Screening of medicinal plant phytochemicals as natural antagonists of p53-MDM2 interaction to reactivate p53 functioning.

In most types of cancer, overexpression of murine double minute 2 (MDM2) often leads to inactivation of p53. The crystal structure of MDM2, with a 109-residue amino-terminal domain, reveals that MDM2 has a core hydrophobic region to which p53 binds as an amphipathic α helix. The interface depends on the steric complementarity between MDM2 and the hydrophobic region of p53. Especially, on p53's triad, amino acids Phe19, Trp23 and Leu26 bind to the MDM2 core. Results from studies suggest that the structural motif of both p53 and MDM2 can be attributed to similarities in the amphipathic α helix. Thus, in the current investigation it is hypothesized that the similarity in the structural motif might be the cause of p53 inactivation by MDM2. Hence, molecular docking and phytochemical screening approaches are appraised to inhibit the hydrophobic cleft of MDM2 and to stop p53-MDM2 interaction, resulting in reactivation of p53 activity. For this purpose, a library of 2295 phytochemicals were screened against p53-MDM2 to find potential candidates. Of these, four phytochemicals including epigallocatechin gallate, alvaradoin M, alvaradoin E and nordihydroguaiaretic acid were found to be potential inhibitors of p53-MDM2 interaction. The screened phytochemicals, derived from natural extracts, may have negligible side effects and can be explored as potent antagonists of p53-MDM2 interactions, resulting in reactivation of the normal transcription of p53.

Designing and molecular docking of cyclic peptides against HCV NS3 protease.

Hepatitis C Virus (HCV) infection is a worldwide serious health issue which contributes towards most of the hepatic morbidities. So far no prophylaxis is available to prevent this virus; therefore, development of antiviral compounds to fight HCV infection is the need of time. Chemically synthesized peptides that are potent immunogenic antigens are being pursued as candidate vaccines against HCV. The present study was planned to identify peptide inhibitors having potential to block the activity of NS3 protein of HCV that will ultimately arrest HCV multiplication. Docking of NS3 with peptides revealed that the majority of the peptides have strong binding affinity for active sites of NS3. Peptide 1, 2, 3 and 6 were found interactive with NS3 active residues while the active sites of NS3 had hydrophobic contact with the rest of peptides. Thus, these peptides bear therapeutic potential of a candidate drug for the prevention of HCV replication. Post docking analysis revealed important binding abilities of peptides with the active sites of NS3 protein, showing the efficiency of peptides as potential peptide inhibitors against HCV. The study revealed that HCV replication can be inhibited by these peptides. HCV replication inhibition potential of these peptides can contribute in reducing the burden of HCV infection and its associated complications worldwide.

HCV Envelope protein 2 sequence comparison of Pakistani isolate and In-silico prediction of conserved epitopes for vaccine development.

BACKGROUND: HCV is causing hundreds of cases yearly in Pakistan and has become a threat for Pakistani population. HCV E2 protein is a transmembrane protein involved in viral attachment and thus can serve as an important target for vaccine development but because of its variability, vaccine development against it has become a challenge. Therefore, this study was designed to isolate the HCV E2 gene from Pakistani HCV infected patients of 3a genotype, to perform In-silico analysis of HCV E2 isolated in Pakistan and to analyze HCV E2 protein sequence in comparison with other E2 proteins belonging to 3a and 1a genotypes to find potential conserved B-cells and T-cell epitopes that can be important in designing novel inhibitory compounds and peptide vaccine against genotype 3a and 1a. PATIENTS AND METHODS: Patients were selected on the basis of elevated serum ALT and AST levels at least for six months, histological examination, and detection of serum HCV RNA anti-HCV antibodies (3rd generation ELISA). RNA isolation, cDNA synthesis, amplification, cloning and sequencing was performed from 4 patient's serum samples in order to get the HCV E2 sequence. HCV E2 protein of Pakistani origin was analyzed using various bioinformatics tools including sequence and structure tools. RESULTS: HCV E1 protein modeling was performed with I-TASSER online server and quality of the model was assessed with ramchandran plot and Z-score. A total of 3 B-cell and 3 T-cell epitopes were found to be highly conserved among HCV 3a and 1a genotype. CONCLUSION: The present study revealed potential conserved B-cell and T-cell epitopes of the HCV E2 protein along with 3D protein modeling. These conserved B-cell and T-cell epitopes can be helpful in developing effective vaccines against HCV and thus limiting threats of HCV infection in Pakistan.

