Structure-aided drug development of potential neuraminidase inhibitors against pandemic H1N1 exploring alternate binding mechanism.

The rate of mutability of pathogenic H1N1 influenza virus is a threat. The emergence of drug resistance to the current competitive inhibitors of neuraminidase, such as oseltamivir and zanamivir, attributes to a need for an alternative approach. The design and synthesis of new analogues with alternate approach are particularly important to identify the potential neuraminidase inhibitors which may not only have better anti-influenza activity but also can withstand challenge of resistance. Five series of scaffolds, namely aurones (1a-1e), pyrimidine analogues (2a-2b), cinnamic acid analogues (3a-3k), chalcones (4a-4h) and cinnamic acid linkages (5a-5c), were designed based on virtual screening against pandemic H1N1 virus. Molecular modelling studies revealed that the designed analogues occupied 430-loop cavity of neuraminidase. Docking of sialic acid in the active site preoccupied with the docked analogues, i.e. in 430-loop cavity, resulted in displacement of sialic acid from its native pose in the catalytic cavity. The favourable analogues were synthesized and evaluated for the cytotoxicity and cytopathic effect inhibition by pandemic H1N1 virus. All the designed analogues resulting in displacement of sialic acid suggested alternate binding mechanism. Overall results indicated that aurones can be measured best among all as potential neuraminidase inhibitor against pandemic H1N1 virus.

A Computational Model for Docking of Noncompetitive Neuraminidase Inhibitors and Probing their Binding Interactions with Neuraminidase of Influenza Virus H5N1.

BACKGROUND: With cases of emergence of drug resistance to the current competitive inhibitors of neuraminidase (NA) such as oseltamivir and zanamavir, there is a present need for an alternative approach in the treatment of avian influenza. With this in view, some flavones and chalcones were designed based on quercetin, the most active naturally occurring noncompetitive inhibitor. OBJECTIVE: We attempt to understand the binding of quercetin to H5N1-NA, and synthetic analogs of quercetin namely flavones and its precursors the chalcones using computational tools. METHODS: Molecular docking was done using Libdock. Molecular dynamics (MD) simulations were performed using Amber14. We synthesized the two compounds; their structures were confirmed by infrared spectroscopy, 1H-NMR, and mass spectrometry. These molecules were then tested for H5N1-NA inhibition and kinetics of inhibition. RESULTS: Molecular docking studies yielded two compounds i.e., 4'-methoxyflavone and 2'-hydroxy-4-methoxychalcone, as promising leads which identified them as binders of the 150-cavity of NA. Furthermore, MD simulation studies revealed that quercetin and the two compounds bind and hold the 150 loop in its open conformation, which ultimately perturbs the binding of sialic acid in the catalytic site. Estimation of the free energy of binding by MM-PBSA portrays quercetin as more potent than chalcone and flavone. These molecules were then determined as non-competitive inhibitors from the Lineweaver-Burk plots rendered from the enzyme kinetic studies. CONCLUSION: We conclude that non-competitive type of inhibition, as shown in this study, can serve as an effective method to block NA and evade the currently seen drug resistance.

Seroprevalence of Measles, Mumps, and Rubella Antibodies in College Students in Mumbai, India.

Measles, Mumps, and Rubella (MMR) are vaccine preventable viral infections, which cause significant mortality and morbidity globally. Increased incidence rates of these infectious diseases are observed in young adults. Information on seroprevalence data on MMR in India is limited. The objective of this study was to determine the prevalence of IgG antibodies against MMR among young adults. This was a descriptive cross-sectional study involving 192 healthy college students from Maharshi Dayanand College, Mumbai. The project was approved by the Institutional Ethics Committee of Haffkine Institute. Between December 2012 and September 2013, blood samples were collected from individuals of age 18-23 years after obtaining written informed consent from them. The quantitative determination of IgG antibodies in serum specimens against MMR was determined using enzyme linked immunosorbent assay. Data on history of vaccination were also collected from participants. Among 192 healthy college students (age 18-23 years), MMR seroprevalence was 91%, 97%, and 88%, respectively. The overall seropositivity of MMR was 79%. The highest level of seronegativity was seen with regards to rubella-specific antibodies in 12% of cases. About 96% of the participants did not know about their vaccination history while none of the participants knew about their history of MMR infections. Despite unknown vaccination status, a majority of college students in our study were found seropositive for all three infections, which indicate natural boosting. However, the proportion of seronegativity for measles and rubella was relatively higher. Especially since the study population belonged to reproductive age group, there is a concern of congenital rubella syndrome in the offspring. Although a larger multicentric study is required to confirm the findings, the results indicate that a dose of measles-rubella (MR) vaccine should be offered to these college students.

