Emergence Of Drug-resistant Variants Research Articles

Mechanistic and dynamic insight into novel IL7 receptor activators as immunotherapy for the treatment of tuberculosis

The bacterium that causes tuberculosis (TB), Mycobacterium tuberculosis, is still a significant global public health problem due to its contagious nature and the emergence of drug-resistant variants. This study leverages network pharmacology and computational drug discovery to identify genes linked to TB using the GSE54992 and GSE62525 datasets. In active tuberculosis patients, it identifies 162 DEGs that are not present in healthy people. The research includes an investigation of gene ontology (GO) and protein–protein interaction (PPI) networks to provide a comprehensive understanding of these DEGs. Crucially, it identifies ten hub genes: IL7R, CCR7, CXCR5, CCR6, CCR9, S1PR1, BACH2, CXCL5, SPIB, and EBF. The research suggests IL7R as a potential therapeutic target because of its essential role in FoxO signalling pathways and cytokine-cytokine receptor interaction. IL7 and its receptor (IL7R) are vital for T cell functionality and essential in combating TB. The study explores novel small-molecule activators for IL7R (IL7Rα), aiming to boost the immune response against TB. The following four compounds were found using molecular dynamics simulation, virtual screening, computational pharmacophore modelling, and MM/GBSA analytical techniques: ZINC02131028, ZINC02135418, ZINC46007405, and ZINC59273354. These activators may enhance the IL7 signalling pathway, potentially strengthening the immune fight against M. tuberculosis. Among these ZINC02131028 showed the best results to activate IL7 by binding to its active site, stabilizing its active conformation and enhancing its interaction with the IL7 receptor (IL7R). The findings encourage experimental validation of these IL7 activators, marking a significant step towards innovative TB treatment strategies.

Characteristics of respiratory tract bacterial microflora detected in patients suffering from community-acquired pneumonia during continuing spread of the new coronavirus infection in Khabarovsk city (december 2020 — march 2021)

Objective of the research to specify respiratory tract bacterial microflora in patients suffering from community-acquired pneumonia (CAP) during initial and repeat examination. To determine local factors affecting microflora in the cohort examined.
 Materials and methods. Surveillance subject 241 patients with CAP differed by their status and age who stayed in two healthcare facilities of the Khabarovsk city. Examination of respiratory smears was performed. Results. Indices of Gram-negative enterobacteria (30.8% [22.639.7%]) and Gram-negative nonfermentable bacteria (14.5% [8.621.7%]), isolated from patients hospitalized in healthcare institution No. 1 and mostly comprised of the elderly (aged over 61 years 82.0%; 74.388.6%) with more severe disease state including patients at the ICU, were higher compared to data obtained from healthcare institution No. 2 (19.8% (13.427.0); 6.1% (2.710.8) respectively). Prevalent pathogen was Klebsiella pneumoniae 13.6% (7.820.6) and 10.7% (6.016.5). Identification of Acinetobacter baumannii complex 6.4% (2.611.7) and 3.1% (0.86.7) should be also noted. A high percentage of drug-resistant bacterial variants was observed and for Klebsiella pneumoniae totaled 66.7% (41.887.4) and 57.1% (32.280.2) at the healthcare institutions No. 1 and No. 2, respectively. A. baumannii complex drug resistant variants were found in 85.7% (52.799.97) at healthcare institution No. 1. All isolates of A. baumannii complex at the healthcare institution No. 2 were drug resistant. High prevalence of Candida spp. was revealed in both healthcare institutions reaching 54.5% (45.263.7) and 58.0% (49.566.3), respectively, with minimal detection rate of classic pathogens such as S. pneumoniae 5.4% (2.010.4) and 5.3% (2.19.8) and H. influenzae 3.6% (0.97.9) and 3.8% (1.27.7), respectively. Repeat examination of 122 patients conducted 710 days later showed diverse changes in microflora spectrum regardless of the healthcare institution that was manifested as loss or emergence of drug-resistant variants as well as simultaneous presence of different variants of the same pathogen.
 Conclusion. The results obtained evidence about complexity and variety of mechanisms underlying microorganism community formation during the course of infectious process in patients. Local factors influencing microflora characteristics of patients at the two healthcare institutions were revealed.