Structural analysis and epitope prediction of HCV E1 protein isolated in Pakistan: an in-silico approach.

BACKGROUND: HCV infection is a major health problem causing acute and chronic hepatitis. HCV E1 protein is a transmembrane protein that is involved in viral attachment and therefore, can serve as an important target for vaccine development. Consequently, this study was designed to analyze the HCV E1 protein sequence isolated in Pakistan to find potential conserved epitopes/antigenic determinants. RESULTS: HCV E1 protein isolated in Pakistan was analyzed using various bio-informatics and immuno-informatics tools including sequence and structure tools. A total of four antigenic B cell epitopes, 5 MHC class I binding peptides and 5 MHC class II binding peptides were predicted. Best designed epitopes were subjected to conservation analyses with other countries. CONCLUSION: The study was conducted to predict antigenic determinants/epitopes of HCV E1 protein of genotype 3a along with the 3D protein modeling. The study revealed potential B-cell and T-cell epitopes that can raise the desired immune response against HCV E1 protein isolated in Pakistan. Conservation analysis can be helpful in developing effective vaccines against HCV and thus limiting threats of HCV infection in Pakistan.

Development of global consensus sequence of HCV glycoproteins involved in viral entry.

BACKGROUND: HCV affects>170 million people worldwide and is a leading cause of liver diseases such as hepatocellular carcinoma. Each year, Pakistan reports hundreds of cases and now it has become a serious health issue. HCV has two transmembrane glycoproteins (E1 and E2) that are involved in virus entry through viral attachment, but because of their hypervariable nature they have become difficult targets for vaccine development. METHODS: A total of 150 protein sequences of E1 and E2 belonging to genotypes 3a and 1a were retrieved from the NCBI protein database and were subjected to conservation and variation analysis using the multiple sequence alignment feature of the CLC workbench. A consensus sequence of each genotype of E1 and E2 was obtained and these consensus sequences were further analyzed to construct a global consensus sequence, which was used to design potentially conserved peptides. RESULTS: From the sequence conservation analysis, highly conserved residues were identified and were used to design peptides. Only two peptides were found to be conserved in the E1 protein of genotypes 3a and 1a and a total of nine conserved peptides were designed for the HCV E2 protein of those genotypes. These designed peptides could serve as useful targets in developing new inhibitory compounds. CONCLUSION: This study was designed to perform conservation and variability analysis of HCV glycoproteins and to find potentially conserved peptides among genotypes 3a and 1a (the most prevalent genotypes in Pakistan) that could serve as useful targets in the development of novel inhibitory compounds, thus reducing the threat of HCV infection in Pakistan.

A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan.

Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue.

Glycyrrhizin as antiviral agent against Hepatitis C Virus.

BACKGROUND: Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression, fatigue, and "flu-like" symptoms. Herbal plants have been used for centuries against different diseases including viral diseases and have become a major source of new compounds to treat bacterial and viral diseases. MATERIAL: The present study was design to study the antiviral effect of Glycyrrhizin (GL) against HCV. For this purpose, HCV infected liver cells were treated with GL at non toxic doses and HCV titer was measured by Quantitative real time RT-PCR. RESULTS AND DISCUSSION: Our results demonstrated that GL inhibit HCV titer in a dose dependent manner and resulted in 50% reduction of HCV at a concentration of 14 ± 2 µg. Comparative studies were made with interferon alpha to investigate synergistic effects, if any, between antiviral compound and interferon alpha 2a. Our data showed that GL exhibited synergistic effect when combined with interferon. Moreover, these results were verified by transiently transfecting the liver cells with HCV 3a core plasmid. The results proved that GL dose dependently inhibit the expression of HCV 3a core gene both at mRNA and protein levels while the GAPDH remained constant. CONCLUSION: Our results suggest that GL inhibit HCV full length viral particles and HCV core gene expression or function in a dose dependent manner and had synergistic effect with interferon. In future, GL along with interferon will be better option to treat HCV infection.

Inhibition of HCV 3a genotype entry through host CD81 and HCV E2 antibodies.

