A guide to oral vaccination: Highlighting electrospraying as a promising manufacturing technique toward a successful oral vaccine development.

Most vaccines approved by regulatory bodies are administered via intramuscular or subcutaneous injections and have shortcomings, such as the risk of needle-associated blood infections, pain and swelling at the injection site. Orally administered vaccines are of interest, as they elicit both systemic and mucosal immunities, in which mucosal immunity would neutralize the mucosa invading pathogen before the onset of an infection. Hence, oral vaccination can eliminate the injection associated adverse effects and enhance the person's compliance. Conventional approaches to manufacturing oral vaccines, such as coacervation, spray drying, and membrane emulsification, tend to alter the structural proteins in vaccines that result from high temperature, organic and toxic solvents during production. Electrohydrodynamic processes, specifically electrospraying, could solve these challenges, as it also modulates antigen release and has a high loading efficiency. This review will highlight the mucosal immunity and biological basis of the gastrointestinal immune system, different oral vaccine delivery approaches, and the application of electrospraying in vaccines development.

SARS-CoV-2: Understanding the Transcriptional Regulation of ACE2 and TMPRSS2 and the Role of Single Nucleotide Polymorphism (SNP) at Codon 72 of p53 in the Innate Immune Response against Virus Infection.

Human ACE2 and the serine protease TMPRSS2 of novel SARS-CoV-2 are primary entry receptors in host cells. Expression of these genes at the transcriptional level has not been much discussed in detail. The ISRE elements of the ACE2 promoter are a binding site for the ISGF3 complex of the JAK/STAT signaling pathway. TMPRSS2, including IFNß, STAT1, and STAT2, has the PARP1 binding site near to TSS either up or downstream promoter region. It is well documented that PARP1 regulates gene expression at the transcription level. Therefore, to curb virus infection, both promoting type I IFN signaling to boost innate immunity and prevention of virus entry by inhibiting PARP1, ACE2 or TMPRSS2 are safe options. Most importantly, our aim is to attract the attention of the global scientific community towards the codon 72 Single Nucleotide Polymorphism (SNP) of p53 and its underneath role in the innate immune response against SARS-CoV-2. Here, we discuss codon 72 SNP of human p53's role in the different innate immune response to restrict virus-mediated mortality rate only in specific parts of the world. In addition, we discuss potential targets and emerging therapies using bioengineered bacteriophage, anti-sense, or CRISPR strategies.

Enhancement of natural killer activity and IFN-γ production in an IL-12-dependent manner by a Brassica rapa L.

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity.

The effect of evolocumab alone and in combination with atorvastatin on atherosclerosis progression and TLRs expression.

Evolocumab, a PCSK-9 inhibitor, is known for its ability to reduce low-density lipoprotein cholesterol (LDL-C). This study aimed to investigate the effects of evolocumab, alone or in combination with atorvastatin, on the progression of atherosclerosis. Fifty male domestic rabbits were randomly assigned to five groups: control, high cholesterol diet, evolocumab vehicle (dimethyl sulfoxide, DMSO), evolocumab alone, and evolocumab plus atorvastatin. Serum levels of interleukin 10 (IL-10), IL-17, IL-1ß, intracellular adhesion molecule (ICAM), and vascular adhesion molecule (VCAM) were measured. Toll-like receptor (TLR) expression on monocytes was evaluated using flow cytometry. Histopathological examination and measurement of intimal thickness (IT) were also conducted. The results revealed that the evolocumab produced a statistically significant (p<0.05) reduction in lipid profile at 5 weeks, with the peak effect occurring at 10 weeks. Furthermore, the inhibitor reduced TLRs at 10 weeks to 10.83±1.8 and intimal thickness to 160.66±9.45. IL-17, IL-1ß, ICAM, and VCAM were significantly reduced by evolocumab treatment, with the improvement of the histopathological changes in the aortic wall. The combination of evolocumab and atorvastatin caused a more statistically significant reduction in TLRs at 10 weeks to 5.08±1.2 and intimal thickness to 121.79±5.3. IL-17, IL-1ß, ICAM, and VCAM were significantly (p<0.05) reduced by the combination, and the histopathological changes in the aortic wall were significantly improved. In conclusion, evolocumab delays the progression of atherosclerosis by modulating inflammatory pathways.

