Development of drugs for Epstein–Barr virus using high-throughput in silico virtual screening

Importance of the field: Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus that is causally associated with endemic forms of Burkitt's lymphoma, nasopharyngeal carcinoma and lymphoproliferative disease in immunosuppressed individuals. On a global scale, EBV infects > 90% of the adult population and is responsible for ～ 1% of all human cancers. To date, there is no efficacious drug or therapy for the treatment of EBV infection and EBV-related diseases.

Areas covered in this review: In this review, we discuss the existing anti-EBV inhibitors and those under development. We discuss the value of different molecular targets, including EBV lytic DNA replication enzymes as well as proteins that are expressed exclusively during latent infection, such as EBV nuclear antigen 1 (EBNA-1) and latent membrane protein 1. As the atomic structure of the EBNA-1 DNA binding domain has been described, it is an attractive target for in silico methods of drug design and small molecule screening. We discuss the use of computational methods that can greatly facilitate the development of novel inhibitors and how in silico screening methods can be applied to target proteins with known structures, such as EBNA-1, to treat EBV infection and disease.

What the reader will gain: The reader is familiarized with the problems in targeting of EBV for inhibition by small molecules and how computational methods can greatly facilitate this process.

Take home message: Despite the impressive efficacy of nucleoside analogs for the treatment of herpesvirus lytic infection, there remain few effective treatments for latent infections. As EBV latent infection persists within and contributes to the formation of EBV-associated cancers, targeting EBV latent proteins is an unmet medical need. High-throughput in silico screening can accelerate the process of drug discovery for novel and selective agents that inhibit EBV latent infection and associated disease.     

Aspirin induces lytic cytotoxicity in Epstein-Barr virus-positive cells

Epstein-Barr virus (EBV) infection in tumor cells is generally restricted to the latent forms of viral infection. Switching the latent form of viral infection into the lytic form may induce tumor cell death. High levels of nuclear factor (NF)-kappaB can inhibit EBV lytic replication, and aspirin has the ability to inhibit NF-kappaB activity. The aims of the current study were to determine the effects of aspirin on inducing EBV lytic infection, and thus to reveal the possibility of targeting EBV-positive cancer cells by aspirin. Our results showed that aspirin depleted NF-kappaB (p65) in the nucleus and reactivated EBV into lytic replication. Cells exhibited decreased viability in a dose- and time-dependent manner when incubated with aspirin. When ganciclovir was used in combination with aspirin to treat EBV-positive B95.8 cells and Raji cells, the cytotoxic effect of aspirin was amplified. We demonstrated that aspirin reduced the viability of EBV-positive B lymphocytes due to its ability to induce EBV lytic replication.     

Sulfated Small Molecules Targeting EBV in Burkitt Lymphoma: From In Silico Screening to the Evidence of In Vitro Effect on Viral Episomal DNA

Epstein–Barr virus (EBV) infects more than 90% of the world population. Following primary infection, Epstein–Barr virus persists in an asymptomatic latent state. Occasionally, it may switch to lytic infection. Latent EBV infection has been associated with several diseases, such as Burkitt lymphoma (BL). To date, there are no available drugs to target latent EBV, and the existing broad‐spectrum antiviral drugs are mainly active against lytic viral infection. Thus, using computational molecular docking, a virtual screen of a library of small molecules, including xanthones and flavonoids (described with potential for antiviral activity against EBV), was carried out targeting EBV proteins. The more interesting molecules were selected for further computational analysis, and sub‐sequently, the compounds were tested in the Raji (BL) cell line, to evaluate their activity against latent EBV. This work identified three novel sulfated small molecules capable of decreasing EBV levels in a BL. Therefore, the in silico screening presents a good approach for the development of new anti‐EBV agents.     

Inhibition of host kinase activity altered by the LMP2A signalosome-a therapeutic target for Epstein-Barr virus latency and associated disease

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection in the majority of the human population. The virus resides in a latent state in B lymphocytes and is associated with a variety of cancers in the human host. In normal individuals, latent infection with EBV typically poses no health risk, but upon immunosuppression, either following organ transplantation or HIV infection, malignancies and lymphoproliferative diseases can result. Latent membrane protein 2A (LMP2A) is a virally encoded membrane protein that is expressed in EBV latent infection and in most of the tumors associated with EBV infection. Previous studies have indicated that LMP2A expression alters the activity of the Src family protein tyrosine kinases, the Syk protein tyrosine kinase, the Btk protein tyrosine kinase, and phosphatidylinositol 3-kinase (PI3-kinase). In this study, inhibitors of each of these kinases were tested using an in vitro system dependent on LMP2A expression for B cell colony formation in IL-7 containing methylcellulose media. Of the inhibitors tested, only piceatannol, a Syk tyrosine kinase inhibitor, demonstrated a specific effect on LMP2A expressing cells and not control cells. These studies provide a basis for targeting LMP2A function in EBV latency and may allow for the identification of novel therapeutics for the treatment or eradication of EBV latent infections and associated proliferative disorders.     