BACKGROUND: HCV causes acute and chronic hepatitis which can eventually lead to permanent liver damage hepatocellular carcinoma and death. HCV glycoproteins play an important role in HCV entry by binding with CD81 receptors. Hence inhibition of virus at entry step is an important target to identify antiviral drugs against HCV. METHODS AND RESULT: The present study elaborated the role of CD81 and HCV glycoprotein E2 in HCV entry using retroviral pseudo-particles of 3a local genotype. Our results demonstrated that HCV specific antibody E2 and host antibody CD81 showed dose- dependent inhibition of HCV entry. HCV E2 antibody showed 50% reduction at a concentration of 1.5 ± 1 µg while CD81 exhibited 50% reduction at a concentration of 0.8 ± 1 µg. In addition, data obtained with HCVpp were also confirmed with the infection of whole virus of HCV genotype 3a in liver cells. CONCLUSION: Our data suggest that HCV specific E2 and host CD81 antibodies reduce HCVpp entry and full length viral particle and combination of host and HCV specific antibodies showed synergistic effect in reducing the viral titer.

Antiviral drugs against hepatitis C virus.

Hepatitis C virus (HCV) infection is a major worldwide problem causes acute and chronic HCV infection. Current treatment of HCV includes pegylated interferon-α (PEG IFN- α) plus ribavirin (RBV) which has significant side effects depending upon the type of genotype. Currently, there is a need to develop antiviral agents, both from synthetic chemistry and Herbal sources. In the last decade, various novel HCV replication, helicase and entry inhibitors have been synthesized and some of which have been entered in different phases of clinical trials. Successful results have been acquired by executing combinational therapy of compounds with standard regime in different HCV replicons. Even though, diverse groups of compounds have been described as antiviral targets against HCV via Specifically Targeted Antiviral Therapy for hepatitis C (STAT-C) approach (in which compounds are designed to directly block HCV or host proteins concerned in HCV replication), still there is a need to improve the properties of existing antiviral compounds. In this review, we sum up potent antiviral compounds against entry, unwinding and replication of HCV and discussed their activity in combination with standard therapy. Conclusively, further innovative research on chemical compounds will lead to consistent standard therapy with fewer side effects.

Antiviral activity of Acacia nilotica against Hepatitis C Virus in liver infected cells.

Hepatitis C virus (HCV) belonging to the family Flaviviridae has infected 3% of the population worldwide and 6% of the population in Pakistan. The only recommended standard treatment is pegylated INF-α plus ribavirin. Due to less compatibility of the standard treatment, thirteen medicinal plants were collected from different areas of Pakistan on the basis of undocumented antiviral reports against different viral infections. Medicinal plants were air dried, extracted and screened out against HCV by infecting HCV inoculums of 3a genotype in liver cells. RT-PCR results demonstrate that acetonic and methanolic extract of Acacia nilotica (AN) showed more than 50% reduction at non toxic concentration. From the above results, it can be concluded that by selecting different molecular targets, specific structure-activity relationship can be achieved by doing mechanistic analysis. So, additional studies are required for the isolation and recognition of antiviral compound in AN to establish its importance as antiviral drug against HCV. For further research, we will scrutinize the synergistic effect of active antiviral compound in combination with standard PEG INF-α and ribavirin which may be helpful in exploring further gateways for antiviral therapy against HCV.

The epidemic of HIV/AIDS in developing countries; the current scenario in Pakistan.

HIV (Human Immunodeficiency virus) causes (acquired immunodeficiency syndrome) AIDS, in which the immune system of body totally fails to develop any defense against the foreign invaders. Infection with HIV occurs by transfer of blood, semen, and breast milk. HIV/AIDS is a global problem and it results nearly 25 million deaths worldwide. Developing countries like Pakistan have issues regarding Public Health. Currently, epidemic of HIV/AIDS is established in Pakistan and there is a threat of an expanded HIV/AIDS outbreak in the country. The major reason is that population is engaging in high-risk practices, low awareness about HIV/AIDS, and treacherous blood transfusion practices. A supplementary threat to Pakistan is India because both sharing a border and India is facing a rapidly growing HIV/AIDS epidemic. Local NGOs, National and International organizations are warning that in near future Pakistan may experiences bad situation regarding HIV/AIDS.In the present article we focused current situation of surveillance of HIV/AIDS, its virology, genotype, diagnostics, high-risk groups, reasons of vulnerability in Pakistani population, and the role of different national and international organizations in this situation.

Lysosomotropic agents as HCV entry inhibitors.