Computational and Crystallographic Analysis of Binding Structures of Inhibitory Compounds for HIV-1 RNase H Activity.

Chemotherapy of human immunodeficiency virus type-1 (HIV-1) has significantly developed over the last three decades. The emergence of drug-resistant variants is, however, still a severe problem. The RNase H activity of HIV-1 reverse transcriptase is an attractive target for a new class of antiviral drugs because there is no approved inhibitor. The nitro-furan-carbonyl and nitro-thiophene-carbonyl groups are potent scaffolds for the HIV-1 RNase H inhibitor. In this work, the binding structures of six inhibitory compounds were obtained by X-ray crystal analysis in a complex with a recombinant protein of HIV-1 RNase H domain. Every inhibitory compound was found to be bound to the catalytic site with the furan- or thiophene-ring coordinated to two divalent metal ions at the binding pocket. All the atoms in nitro, furan, carbonyl, and two metals were aligned in the nitro-furan derivatives. The straight line connecting nitro and carboxyl groups was parallel to the plane made by two metal ions and a furan O atom. The binding modes of the nitro-thiophene derivatives were slightly different from those of the nitro-furan ones. The nitro and carbonyl groups deviated from the plane made by two metals and a thiophene S atom. Molecular dynamics simulations suggested that the furan O or thiophene S atom and carbonyl O atom were firmly coordinated to the metal ions. The simulations made the planar nitro-furan moiety well aligned to the line connecting the two metal ions. In contrast, the nitro-thiophene derivatives were displaced from the initial positions after the simulations. The computational findings will be a sound basis for developing potent inhibitors for HIV-1 RNase H activity.

Circulating macrophage inflammatory protein-1β/IL-12p40 ratio predicts sofosbuvir-based treatment outcome in HCV- genotype 4 patients.

Sera of 450 treatment-naïve chronic HCV patients and 50 healthy individuals were collected. Liver transaminases, total bilirubin and albumin were biochemically tested, viral RNA was quantified, and circulating MIP-1β and IL-12p40 were estimated using human anti-MIP-1β and IL-12p40 antibodies in Sandwich ELISA. No difference was observed in the baseline chemokines levels between responders and relapsers, but the later had a significantly higher MIP-1β/IL-12p40 ratio (P< 0.0001). Multivariate regression analysis of baseline characteristics showed that gender, age, viral load, albumin level and chemokine ratios can significantly predict treatment outcome (P= 0.0114, 0.0095, 0.042, 0.0004 and < 0.0001; respectively). Accordingly, a predictive threshold of baseline chemokine ratio was calculated and it showed an AUC of 0.6917 (P= 0.0108; 95% CI: 0.5566 to 0.8268). The calculated threshold for predicting virologic response was 8.245, with positive and negative predictive values of 92.98% and 100%; respectively. The chemokine ratios had significant correlations with liver transaminases in treated groups whether pre or post-treatment. Baseline MIP-1β/IL-12p40 ratio represents a non-invasive prognostic biomarker that would provide shorter treatment duration and minimizes the emergence of drug-resistant variants in HCV genotype 4-patients.

Synthesis, inhibitory activity and oral dosing formulation of AV5124, the structural analogue of influenza virus endonuclease inhibitor baloxavir.

