular hubs regulating cell proliferation and survival and are among the most frequently mutated oncogenes in human cancers. In recent years, significant efforts have been made to develop Ras-targeting anticancer strategies. Although promising preclinical results were reported for approaches directed against specific KRas mutant variants (e.g., G12C), more effective tools for inhibiting the spectrum of common KRas mutants and/or aberrantly activated wild-type Ras oncoproteins are required to bring Ras-blocking strategies from the bench to the bedside. We discovered the Ras-Rap1-Specific Protease (RRSP) from bacterium Vibrio vulnificus. RRSP site-specifically cleaves the major Ras isoforms (H, N and K) and the most common mutated Ras oncoproteins (G12V, G13D and Q61R) within the Switch I region. In order to bring RRSP into cancer cells, the Melnyk’s group fused RRSP with diphtheria toxin binding subunit B (DTB). DTB is a novel tool for delivery of cargo proteins into the cytosol of target cells via binding to its receptor hHB-EGF (human heparin-binding epidermal growth factor-like growth factor), which is expressed on most human cell types. Successful RRSP intracellular delivery was assessed via Western blot as indicated by Ras cleavage and pERK1/2 dephosphorylation. In MDA-MB-436 (triple-negative breast cancer, KRAS WT) cells with hyperactive Ras signaling, RRSP-DTB efficiently cleaved Ras at concentrations as low as 1 pM. As a consequence of Ras cleavage, we observed pERK1/2 dephosphorylation and decrease in cell viability as shown by crystal violet assay. Similar results were observed in MDA-MB-231 (triple-negative breast cancer, KRAS G13D) cells. A catalytically dead mutant of RRSP carrying an amino acid substitution in its active site (H451A RRSP-DTB) did not show Ras clipping nor decrease in pERK1/2 expression. We also found that RRSP-DTB reduced viability and proliferation of HCT-116 (colorectal, KRAS G13D) cells in 2-dimensional monolayers and 3-dimensional (3D) spheroids in a time- and dose-dependent manner. Notably, 3D spheroids from HCT-116 cells showed a significant reduction in size following RRSP-DTB but not H451A RRSP-DTB treatment. Also, in vivo experiments are currently ongoing to corroborate our in vitro findings. All together our results revealed that, by physically clipping Ras, bacterial toxin RRSP strongly reduces viability and proliferation of Ras-dependent human cancer cell lines bearing either wild-type or mutant KRAS. This work supports further development of RRSP as a potential anticancer therapeutic for a wide variety of Ras-driven tumors. Citation Format: Vania Vidimar, Minyoung Park, Roman A. Melnyk, Karla J.F. Satchell. Ras clipping by bacterial toxin RRSP reduces viability and proliferation of Ras-dependent cancer cell lines in 2D and 3D in vitro models [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B38.

Mutation-Associated Phenotypic Heterogeneity in Novel and Canonical PIK3CA Helical and Kinase Domain Mutants.

Phosphatidylinositol 3-kinase, catalytic subunit alpha (PIK3CA) is an oncogene often mutated in colorectal cancer (CRC). The contribution of PIK3CA mutations in acquired resistance to anti-epidermal growth factor receptor (EGFR) therapy is well documented, but their prognostic and predictive value remain unclear. Domain- and exon-specific mutations are implicated in either favorable or poor prognoses, but there is paucity in the number of mutations characterized outside of the mutational hotspots. Here, two novel non-hotspot mutants—Q661K in exon 13 and C901R in exon 19—were characterized alongside the canonical exon 9 E545K and exon 20 H1047R mutants in NIH3T3 and HCT116 cells. Q661K and E545K both map to the helical domain, whereas C901R and H1047R map to the kinase domain. Results showed variable effects of Q661K and C901R on morphology, cellular proliferation, apoptosis resistance, and cytoskeletal reorganization, with both not having any effect on cellular migration. In comparison, E545K markedly promoted proliferation, survival, cytoskeletal reorganization, migration, and spheroid formation, whereas H1047R only enhanced the first three. In silico docking suggested these mutations negatively affect binding of the p85 alpha regulatory subunit to PIK3CA, thereby relieving PIK3CA inhibition. Altogether, these findings support intra-domain and mutation-specific variability in oncogenic readouts, with implications in degree of aggressiveness.

Neonatal Clonazepam Administration Induced Long-Lasting Changes in GABAA and GABAB Receptors.