Emerging Pharmacotherapies in Alcohol-Associated Hepatitis.

The incidence of alcoholic-associated hepatitis (AH) is increasing. The treatment options for severe AH (sAH) are scarce and limited to corticosteroid therapy which showed limited mortality benefit in short-term use only. Therefore, there is a dire need for developing safe and effective therapies for patients with sAH and to improve their high mortality rates.This review article focuses on the current novel therapeutics targeting various mechanisms in the pathogenesis of alcohol-related hepatitis. Anti-inflammatory agents such as IL-1 inhibitor, Pan-caspase inhibitor, Apoptosis signal-regulating kinase-1, and CCL2 inhibitors are under investigation. Other group of agents include gut-liver axis modulators, hepatic regeneration, antioxidants, and Epigenic modulators. We describe the ongoing clinical trials of some of the new agents for alcohol-related hepatitis. Conclusion: A combination of therapies was investigated, possibly providing a synergistic effect of drugs with different mechanisms. Multiple clinical trials of novel therapies in AH remain ongoing. Their result could potentially make a difference in the clinical course of the disease. DUR-928 and granulocyte colony-stimulating factor had promising results and further trials are ongoing to evaluate their efficacy in the large patient sample.

Association of Toll-like receptor-4 polymorphism with SARS CoV-2 infection in Kurdish Population.

Genetic variations are critical for understanding clinical outcomes of infections including server acute respiratory syndrome coronavirus 2 (SARS CoV-2). The immunological reactions of human immune genes with SARS CoV-2 have been under investigation. Toll-like receptors (TLRs), a group of proteins, are important for microbial detections including bacteria and viruses. TLR4 can sense both bacterial lipopolysaccharides (LPS) and endogenous oxidized phospholipids triggered by Covid-19 infection. Two TLR4 single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile have been linked to infectious diseases. No studies have focused on these SNPs in association with Covid-19. This study aims to reveal the association between Covid-19 infection with these SNPs by comparing a group of patients and a general population. Restriction fragment length polymorphisms (RFLP) were used to identify the TLR4 SNPs in both the general population (n = 114) and Covid-19 patient groups (n = 125). The results found no association between the TLR4 polymorphisms and Covid-19 infections as the data showed no statistically significant difference between the compared groups. This suggested that these TLR4 SNPs may not be associated with Covid-19 infections.

Vitamin D status can affect COVID-19 outcomes also in pediatric population.

Background: vitamin D influences the immune system and the inflammatory response. It is known that vitamin D supplementation reduces the risk of acute respiratory tract infection. In the last two years, many researchers have investigated vitamin D's role in the pathophysiology of COVID-19 disease. Results: the findings obtained from clinical trials and systematic reviews highlight that most patients with COVID-19 have decreased vitamin D levels and low levels of vitamin D increase the risk of severe disease. This evidence seems to be also confirmed in the pediatric population. Conclusions: further studies (systematic review and meta-analysis) conducted on children are needed to confirm that vitamin D affects COVID-19 outcomes and to determine the effectiveness of supplementation and the appropriate dose, duration and mode of administration.

Molecular and Cellular Mediators of the Gut-Liver Axis in the Progression of Liver Diseases.

The gut-liver axis covers the bidirectional communication between the gut and the liver, and thus includes signals from liver-to-gut (e.g., bile acids, immunoglobulins) and from gut-to-liver (e.g., nutrients, microbiota-derived products, and recirculating bile acids). In a healthy individual, liver homeostasis is tightly controlled by the mostly tolerogenic liver resident macrophages, the Kupffer cells, capturing the gut-derived antigens from the blood circulation. However, disturbances of the gut-liver axis have been associated to the progression of varying chronic liver diseases, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and primary sclerosing cholangitis. Notably, changes of the gut microbiome, or intestinal dysbiosis, combined with increased intestinal permeability, leads to the translocation of gut-derived bacteria or their metabolites into the portal vein. In the context of concomitant or subsequent liver inflammation, the liver is then infiltrated by responsive immune cells (e.g., monocytes, neutrophils, lymphoid, or dendritic cells), and microbiota-derived products may provoke or exacerbate innate immune responses, hence perpetuating liver inflammation and fibrosis, and potentiating the risks of developing cirrhosis. Similarly, food derived antigens, bile acids, danger-, and pathogen-associated molecular patterns are able to reshape the liver immune microenvironment. Immune cell intracellular signaling components, such as inflammasome activation, toll-like receptor or nucleotide-binding oligomerization domain-like receptors signaling, are potent targets of interest for the modulation of the immune response. This review describes the current understanding of the cellular landscape and molecular pathways involved in the gut-liver axis and implicated in chronic liver disease progression. We also provide an overview of innovative therapeutic approaches and current clinical trials aiming at targeting the gut-liver axis for the treatment of patients with chronic liver and/or intestinal diseases.