The development and clinical implementation of the cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil was a breakthrough in influenza therapy, but it was associated with the emergence of drug-resistant variants. To design and synthesize structural analogues of CEN inhibitors and evaluate their safety, pharmacokinetics and antiviral potency in vitro and in vivo. The drug candidate AV5124 and its active metabolite AV5116 were synthesized based on pharmacophore modelling. Stability in plasma and microsomes, plasma protein binding, cytotoxicity and antiviral activities were assessed in vitro. Pharmacokinetics after IV or oral administration were analysed in CD-1 mice. Acute toxicity and protective efficacy against lethal A(H1N1)pdm09 influenza virus challenge were examined in BALB/c mice. Pharmacophore model-assisted, 3D molecular docking predicted key supramolecular interactions of the metal-binding group and bulky hydrophobic group of AV5116 with the CEN binding site (Protein Data Bank code: 6FS6) that are essential for high antiviral activity. AV5116 inhibited influenza virus polymerase complexes in cell-free assays and replication of oseltamivir-susceptible and -resistant influenza A and B viruses at nanomolar concentrations. Notably, AV5116 was equipotent or more potent than baloxavir acid (BXA) against WT (I38-WT) viruses and viruses with reduced BXA susceptibility carrying an I38T polymerase acidic (PA) substitution. AV5116 exhibited low cytotoxicity in Madin-Darby canine kidney cells and lacked mitochondrial toxicity, resulting in favourable selective indices. Treatment with 20 or 50 mg/kg AV5124 prevented death in 60% and 100% of animals, respectively. Overall, AV5124 and A5116 are promising inhibitors of the influenza virus CEN and warrant further development as potent anti-influenza agents.

Performance of Radial Distribution Function-Based Descriptors in the Chemoinformatic Studies of HIV-1 Protease

Aim: This letter investigates the role of radial distribution function-based descriptors for in silico design of new drugs. Methodology: The multiple linear regression models for HIV-1 protease and its complexes with a series of inhibitors were constructed. A detailed analysis of major atomic contributions to the radial distribution function descriptor weighted by the number of valence shell electrons identified residues Arg8, Asp29 and residues of the catalytic triad as crucial for the correlation with the inhibition constant, together with residues Asp30 and Ile50, whose mutations are known to cause an emergence of drug resistant variants. Conclusion: This study demonstrates an easy and fast assessment of the activity of potential drugs and the derivation of structural information of their complexes with the receptor or enzyme.

Molecular Identification of Transmitted/Founder Hepatitis C Viruses and Their Progeny by Single Genome Sequencing.

Chronic hepatitis C virus (HCV) infection exists as a complex mixture of genetically distinct viruses, commonly referred to as a "quasispecies." Quasispecies complexity can vary substantially during the course of natural infection as a consequence of viral population "bottlenecking." This occurs at the time of transmission from one individual to the next and during the course of chronic infection of an individual when adaptive immune responses eliminate certain viruses but allow others to escape and expand. Antiviral treatment with drugs that fail to eradicate virus can also lead to virus population bottlenecks and emergence of drug-resistant variants. Single genome sequencing (SGS) combined with mathematical modeling and phylogenetic inference is a recently described approach for characterizing the HCV quasispecies in unprecedented detail, allowing for the first time the retention of genetic linkage across genes and near full-length genomes and precise identificationof transmitted/founder (T/F) genomes. Here, we describe the methodological approach to SGS and show how this strategy allows for the precise and unambiguous molecular identification of transmitted viruses as well as those that repopulate the body after drug or immune-mediated selective sweeps. This is an enabling experimental strategy that allows for a precise genetic, biologic, and antigenic characterization of HCV viruses that are responsible for transmission and persistence. Such an approach can be particularly valuable to future HCV vaccine design efforts, as it has been for human immunodeficiency virus type 1 (HIV-1).

Silencing HCV Replication in Its Reservoir.

BACKGROUND:HCV infection and its complications are among the leading public health challenges; the emergence of drug-resistant variants are expected to be a major problem. A novel combinatorial small interfering RNA (siRNA) could be a novel triple therapy that could be suitable for genotype 4. Although HCV is a hepatotropic virus, there is reliable evidence about its replication in peripheral blood mononuclear cells (PBMC) of chronically infected patients; these cells act as an extra-hepatic reservoir for viral recurrence and persistence. The patients with HCV-RNA in PBMC showed a significantly lower response to therapy that supports to be one of the factors influencing therapeutic response. Almost all regions of HCV show potential for siRNA target with relative efficiencies of individual siRNA sequences.AIM:This study aims to test the efficacy of siRNA against HCV-4 replication in PBMC in vitro, to introduce an alternative therapeutic option for HCV-4 suitable to eradicate it from both hepatic and extra-hepatic reservoirs.METHODS:Efficacy of synthesised siRNA molecule that targets 5/UTR of domain IIIC within IRES of HCV RNA to eradicate HCV intra-PBMC in vitro was tested and compared with IFN/RBV in vitro, by using both qRT-PCR and western blot. Sixty genotype-4 chronic HCV patients who are naïve for any HCV treatment were enrolled and tested for the presence of HCV intra-PBMC using qRT-PCR before and after siRNA treatment in vitro.RESULTS:Real-time PCR analysis showed a significant reduction of HCV RNA levels after 24hr post-HCV-positive-PBMCs treatment by siRNA with cell vitality reached up to 98%. Besides a complete inhibition of NS5A viral protein expression, that is functionally essential for viral assembly, replication and egress.CONCLUSION:So, Targeting HCV infection using RNA interference technology might be a reliable therapeutic option for chronic HCV patients with HCV minus strand within PBMCs.