Benzodiazepines (BZDs) are widely used in patients of all ages. Unlike adults, neonatal animals treated with BZDs exhibit a variety of behavioral deficits later in life; however, the mechanisms underlying these deficits are poorly understood. This study aims to examine whether administration of clonazepam (CZP; 1 mg/kg/day) in 7–11-day-old rats affects Gama aminobutyric acid (GABA)ergic receptors in both the short and long terms. Using RT-PCR and quantitative autoradiography, we examined the expression of the selected GABAA receptor subunits (α1, α2, α4, γ2, and δ) and the GABAB B2 subunit, and GABAA, benzodiazepine, and GABAB receptor binding 48 h, 1 week, and 2 months after treatment discontinuation. Within one week after CZP cessation, the expression of the α2 subunit was upregulated, whereas that of the δ subunit was downregulated in both the hippocampus and cortex. In the hippocampus, the α4 subunit was downregulated after the 2-month interval. Changes in receptor binding were highly dependent on the receptor type, the interval after treatment cessation, and the brain structure. GABAA receptor binding was increased in almost all of the brain structures after the 48-h interval. BZD-binding was decreased in many brain structures involved in the neuronal networks associated with emotional behavior, anxiety, and cognitive functions after the 2-month interval. Binding of the GABAB receptors changed depending on the interval and brain structure. Overall, the described changes may affect both synaptic development and functioning and may potentially cause behavioral impairment.

In-silico analysis of ligand-receptor binding patterns of α-MMC, TCS and MAP30 protein to LRP1 receptor

Alpha-momorcharin (α-MMC), trichosanthin (TCS), and momordica anti-HIV protein of 30 kD (MAP30) are potential anti-tumor drug candidates but have cytotoxicity to normal cells. The binding of these proteins to LRP1 receptor and the subsequent endocytosis are essential to their cytotoxicity, but this binding process remains largely unknown. This study, in-silico analysis of the binding patterns, was conducted via the protein-protein docking software, ZDOCK 3.0.2 package, to better understand the binding process. Specifically, α-MMC, TCS and MAP30 were selected and bound to binding subunits CR56 and CR17 of LRP1. After docking, the 10 best docking solutions are retained based on the default ZDOCK scores and used for structural assessment. Our results showed that, α-MMC bound to LRP1 stably at the amino acid residues 1–20, at which 8 residues formed 21 hydrogen bonds with 15 residues of CR56 and 10 residues formed 15 hydrogen bonds with 12 residues of CR17. In contrast, TCS and MAP30 bound mainly to LRP1 at the residues 1–57/79-150 and residues 58–102, respectively, which were functional domains of TCS and MAP30. Since residues 1–20 are outside the functional domain of α-MMC, α-MMC is considered more suitable to attenuate by mutating the receptor binding site. Thus, our analysis lays the foundation for future genetic engineering work on α-MMC, and makes important contributions to its potential clinical use in cancer treatment.

SPOP as an Emerging Key Player in Breast Cancer Oncogenesis

Breast cancer is currently the second leading cause of cancer deaths among American women thereby highlighting the compelling need to uncover novel and superior treatment regimens for women suffering from this disease. Recent advances in genome‐wide studies have allowed for the identification of cancer‐related mutational landscapes, which allows for the identification of oncogenic alterations that are unique to particular cancer patients. Through this technology it has been uncovered that up to 37% of breast cancer patients have downregulated expression of the E3 ubiquitin ligase substrate binding subunit Speckle‐type POZ Protein (SPOP) thereby prompting our lab to study the mechanistic basis behind breast oncogenesis when SPOP expression is low. Previous findings from our lab and others have shown that SPOP targets GLI3 for ubiquitination and subsequent degradation which subsequently promotes SHH signaling in prostate cancer patients. Since SHH hedgehog signaling is often found to be hyper‐activated in advanced breast cancer, we hypothesize that downregulated SPOP promotes GLI3‐dependent Sonic Hedgehog (SHH) signaling in breast cancer patients to promote tumorigenesis and that this signaling pathway could prove to be an attractive therapeutic target for SPOP downregulated breast cancer. To study the effect of downregulated SPOP, a lentivirus carrying an shRNA against SPOP was generated and infected into MCF‐7 cells. Proliferation assays were utilized to confirm that downregulated SPOP plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated SPOP on the expression of GLI3‐dependent SHH target genes. Finally, a natural compound library was utilized to find a novel therapeutic strategy for SPOP downregulated breast cancer. Our findings confirmed that downregulation of SPOP increases proliferation of MCF‐7 cells in vitro. Furthermore, quantitative PCR confirmed that GLI3 target genes are upregulated in MCF‐7 cells when SPOP expression is low. Finally, our natural compound library screen identified novel therapeutic compounds that specifically target SPOP downregulated MCF‐7 cells in a manner that involves disruption of GLI3‐dependent SHH signaling. These findings therefore provide critical insight into novel treatment strategies for patients suffering from SPOP downregulated breast cancer.

A bivalent vaccine confers immunogenicity and protection against Shigella flexneri and enterotoxigenic Escherichia coli infections in mice

Vaccine studies for Shigella flexneri and enterotoxigenic Escherichia coli have been impaired by the lack of optimal animal models. We used two murine models to show that a S. flexneri 2a bivalent vaccine (CVD 1208S-122) expressing enterotoxigenic Escherichia coli colonization factor antigen-I (CFA/I) and the binding subunits A2 and B of heat labile-enterotoxin (LTb) is immunogenic and protects against weight loss and diarrhea. These findings document the immunogenicity and pre-clinical efficacy effects of CVD 1208S-122 vaccine and suggest that further work can help elucidate relevant immune responses and ultimately its clinical efficacy in humans.