Aberrant Expression of TLR2, TLR7, TLR9, Splicing Variants of TLR4 and MYD88 in Chronic Lymphocytic Leukemia Patients.

Functional toll-like receptors (TLRs) could modulate anti-tumor effects by activating inflammatory cytokines and the cytotoxic T-cells response. However, excessive TLR expression could promote tumor progression, since TLR-induced inflammation might stimulate cancer cells expansion into the microenvironment. Myd88 is involved in activation NF-κB through TLRs downstream signaling, hence in the current study we provided, for the first time, a complex characterization of expression of TLR2, TLR4, TLR7, TLR9, and MYD88 as well as their splicing forms in two distinct compartments of the microenvironment of chronic lymphocytic leukemia (CLL): peripheral blood and bone marrow. We found correlations between MYD88 and TLRs expressions in both compartments, indicating their relevant cooperation in CLL. The MYD88 expression was higher in CLL patients compared to healthy volunteers (HVs) (0.1780 vs. 0.128, p < 0.0001). The TLRs expression was aberrant in CLL compared to HVs. Analysis of survival curves revealed a shorter time to first treatment in the group of patients with low level of TLR4(3) expression compared to high level of TLR4(3) expression in bone marrow (13 months vs. 48 months, p = 0.0207). We suggest that TLRs expression is differentially regulated in CLL but is similarly shared between two distinct compartments of the microenvironment.

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes.

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

Using CRISPR to enhance T cell effector function for therapeutic applications.

T cells are critical to fight pathogenic microbes and combat malignantly transformed cells in the fight against cancer. To exert their effector function, T cells produce effector molecules, such as the pro-inflammatory cytokines IFN-γ, TNF-α and IL-2. Tumors possess many inhibitory mechanisms that dampen T cell effector function, limiting the secretion of cytotoxic molecules. As a result, the control and elimination of tumors is impaired. Through recent advances in genomic editing, T cells can now be successfully modified via CRISPR/Cas9 technology. For instance, engaging (post-)transcriptional mechanisms to enhance T cell cytokine production, the retargeting of T cell antigen specificity or rendering T cells refractive to inhibitory receptor signaling can augment T cell effector function. Therefore, CRISPR/Cas9-mediated genome editing might provide novel strategies for cancer immunotherapy. Recently, the first-in-patient clinical trial was successfully performed with CRISPR/Cas9-modified human T cell therapy. In this review, a brief overview of currently available techniques is provided, and recent advances in T cell genomic engineering for the enhancement of T cell effector function for therapeutic purposes are discussed.

Edaravone protects rat astrocytes from oxidative or neurotoxic inflammatory insults by restoring Akt/Bcl-2/Caspase-3 signaling axis.

Astrocytes are the major glia cells in the central nervous system (CNS). Increasing evidence indicates that more than to be safe-guard and supporting cells for neurons, astrocytes play a broad spectrum of neuroprotective and pathological functions. Thus, they are compelling models to decipher mechanistic insights of glia cells to CNS insults and for the development of drugs. Edaravone is a free radical scavenger with the capacity to eliminate hydroxyl radicals and lipid peroxides. In this study, we examined the neuroprotective effects of edaravone in rat astrocytes challenged by hydrogen peroxide (H2O2) or bacterial lipopolysaccharides (LPS), respectively. We discovered that edaravone attenuated H2O2-induced oxidative stress by reactivating the Akt signaling axis and antagonistically restoring the expression of apoptosis associated regulators such as Bcl-2 and Caspase-3. Consistently, inhibition of Akt signaling by LY294002 attenuated the anti-oxidative activity of edaravone. In addition, edaravone mitigated LPS-induced morphological changes in astrocytes and alleviated the inflammatory activation and expression of TNF-α, IL-1ß, IL-6 and NOS2. In summary, our data suggested that edavarone effectively protects astrocytes from oxidative stress or infectious insults, which may pave a new avenue for its application in preclinical research and human disease therapeutics.