Taking advantage of drug resistance, a new approach in the war on cancer

Identification of the driver mutations in cancer has resulted in the development of a new category of molecularly targeted anti-cancer drugs. However, as was the case with conventional chemotherapies, the effectiveness of these drugs is limited by the emergence of drug-resistant variants. While most cancer therapies are given in combinations that are designed to avoid drug resistance, we discuss here therapeutic approaches that take advantage of the changes in cancer cells that arise upon development of drug resistance. This approach is based on notion that drug resistance comes at a fitness cost to the cancer cell that can be exploited for therapeutic benefit.We discuss the development of sequential drug therapies in which the first therapy is not given with curative intent, but to induce a major new sensitivity that can be targeted with a second drug that selectively targets the acquired vulnerability. This concept of collateral sensitivity has hitherto not been used on a large scale in the clinic and holds great promise for future cancer therapy.

Design of Highly Potent, Dual-Acting and Central-Nervous-System-Penetrating HIV-1 Protease Inhibitors with Excellent Potency against Multidrug-Resistant HIV-1 Variants.

Herein we report the design, synthesis, X-ray structural, and biological studies of an exceptionally potent HIV-1 protease inhibitor, compound 5 ((3S,7aS,8S)-hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-yl ((2S,3R)-4-((2-(cyclopropylamino)-N-isobutylbenzo[d]thiazole)-6-sulfonamido)-1-(3,5-difluorophenyl)-3-hydroxybutan-2-yl)carbamate). Using structure-based design, we incorporated an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran as the P2-ligand, a cyclopropylaminobenzothiazole as the P2'-ligand, and a 3,5-difluorophenylmethyl group as the P1-ligand. The resulting inhibitor 5 exhibited exceptional HIV-1 protease inhibitory and antiviral potency at the picomolar level. Furthermore, it displayed antiviral IC50 values in the picomolar range against a wide panel of highly multidrug-resistant HIV-1 variants. The inhibitor shows an extremely high genetic barrier against the emergence of drug-resistant variants. It also showed extremely potent inhibitory activity toward dimerization as well as favorable central nervous system penetration. We determined a high-resolution X-ray crystal structure of the complex between inhibitor 5 and HIV-1 protease, which provides molecular insight into the unprecedented activity profiles observed.

A novel central nervous system-penetrating protease inhibitor overcomes human immunodeficiency virus 1 resistance with unprecedented aM to pM potency.

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

Synthesis and In Vitro Anti-Influenza Virus Evaluation of Novel Sialic Acid (C-5 and C-9)-Pentacyclic Triterpene Derivatives

The emergence of drug resistant variants of the influenza virus has led to a great need to identify novel and effective antiviral agents. In our previous study, a series of sialic acid (C-2 and C-4)-pentacyclic triterpene conjugates have been synthesized, and a five-fold more potent antiviral activity was observed when sialic acid was conjugated with pentacyclic triterpene via C-4 than C-2. It was here that we further reported the synthesis and anti-influenza activity of novel sialic acid (C-5 and C-9)-pentacyclic triterpene conjugates. Their structures were confirmed by ESI-HRMS, 1H-NMR, and 13C-NMR spectroscopic analyses. Two conjugates (26 and 42) showed strong cytotoxicity to MDCK cells in the CellTiter-Glo assay at a concentration of 100 μM. However, they showed no significant cytotoxicity to HL-60, Hela, and A549 cell lines in MTT assay under the concentration of 10 μM (except compound 42 showed weak cytotoxicity to HL-60 cell line (10 μM, ~53%)). Compounds 20, 28, 36, and 44 displayed weak potency to influenza A/WSN/33 (H1N1) virus (100 μM, ~20–30%), and no significant anti-influenza activity was found for the other conjugates. The data suggested that both the C-5 acetylamide and C-9 hydroxy of sialic acid were important for its binding with hemagglutinin during viral entry into host cells, while C-4 and C-2 hydroxy were not critical for the binding process and could be replaced with hydrophobic moieties. The research presented herein had significant implications for the design of novel antiviral inhibitors based on a sialic acid scaffold.