Guselkumab in patients with active psoriatic arthritis who were biologic-naive or had previously received TNFα inhibitor treatment (DISCOVER-1): a double-blind, randomised, placebo-controlled phase 3 trial

Many patients with psoriatic arthritis have an inadequate response to tumor necrosis factor (TNF) inhibitors. Guselkumab, a specific inhibitor of interleukin-23 (IL-23) via IL-23 p19 subunit binding, significantly improved psoriatic arthritis signs and symptoms with an acceptable safety profile in a phase 2 trial. This multicentre, double-blind, randomised, placebo-controlled, phase 3 trial was done at 86 sites in 13 countries across Asia, Australasia, Europe, and North America and enrolled adults with active psoriatic arthritis (at least three swollen and three tender joints; and C-reactive protein ≥0·3 mg/dL) despite standard therapies. Eligibility criteria included inadequate response to or intolerance of standard treatment, including at least 4 months of apremilast, at least 3 months of non-biologic disease-modifying antirheumatic drugs (DMARDs), or at least 4 weeks of non-steroidal anti-inflammatory drugs for psoriatic arthritis. About 30% of study participants could have previously received one or two TNF inhibitors. Patients were randomly assigned (1:1:1, computer-generated permuted blocks; stratified by baseline DMARD and previous TNF inhibitor use) to subcutaneous guselkumab 100 mg every 4 weeks; guselkumab 100 mg at weeks 0, 4, then every 8 weeks; or matching placebo. The primary endpoint was American College of Rheumatology 20% improvement (ACR20) at week 24 in all patients per assigned treatment group using non-responder imputation. Safety was assessed in all patients per treatment received. This trial is registered at ClinicalTrials.gov, NCT03162796 (active, not recruiting). From Aug 28, 2017, to Aug 17, 2018, we screened 624 patients, of whom 381 were randomly assigned and treated with guselkumab every 4 weeks (n=128), guselkumab every 8 weeks (n=127), or placebo (n=126). 362 patients continued study treatment up to week 24. The primary endpoint was met: ACR20 at week 24 was achieved by significantly greater proportions of patients in the guselkumab every 4 weeks group (76 [59%] of 128 [95% CI 50-68]) and every 8 weeks group (66 [52%] of 127 [43-61]) than in the placebo group (28 [22%] of 126 [15-30]), with percentage differences versus placebo of 37% (95% CI 26-48) for the every 4 weeks group and 30% (19-41) for the every 8 weeks group (both p<0·0001). Serious adverse events up to week 24 occurred in no patients receiving guselkumab every 4 weeks, four (3%) patients receiving guselkumab every 8 weeks, and five (4%) patients receiving placebo. Up to week 24, one patient in the placebo group died from cardiac failure and two had serious infections; no guselkumab-treated patient died or had serious infections. Guselkumab demonstrated a favourable benefit-risk profile and might be an effective treatment option for patients with active psoriatic arthritis. Janssen Research and Development.

Structure-based virtual screening of influenza virus RNA polymerase inhibitors from natural compounds: Molecular dynamics simulation and MM-GBSA calculation

The resistances of matrix protein 2 (M2) protein inhibitors and neuraminidase inhibitors for influenza virus have attracted much attention and there is an urgent need for new drug. The antiviral drugs that selectively act on RNA polymerase are less prone to resistance and possess fewer side effects on the patient. Therefore, there is increased interest in screening compounds that can inhibit influenza virus RNA polymerase. Three natural compounds were found by using molecular docking-based virtual screening, which could bind tightly within the polymerase acidic protein-polymerase basic protein 1 (PA-PB1) subunit of influenza virus polymerase. Firstly, their drug likeness properties were evaluated, which showed that the hepatotoxicity values of all the three compounds indicating they had less or no hepatotoxicity, and did not have the plasma protein biding (PPB) ability, the three compounds needed to be modified in some aspects, like bulky molecular size. The stability of the complexes of PA-hits was validated through molecular dynamics (MD) simulation, revealing compound 2 could form more stable complex with PA subunit. The torsional conformations of each rotatable bond of the ligands in PA subunit were also monitored, to investigate variation in the ligand properties during the simulation, compound 3 had fewer rotatable bonds, indicating that the molecule had stronger rigidity. The bar charts of protein–ligand contacts and contacts over the course of trajectory showed that four key residues (Glu623, Lys643, Asn703 and Trp706) of PA subunit that participated in hydrogen-bond, water bridge and hydrophobic interactions with the hit compounds. Finally, the binding free energy and contributed energies were calculated by using MM-GBSA method. Out of the three compounds, compound 1 showed the lowest total binding free energy. Among all the interactions, the contribution of the covalent binding and the van der Waals energy were more than other items, compound 1 formed more stable hydrogen bonds with the residues of PA subunit binding pocket. This study smoothed the path for the development of novel lead compounds with improved binding properties, high drug likeness, and low toxicity to humans for the treatment of influenza, which provided a good basis for further research on novel and effective influenza virus PA-PB1 interaction inhibitors.