HCV has two envelop proteins named as E1 and E2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. Fusion of the HCV envelope proteins is triggered by low pH within the endosome. Lysosomotropic agents (LA) such as Chloroquine and Ammonium chloride (NH4Cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular pH. To investigate the antiviral effect of LA (Chloroquine and NH4Cl) on pH dependent endocytosis, HCV pseudoparticles (HCVpp) of 1a and 3a genotype were produced and used to infect liver cells. The toxicological effects of Chloroquine and NH4Cl were tested in liver cells through MTT cell proliferation assay. For antiviral screening of Chloroquine and NH4Cl, liver cells were infected with HCVpp of 3a and 1a genotype in the presence or absence of different concentrations of Chloroquine and NH4Cl and there luciferase activity was determined by using a luminometer. The results demonstrated that Chloroquine and NH4Cl showed more than 50% reduction of virus infectivity at 50 µM and 10 mM concentrations respectively. These results suggest that inhibition of HCV at fusion step by increasing the lysosomal pH will be better option to treat chronic HCV.

An overview of HCV molecular biology, replication and immune responses.

Hepatitis C virus (HCV) causes acute and chronic hepatitis which can eventually lead to permanent liver damage, hepatocellular carcinoma and death. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. In this review, we summarize molecular virology, replication and immune responses against HCV and discussed how HCV escape from adaptive and humoral immune responses. This advance knowledge will be helpful for development of vaccine against HCV and discovery of new medicines both from synthetic chemistry and natural sources.

siRNAs: potential therapeutic agents against hepatitis C virus.

Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression and "flu-like" symptoms. Needless to mention, the effectiveness of interferon therapy is predominantly, if not exclusively, limited to virus type 3a and 3b whereas in Europe and North America the majority of viral type is 1a and 2a. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Several reports have indicated the efficiency and specificity of synthetic and vector based siRNAs inhibiting HCV replication. In the present review, we focused that combination of siRNAs against virus and host genes will be a better option to treat HCV.

Inhibition of hepatitis C virus 3a genotype entry through Glanthus Nivalis Agglutinin.

BACKGROUND: Hepatitis C Virus (HCV) has two envelop proteins E1 and E2 which is highly glycosylated and play an important role in cell entry. Inhibition of virus at entry step is an important target to find antiviral drugs against HCV. Glanthus Nivalis Agglutinin (GNA) is a mannose binding lectin which has tendency for specific recognition and reversible binding to the sugar moieties of a wide variety of glycoproteins of enveloped viruses. RESULTS: In the present study, HCV pseudoparticles (HCVpp) for genotype 3a were produced to investigate the ability of GNA to block the HCV entry. The results demonstrated that GNA inhibit the infectivity of HCVpp and HCV infected serum in a dose-dependent manner and resulted in 50% reduction of virus at 1 ± 2 µg concentration. Molecular docking of GNA and HCV glycoproteins (E1 and E2) showed that GNA inhibit HCV entry by binding N-linked glycans. CONCLUSION: These results demonstrated that targeting the HCV glycans is a new approach to develop antiviral drugs against HCV.

Anti-apoptotic effect of HCV core gene of genotype 3a in Huh-7 cell line.

BACKGROUND: Hepatitis C virus (HCV) Core protein regulates multiple signaling pathways and alters cellular genes expression responsible for HCV induced pathogenesis leading to hepatocellular carcinoma (HCC). Prevalence of HCV genotype 3a associated HCC is higher in Pakistan as compare to the rest of world; however the molecular mechanism behind this is still unclear. This study has been designed to evaluate the effect of HCV core 3a on apoptosis and cell proliferation which are involved in HCC METHODOLOGY: We examined the in vitro effect of HCV Core protein of genotype 3a and 1a on cellular genes involved in apoptosis by Real time PCR in liver cell line (Huh-7). We analyzed the effect of HCV core of genotype 1a and 3a on cell proliferation by MTT assay and on phosphrylation of Akt by western blotting in Huh-7 cells. RESULTS: The HCV 3a Core down regulates the gene expression of Caspases (3, 8, 9 and 10), Cyto C and p53 which are involved in apoptosis. Moreover, HCV 3a Core gene showed stronger effect in regulating protein level of p-Akt as compared to HCV 1a Core accompanied by enhanced cell proliferation in Huh-7 cell line. CONCLUSION: From the current study it has been concluded that reduced expression of cellular genes involved in apoptosis, increased p-Akt (cell survival gene) and enhanced cell proliferation in response to HCV 3a core confirms anti apoptotic effect of HCV 3a Core gene in Huh-7 that may lead to HCC.

NS4A protein as a marker of HCV history suggests that different HCV genotypes originally evolved from genotype 1b.