Novel evidence of microglial immune response in impairment of Dengue infection of CNS.

Dengue, the most rampant zoonotic viral disease in tropics, contributes to 14% of acute febrile illness cases globally. Encephalitis in primary Dengue fever, with/without haemorrhage has been reported occasionally. Our study presents novel evidence for this rarity at the molecular level. Murine microglia (BV2) were infected in-vitro with Dengue virus (DENV) serotypes (1-4) and their immune response was evaluated. Gene expressions of TNF-α, IL-10, IFN-γ, and IL1-ß constituted the pro-inflammatory response, levels of MCP-1 and IL-6 represented the regulatory mechanism and changes in the levels of Occludin, MMP-2, MMP-9 and TIMP-1 encompassed the break-down of the blood-brain barrier (BBB). Cytokine response was studied using RT-PCR, with relative fold change assessed using ΔΔCt method. We observed that DENV1 increased vascular permeability and trans-membrane transport, while DENV2 resulted in oxidative stress. DENV3 infection presented with impaired immune response and DENV4 manifested a chaotropic response of the BBB protein genes. However, no serotype was able to breakdown the BBB, thus validating the low prevalence of encephalitis in dengue. Our study is the first reported evidence of the microglial immune response resisting the entry of DENV into the CNS. It also supports the theory that primary Dengue infection results in the acute inflammation of the microglia, and the host immune response plays a critical role in development of encephalitis.

Drug susceptibility of influenza A/H3N2 strains co-circulating during 2009 influenza pandemic: first report from Mumbai.

OBJECTIVE: From its first instance in 1977, resistance to amantadine, a matrix (M2) inhibitor has been increasing among influenza A/H3N2, thus propelling the use of oseltamivir, a neuraminidase (NA) inhibitor as a next line drug. Information on drug susceptibility to amantadine and neuraminidase inhibitors for influenza A/H3N2 viruses in India is limited with no published data from Mumbai. This study aimed at examining the sensitivity to M2 and NA inhibitors of influenza A/H3N2 strains isolated from 2009 to 2011 in Mumbai. METHODS: Nasopharyngeal swabs positive for influenza A/H3N2 virus were inoculated on Madin-Darby canine kidney (MDCK) cell line for virus isolation. Molecular analysis of NA and M2 genes was used to detect known mutations contributing to resistance. Resistance to neuraminidase was assayed using a commercially available chemiluminescence based NA-Star assay kit. RESULTS: Genotypically, all isolates were observed to harbor mutations known to confer resistance to amantadine. However, no know mutations conferring resistance to NA inhibitors were detected. The mean IC50 value for oseltamivir was 0.25 nM. One strain with reduced susceptibility to the neuraminidase inhibitor (IC50=4.08 nM) was isolated from a patient who had received oseltamivir treatment. Phylogenetic analysis postulate the emergence of amantadine resistance in Mumbai may be due to genetic reassortment with the strains circulating in Asia and North America. CONCLUSIONS: Surveillance of drug susceptibility helped us to identify an isolate with reduced sensitivity to oseltamivir. Therefore, we infer that such surveillance would help in understanding possible trends underlying the emergence of resistant variants in humans.

Brain-Derived Neurotrophic Factor in children with Autism Spectrum Disorder.