The effective rate of influenza reassortment is limited during human infection.

We characterise the evolutionary dynamics of influenza infection described by viral sequence data collected from two challenge studies conducted in human hosts. Viral sequence data were collected at regular intervals from infected hosts. Changes in the sequence data observed across time show that the within-host evolution of the virus was driven by the reversion of variants acquired during previous passaging of the virus. Treatment of some patients with oseltamivir on the first day of infection did not lead to the emergence of drug resistance variants in patients. Using an evolutionary model, we inferred the effective rate of reassortment between viral segments, measuring the extent to which randomly chosen viruses within the host exchange genetic material. We find strong evidence that the rate of effective reassortment is low, such that genetic associations between polymorphic loci in different segments are preserved during the course of an infection in a manner not compatible with epistasis. Combining our evidence with that of previous studies we suggest that spatial heterogeneity in the viral population may reduce the extent to which reassortment is observed. Our results do not contradict previous findings of high rates of viral reassortment in vitro and in small animal studies, but indicate that in human hosts the effective rate of reassortment may be substantially more limited.

The trend of transmitted drug resistance in newly diagnosed antiretroviral-naive HIV/AIDS patients during 1999–2012 in South Korea

BackgroundThe use of antiretroviral drugs has reduced the mortality and morbidity of patients with HIV/AIDS. More than 20 antiretroviral drugs have been used in patients with HIV/AIDS since zidovudine was first introduced in 1991 in South Korea. ObjectivesTo investigate and estimate the annual prevalence of transmitted drug resistance and drug-resistant variants of HIV-1 in newly diagnosed antiretroviral-naive patients in South Korea during 1999–2012. Study designPlasma specimens were collected from 928 antiretroviral-naive patients during 1999–2012. Mutations in the protease and reverse transcriptase sections of the HIV-1 pol gene were identified using the Stanford HIV Drug Resistance Database (Stanford DB). ResultsAmong 928 HIV-1 isolates from antiretroviral-naive patients, 45 (4.8%) showed ‘intermediate’ or ‘resistant’ drug resistance. The predicted prevalence of drug resistance among isolates was 2.2%, 2.7%, and 0.3% for resistance to nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors, respectively. ConclusionsThere was no significant increase in the prevalence of drug resistance among antiretroviral-naive patients infected with HIV-1 during 1999–2012 in South Korea, although there was a slight increase during 2009–2012. The emergence of drug-resistant variants will continue to be monitored by national surveys.