BACKGROUND: The 9.6 kb long RNA genome of Hepatitis C virus (HCV) is under the control of RNA dependent RNA polymerase, an error-prone enzyme, for its transcription and replication. A high rate of mutation has been found to be associated with RNA viruses like HCV. Based on genetic variability, HCV has been classified into 6 different major genotypes and 11 different subtypes. However this classification system does not provide significant information about the origin of the virus, primarily due to high mutation rate at nucleotide level. HCV genome codes for a single polyprotein of about 3011 amino acids which is processed into structural and non-structural proteins inside host cell by viral and cellular proteases. RESULTS: We have identified a conserved NS4A protein sequence for HCV genotype 3a reported from four different continents of the world i.e. Europe, America, Australia and Asia. We investigated 346 sequences and compared amino acid composition of NS4A protein of different HCV genotypes through Multiple Sequence Alignment and observed amino acid substitutions C22, V29, V30, V38, Q46 and Q47 in NS4A protein of genotype 1b. Furthermore, we observed C22 and V30 as more consistent members of NS4A protein of genotype 1a. Similarly Q46 and Q47 in genotype 5, V29, V30, Q46 and Q47 in genotype 4, C22, Q46 and Q47 in genotype 6, C22, V38, Q46 and Q47 in genotype 3 and C22 in genotype 2 as more consistent members of NS4A protein of these genotypes. So the different amino acids that were introduced as substitutions in NS4A protein of genotype 1 subtype 1b have been retained as consistent members of the NS4A protein of other known genotypes. CONCLUSION: These observations indicate that NS4A protein of different HCV genotypes originally evolved from NS4A protein of genotype 1 subtype 1b, which in turn indicate that HCV genotype 1 subtype 1b established itself earlier in human population and all other known genotypes evolved later as a result of mutations in HCV genotype 1b. These results were further confirmed through phylogenetic analysis by constructing phylogenetic tree using NS4A protein as a phylogenetic marker.

Therapeutic Potential of Umbilical Cord Stem Cells for Liver Regeneration.

The liver is a vital organ for life and the only internal organ that is capable of natural regeneration. Although the liver has high regeneration capacity, excessive hepatocyte death can lead to liver failure. Various factors can lead to liver damage including drug abuse, some natural products, alcohol, hepatitis, and autoimmunity. Some models for studying liver injury are APAP-based model, Fas ligand (FasL), D-galactosamine/endotoxin (Gal/ET), Concanavalin A, and carbon tetrachloride-based models. The regeneration of the liver can be carried out using umbilical cord blood stem cells which have various advantages over other stem cell types used in liver transplantation. UCB-derived stem cells lack tumorigenicity, have karyotype stability and high immunomodulatory, low risk of graft versus host disease (GVHD), low risk of transmitting somatic mutations or viral infections, and low immunogenicity. They are readily available and their collection is safe and painless. This review focuses on recent development and modern trends in the use of umbilical cord stem cells for the regeneration of liver fibrosis.

Medicinal plants against hepatitis C virus.

Hepatitis C virus (HCV) is a global health concern which is responsible for most of the liver diseases. Currently, there is no vaccine available for prevention of HCV infection due to the high degree of strain variation. The current standard of care is a combination of pegylated interferon α with ribavirin and boceprevir/telaprevir. This treatment was partially effective and had significant side effects. Hence, there is a need to develop new antiviral agents that interfere with different stages of the HCV life cycle. Recent advances in the understanding of both the cellular and molecular mechanisms of HCV replication have provided the basis for novel therapeutic strategies. Several hundred plant species and their phyto-constituents have been isolated for screening against HCV, and some have been shown to have great medicinal value in preventing and/or ameliorating viral diseases in pre-clinical and clinical trials. This review summarizes medicinal plants and their phytochemicals which inhibit different stages of HCV life cycle and discuss their potential use in HCV therapy.

RNAi: antiviral therapy against dengue virus.

Dengue virus infection has become a global threat affecting around 100 countries in the world. Currently, there is no licensed antiviral agent available against dengue. Thus, there is a strong need to develop therapeutic strategies that can tackle this life threatening disease. RNA interference is an important and effective gene silencing process which degrades targeted RNA by a sequence specific process. Several studies have been conducted during the last decade to evaluate the efficiency of siRNA in inhibiting dengue virus replication. This review summarizes siRNAs as a therapeutic approach against dengue virus serotypes and concludes that siRNAs against virus and host genes can be next generation treatment of dengue virus infection.