Correlation between IL28B/TLR4 genetic variants and HCC development with/without DAAs treatment in chronic HCV patients.

In Egypt, Sofosbuvir (SOF) in combination with Dataclasvir (DCV) is the broadly used DAAs with excellent therapeutic profile. This study is designed to explore the relation between IL28B/TLR4 genetic variants and each of the followings; HCC development post SOF/DCV treatment, progression to HCC in naïve patients and SOF/DCV therapy outcome. A total of 493 blood samples were collected (controls (n = 70); HCV patients treated with SOF/DCV (n = 252) of whom 65 patients developed HCC, 187 patients didn't develop HCC (125 responders, 62 relapsers); naïve HCV patients (n = 171) had early (n = 48), late liver fibrosis (n = 21) and HCC (n = 102)). Both SNPs were genotyped using a TaqMan 5' allelic discrimination assay. At IL28B rs12979860 SNP, the C allele was significantly correlating with the response rate more than T allele (OR 1.9, 95% CI 1.29-2.9, p = 0.004), while at TLR4 rs4986791 SNP, no association was found (OR 6.5, 95% 0.57-75.28, p = 0.09). Both SNPs couldn't detect the probability for HCC emergence after treatment. In naïve patients, the protective alleles were detected in their lowest frequency in HCC patients (p = 0.1, for rs12979860 and, p = 0.001 for rs4986791). SOF/DCV combination improved SVR rates in HCV genotype 4a infected patients regardless of IL28B genotype, with the best rates in those lacking the T allele.

Neuroinflammation and glial cell activation in mental disorders.

Mental disorders (MDs) are highly prevalent and potentially debilitating complex disorders which causes remain elusive. Looking into deeper aspects of etiology or pathophysiology underlying these diseases would be highly beneficial, as the scarce knowledge in mechanistic and molecular pathways certainly represents an important limitation. Association between MDs and inflammation/neuroinflammation has been widely discussed and accepted by many, as high levels of pro-inflammatory cytokines were reported in patients with several MDs, such as schizophrenia (SCZ), bipolar disorder (BD) and major depression disorder (MDD), among others. Correlation of pro-inflammatory markers with symptoms intensity was also reported. However, the mechanisms underlying the inflammatory dysfunctions observed in MDs are not fully understood yet. In this context, microglial dysfunction has recently emerged as a possible pivotal player, as during the neuroinflammatory response, microglia can be over-activated, and excessive production of pro-inflammatory cytokines, which can modify the kynurenine and glutamate signaling, is reported. Moreover, microglial activation also results in increased astrocyte activity and consequent glutamate release, which are both toxic to the Central Nervous System (CNS). Also, as a result of increased microglial activation in MDs, products of the kynurenine pathway were shown to be changed, influencing then the dopaminergic, serotonergic, and glutamatergic signaling pathways. Therefore, in the present review, we aim to discuss how neuroinflammation impacts on glutamate and kynurenine signaling pathways, and how they can consequently influence the monoaminergic signaling. The consequent association with MDs main symptoms is also discussed. As such, this work aims to contribute to the field by providing insights into these alternative pathways and by shedding light on potential targets that could improve the strategies for pharmacological intervention and/or treatment protocols to combat the main pharmacologically unmatched symptoms of MDs, as the SCZ.

Transcriptome Modifications in Porcine Adipocytes via Toll-Like Receptors Activation.

Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10, and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2, and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.

Genetics of Malaria Inflammatory Responses: A Pathogenesis Perspective.