767. A Novel Humanized Mouse Model for Testing Anti-HIV T-Cell Gene Therapy Strategies

Despite major advances in the therapeutic treatment of individuals infected with HIV using anti-retroviral therapies (ART), patients are not cured. Deviation from standard drug administration can lead to rapid rebound of viremia, and the emergence of drug-resistant variants. New approaches for treatment of HIV include a range of techniques from gene therapies to vaccine studies. Many of these methods utilize animal models to test the efficacy of treatment prior to advancing to clinical settings. Humanized mouse models have been invaluable for such strategies. Immune compromised mice can engraft with human hematopoietic stem cells (HSC), give rise to a functional human immune system, and maintain an HIV infection. A novel concept for treatment involves engineering a specific immune response against infected cells using chimeric antigen receptors (CAR) to viral epitopes on infected cells. These CAR modified T cells have been used in the preliminary treatment of some cancers, where modified cells lead to the elimination of target cancer cells. A similar approach is being investigated now for HIV. While progress has been made on creating engineered receptors, there has not been a good pre-clinical animal model to test them in. We address this problem by building on the well characterized NOD-scid-gamma (NSG) animal model of HIV treatment, and adapted it to test the functionality of T cell-based immunotherapies. To better model the clinical setting, we use adult apheresis HSCs from healthy mobilized donors as the source for engraftment instead of cord blood or fetal tissue. Neonatal NSG mice injected with these harvested HSCs give rise to over 90% CD3 T-cells in the human fraction circulating in the blood unlike other models which don't readily develop T-cells. Instead of using additional sources for T-cell collection, we harvested CD3 cells directly from the mice, cultured, expanded, and transduced them using GFP expressing lentiviral vectors, and re-infused them back into the mice. The modified CD3 cells were detected in the peripheral blood with frequencies reaching 50% of total CD3 cells without the symptoms of graft-vs-host disease. These cells persisted for over two months and were also found in all lymphoid tissues analyzed at necropsy. To assess safety of vector modified cells, we utilized integration site (IS) analysis to measure the clonal repertoire of injected cells. While over 500 unique clones were detected, we did not observe indications of clonal outgrowth in animals over the course of the experiment. Mice receiving the engrafted HSCs and transduced CD3 cells were capable of sustaining HIV infection with titers between 105-107 viral copies/mL and demonstrated significant CD4 depletion in both blood and lymphoid tissues. With the establishment of this clinically relevant model for testing the effectiveness of T-cell based therapies to treat HIV, we are beginning to test anti-HIV engineered cells. By building upon the existing animal models and combining T-cell gene therapy approaches with IS to monitor for vector safety, we have established a novel method for testing new anti-HIV cure therapies.

Early HAART Initiation May Not Reduce Actual Reproduction Number and Prevalence of MSM Infection: Perspectives from Coupled within- and between-Host Modelling Studies of Chinese MSM Populations.

Having a thorough understanding of the infectivity of HIV, time of initiating treatment and emergence of drug resistant virus variants is crucial in mitigating HIV infection. There are many challenges to evaluating the long-term effect of the Highly Active Antiretroviral Therapy (HAART) on disease transmission at the population level. We proposed an individual based model by coupling within-host dynamics and between-host dynamics and conduct stochastic simulation in the group of men who have sex with men (MSM). The mean actual reproduction number is estimated to be 3.6320 (95% confidence interval: [3.46, 3.80]) for MSM group without treatment. Stochastic simulations show that given relatively high (low) level of drug efficacy after emergence of drug resistant variants, early initiation of treatment leads to a less (greater) actual reproduction number, lower (higher) prevalence and less (more) incidences, compared to late initiation of treatment. This implies early initiation of HAART may not always lower the actual reproduction number and prevalence of infection, depending on the level of treatment efficacy after emergence of drug resistant virus variants, frequency of high-risk behaviors and etc. This finding strongly suggests early initiation of HAART should be implemented with great care especially in the settings where the effective drugs are limited. Coupling within-host dynamics with between-host dynamics can provide critical information about impact of HAART on disease transmission and thus help to assist treatment strategy design and HIV/AIDS prevention and control.

Treatment with a Nucleoside Polymerase Inhibitor Reduces Shedding of Murine Norovirus in Stool to Undetectable Levels without Emergence of Drug-Resistant Variants.

Prolonged norovirus shedding may occur in certain patients, such as organ transplant recipients. We established a mouse model for persistent norovirus infection (using the mouse norovirus MNV.CR6 strain). The nucleoside viral polymerase inhibitor 2'-C-methylcytidine (2CMC), but not favipiravir (T-705), reduced viral shedding to undetectable levels. Viral rebound was observed after stopping treatment, which was again effectively controlled by treatment with 2CMC. No drug-resistant variants emerged.