BACKGROUND: Autism Spectrum Disorder (ASD) is a complex neurobehavioral syndrome with no known biomarker so far for early detection. It has been challenging, both to classify typical autism and associate a suitable biomarker with clinical phenotype spectrum. Brain-derived neurotrophic factor (BDNF) has emerged as a key neurotrophin regulating synaptic plasticity, neuronal differentiation and survival. PURPOSE: Recently, BDNF depletion is reported in neurodegenerative as well as in psychiatric disorders, associated with severity of neurological dysfunction. Role of BDNF as a biomarker in ASD is gaining significance. Pre-clinical results have linked BDNF depletion in autism and mental retardation, however, with conflicting findings. METHODS: In view of this, a preliminary study was carried out to measure serum BDNF levels in 48 children with ASD and mental retardation, and 29 age-matched controls. RESULTS: Serum BDNF levels were found significantly higher (p<0.001) in atypical autistic subjects (clinically milder phenotype) as compared to controls, but not in typical ASD cases (clinically severe phenotype). BDNF levels were significantly lower in females with typical/Rett Syndrome (p<0.05), but not in males with typical autism (p>0.1), as compared to controls. Lower BDNF levels indicate impairment in neuroprotective mechanism, while higher levels may imply a manifested protective response. CONCLUSION: Our study highlights the differential BDNF response based on the severity of neurobehavioral deficit, indicating a possible neuroprotective role of this molecule and supporting its exploration in targeted therapy in ASD.

Molecular analysis of genetic variation in angiotensin I-converting enzyme identifies no association with sporting ability: First report from Indian population.

INTRODUCTION: A polymorphism in the angiotensin-converting enzyme (ACE) gene was the first performance enhancing polymorphisms (PEPs) to be identified and correlated with athletic abilities. This polymorphism (rs. 5186) is the absence (deletion; D allele), rather than the presence (insertion, I allele) of 287bp Alu repeat element in intron 16. However, the association of ACE I/D polymorphism in sports abilities have been contradicted and debated. No study has evaluated the ACE gene polymorphism in Indian athletes so far. Hence, the genotype distribution and allelic frequency of ACE gene in selected Indian athletic and non-athletic population was studied. MATERIALS AND METHODS: A total of 147 athletes and 131 controls were genotyped for the ACE gene polymorphism using PCR. RESULTS: No significant association was observed between the allelic frequencies of ACE gene in controls and athletes on a whole, as well as after sub-categorizing the athletes based on the type of sport they played (P > 0.1). However, a higher representation of I allele was observed in the athletes. CONCLUSION: ACE genotyping studies need to focus on truly elite athletes of a single sporting discipline, to be able to find an association. The ACE I/D polymorphism may not be considered a marker for human performance, but can be further studied in combination with other potent performance enhancing polymorphisms.

Estimation of mRNA levels of interleukin (IL)-10, tumor necrosis factor (TNF)-α, IL-4 and interferon (IFN)-γ in HIV infected children in Mumbai.

Cytokines, viral load and opportunistic infections play an important role in HIV-disease progression. Hundred children vertically infected with HIV were enrolled to determine mRNA levels of TNF-α, IL-10, IL-4 and IFN-γ. These levels were estimated by amplifying cytokine mRNA from peripheral blood mononuclear cells. Severity of HIV was staged by the reduction in CD(4) (+) T cells and the onset of opportunistic infections. IL-10 mRNA levels were observed to increase with the severity. Despite the rising IL-10 mRNA levels, TNF-α mRNA levels increased with severity of HIV and decrease in CD(4) (+) T cell counts. IL-4 mRNA levels increased with the reduction in CD(4) (+) T cell numbers. Depleting mRNA levels of IFN-γ contributed to the worsening of HIV disease. Increase in TNF-α and IL-4 levels appended to the disease severity by upregulation of the viral replication. Increased IL-10 levels and decreased IFN-γ levels predisposed the children to HIV associated opportunistic infections, which in return contributed to cytokine disarray.