Despite significant progress in combating malaria in recent years the burden of severe disease and death due to Plasmodium infections remains a global public health concern. Only a fraction of infected people develops severe clinical syndromes motivating a longstanding search for genetic determinants of malaria severity. Strong genetic effects have been repeatedly ascribed to mutations and allelic variants of proteins expressed in red blood cells but the role of inflammatory response genes in disease pathogenesis has been difficult to discern. We revisited genetic evidence provided by inflammatory response genes that have been repeatedly associated to malaria, namely TNF, NOS2, IFNAR1, HMOX1, TLRs, CD36, and CD40LG. This highlighted specific genetic variants having opposing roles in the development of distinct malaria clinical outcomes and unveiled diverse levels of genetic heterogeneity that shaped the complex association landscape of inflammatory response genes with malaria. However, scrutinizing genetic effects of individual variants corroborates a pathogenesis model where pro-inflammatory genetic variants acting in early infection stages contribute to resolve infection but at later stages confer increased vulnerability to severe organ dysfunction driven by tissue inflammation. Human genetics studies are an invaluable tool to find genes and molecular pathways involved in the inflammatory response to malaria but their precise roles in disease pathogenesis are still unexploited. Genome editing in malaria experimental models and novel genotyping-by-sequencing techniques are promising approaches to delineate the relevance of inflammatory response gene variants in the natural history of infection thereby will offer new rational angles on adjuvant therapeutics for prevention and clinical management of severe malaria.

Nonsaponin fraction of Korean Red Ginseng attenuates cytokine production via inhibition of TLR4 expression.

BACKGROUND: Ginsenosides of Korean Red Ginseng extracts (RGE) and its saponin components suppress secretion of inflammasome-mediating cytokines, whereas the nonsaponin fraction (NS) of RGE oppositely stimulates cytokine secretion. Although direct exposure of NS to macrophages in mice induces cytokine production, oral administration of NS has not been studied in inflammasome-related disease in animal models. METHODS: Mice were fed RGE or NS for 7 days and then developed peritonitis. Peritoneal cytokines were measured, and peritoneal exudate cells (PECs) were collected to assay expression levels of a set of toll-like receptors (TLRs) and cytokines in response to NS ingestion. In addition, the role of intestinal bacteria in NS-fed mice was assessed. The effect of preexposure to NS in bone marrow-derived macrophages (BMDMs) on cytokine production was further confirmed. RESULTS: NS ingestion attenuated secretion of peritoneal cytokines resulting from peritonitis. In addition, the isolated PECs from NS-fed mice presented lower TLR transcription levels than PECs from control diet-fed mice. BMDMs treated with NS showed downregulation of TLR4 mRNA and protein expression, which was mediated by the TLR4-MyD88-NFκB signal pathway. BMDMs pretreated with NS produced less cytokines in response to TLR4 ligands. CONCLUSION: NS administration directly inhibits TLR4 expression in inflammatory cells such as macrophages, thereby reducing secretion of cytokines during peritonitis.

Kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating NF-κB/MAPK signaling and protecting chondrocytes.

The objective was to investigate the effect of kinsenoside (Kin) treatments on macrophage polarity and evaluate the resulting protection of chondrocytes to attenuate osteoarthritis (OA) progression. RAW264.7 macrophages were polarized to M1/M2 subtypes then administered with different concentrations of Kin. The polarization transitions were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), confocal observation and flow cytometry analysis. The mechanism of Kin repolarizing M1 macrophages was evaluated by Western blot. Further, macrophage conditioned medium (CM) and IL-1ß were administered to chondrocytes. Micro-CT scanning and histological observations were conducted in vivo on anterior cruciate ligament transection (ACLT) mice with or without Kin treatment. We found that Kin repolarized M1 macrophages to the M2 phenotype. Mechanistically, Kin inhibited the phosphorylation of IκBα, which further reduced the downstream phosphorylation of P65 in nuclear factor-κB (NF-κB) signaling. Moreover, Kin inhibited mitogen-activated protein kinases (MAPK) signaling molecules p-JNK, p-ERK and p-P38. Additionally, Kin attenuated macrophage CM and IL-1ß-induced chondrocyte damage. In vivo, Kin reduced the infiltration of M1 macrophages, promoted M2 macrophages in the synovium, inhibited subchondral bone destruction and reduced articular cartilage damage induced by ACLT. All the results indicated that Kin is an effective therapeutic candidate for OA treatment.

Structural insights into pharmacophore-assisted in silico identification of protein-protein interaction inhibitors for inhibition of human toll-like receptor 4 - myeloid differentiation factor-2 (hTLR4-MD-2) complex.

Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS-TLR4-MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein-protein interaction (PPI) in TLR4-MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4-MD-2) and dimerization (MD-2-TLR4*) protein-protein interaction interfaces in TLR4-MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4-MD-2 protein-protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in µM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4-MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.