Identification of novel membrane-associated prostaglandin E synthase-1 (mPGES-1) inhibitors with anti-influenza activities in vitro

Influenza A virus (IAV) is a major public health concern that leads to high morbidity and mortality worldwide. Despite various vaccination programs and development of drugs targeting essential viral proteins, the emergence of drug-resistant variants has been frequently reported and the therapeutic options are limited. Because exaggerated inflammation is considered as an important factor in disease pathogenesis, immunomodulatory agents that effectively suppress cytokine responses are needed for the treatment of IAV infection. Membrane-associated prostaglandin E synthase-1 (mPGES-1) is an enzyme responsible for the production of prostaglandin E2 (PGE2) that is the best-characterized immune modulatory lipid in vitro and in vivo models of inflammation. In the present study, we tested the anti-influenza activities of mPGES-1 inhibitors, using a phenotype-based assay involving image analyses. Seven primary hits among 49 compounds targeting mPGES-1 exhibited anti-influenza activities against A/Puerto Rico/8/1934 (H1N1) in a dose-dependent manner. The most effective hit, MPO-0047, suppressed influenza-induced p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) activation. We also showed that mRNA levels of TNF-α, IL-8, CCL5/RANTES, and CXCL10/IP-10 were significantly reduced by the treatment of influenza-infected cells with MPO-0047. Exogenous PGE2 reversed the inhibitory effects of MPO-0047. Our results showed that this selective mPGES-1 inhibitor has anti-influenza effects by inhibiting PGE2 production, which suppresses the induction of pro-inflammatory genes. Taken together our data revealed that mPGES-1 inhibitor has the potential for further development as an influenza therapeutic agent.

A live attenuated vaccine prevents replication and transmission of H7N9 virus in mammals.

The continued spread of the newly emerged H7N9 viruses among poultry in China, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. An MF59-adjuvant H7N9 inactivated vaccine is reported to be well-tolerated and immunogenic in humans; however a study in ferrets indicated that while a single dose of the inactivated H7N9 vaccine reduced disease severity, it did not prevent virus replication and transmission. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H7N9 vaccine (H7N9/AAca) that contains wild-type HA and NA genes from AH/1, and the backbone of the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AAca). H7N9/AAca was attenuated in mice and ferrets, and induced robust neutralizing antibody responses in rhesus mice, ferrets, and guinea pigs immunized once or twice intranasally. The animals immunized twice were completely protected from H7N9 virus challenge. Importantly, the animals vaccinated once were fully protected from transmission when exposed to or in contact with the H7N9 virus-inoculated animals. These results demonstrate that a cold-adapted H7N9 vaccine can prevent H7N9 virus transmission; they provide a compelling argument for further testing of this vaccine in human trials.

Molecular principles behind Boceprevir resistance due to mutations in hepatitis C NS3/4A protease

The hepatitis C virus (HCV) infection is a primary cause of chronic hepatitis which eventually progresses to cirrhosis and in some instances might advance to hepatocellular carcinoma. According to the WHO report, HCV infects 130–150million people globally and every year 350,000 to 500,000 people die from hepatitis C virus infection. Great achievement has been made in viral treatment evolution, after the development of HCV NS3/4A protease inhibitor (Boceprevir). However, efficacy of Boceprevir is compromised by the emergence of drug resistant variants. The molecular principle behind drug resistance of the protease mutants such as (V36M, T54S and R155K) is still poorly understood. Therefore in this study, we employed a series of computational strategies to analyze the binding of antiviral drug, Boceprevir to HCV NS3/4A protease mutants. Our results clearly demonstrate that the point mutations (V36M, T54S and R155K) in protease are associated with lowering of its binding affinity with Boceprevir. Exhaustive analysis of the simulated Boceprevir-bound wild and mutant complexes revealed variations in hydrophobic interactions, hydrogen bond occupancy and salt bridge interactions. Also, substrate envelope analysis scrutinized that the studied mutations reside outside the substrate envelope which may affect the Boceprevir affinity towards HCV protease but not the protease enzymatic activity. Furthermore, structural analyses of the binding site volume and flexibility show impairment in flexibility and stability of the binding site residues in mutant structures. In order to combat Boceprevir resistance, renovation of binding interactions between the drug and protease may be valuable. The structural insight from this study reveals the mechanism of the Boceprevir resistance and the results can be valuable for the design of new PIs with improved efficiency.