Viral latent proteins as targets for Kaposi's sarcoma and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) induced lymphoma

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is present in all Kaposi's sarcoma tumor cells as well as in several lymphomas that are linked to this agent. Every tumor cell expresses the viral latent protein LANA, which is required for KSHV latent replication and proper segregation of the viral episome. In certain tumors, other latent KSHV proteins (LANA-2/vIRF3, v-cyclin, v-IL6) are expressed as well. Since all herpesviruses persist for life in infected individuals, only eradication of latent virus can cure infection. The KSHV latent genes serve as bona fide tumor markers, but do they also provide targets for anti-tumor and/or anti-viral drugs? To decide this question we review the known biochemical interactions between KSHV latent proteins and their viral and cellular partners. Recent epidemiological studies show that KSHV lytic replication precedes KSHV associated cancers. Gancilovir has been linked to KS tumor regression, which implicates the KSHV-encoded polymerase as a potential intervention point. Yet, KSHV specific transactivators might represent more specific targets, as they have no cellular homologs. In particular Rta/orf50 is necessary and sufficient for lytic replication and deserves serious consideration as a target for KSHV-specific antivirals.     

Frequent double infection with Epstein-Barr virus and human herpesvirus-6 in patients with acute infectious mononucleosis.

Clinical infectious mononucleosis (IM) represents a benign self-limited form of lymphoproliferative disease which is usually caused by infection with Epstein-Barr virus (EBV). Microscopic characteristics of this lymphoproliferative disorder, however, are not ultimately specific for EBV infection, but can also be seen in infections with other lymphotropic viruses, especially of the herpesvirus family. Human herpesvirus-6 (HHV-6) infection can apparently be associated with a number of diseases also seen in EBV infection. Also, postinfectious chronic fatigue syndrome (PICFS) which may follow IM is in more than 60% of the cases accompanied by persistent active HHV-6 infection. We thus screened serologically 215 cases of acute IM for evidence for infection with EBV, HHV-6 and CMN. Patients were tentatively grouped into those having primary infection or reactivated (probably non-primary) infections. Cases were followed for two years to monitor changes in titers. Of all 215 cases, 211 (98.1%) were positive for EBV, 137 (63.7%) for primary infections, 21 (9.8%) for reactivated infection, and 53 (24.6%) for latent EBV. Thirty-three (15.3%) cases had primary HHV-6 infection, 63 (29.3%) active or reactivated HHV-6 infection, and 71 (33.9%) latent HHV-6. Double active EBV and HHV-6 infection, including primary and reactivated infections, amounted to 89 (39.5%) cases. Cytomegalovirus (CMV) antibody titers were found in 81 (37%) cases, 48 (22.3%) of which indicated latent infection and 33 (15.3%) active infection. Only two cases had evidence of active CMV infection alone, 1 cases of active CMV and HHV-6 infection. Serologic titers in 12 (5.6%) cases indicated combined active infection with CMV, EBV and HHV-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epstein–Barr virus: novel patented therapeutics

Importance of the field: Therapeutic strategies against Epstein–Barr virus (EBV) infection and associated cancers are still limited. Due to the primary role of this virus in the pathogenesis of these diseases, molecules interfering with EBV protein functions may represent effective therapeutic tools to treat EBV-associated diseases.

Areas covered in this review: Active immunotherapeutic protocols targeting EBV and EBV-associated cancers are reviewed first; then, we reviewed adoptive cellular therapies, innovative pharmacological approaches and other non-conventional therapeutic molecules.

What the reader will gain: The published literature, in particular patent publications on EBV therapeutic options, has been searched for relevant information. This information has been organized and discussed.

Take home message: Many promising therapeutic opportunities are identified. Among these, vaccination protocols and adoptive cellular therapies are the most advanced and reached Phase I/II clinical trials. Pharmacological approaches are at the moment limited to the laboratory phase.     

抗疱疹病毒感染药物的研究进展

疱疹病毒感染类疾病高发,可形成终生感染,给人类健康和财产安全带来了极大的损失。然而,临床上批准常用的治疗疱疹病毒感染的药物仅几种核苷类似物,这些药物的应用往往还会引发耐药性和不良反应。因而,克服抗疱疹病毒药物存在的问题,探索新型的抗疱疹病毒药物迫在眉睫。总结了临床常用抗疱疹病毒感染药物、靶向病原体的抗疱疹病毒抑制剂和靶向宿主的抗疱疹病毒抑制剂,介绍了联合用药抗病毒新型策略,以期为科研人员在抗疱疹病毒药物方面的研究提供参考。     

Regulatory T cells and EBV associated malignancies

It has been shown that the T-regulatory cells (Tregs) not only play a key role in the establishment and maintenance of peripheral tolerance to prevent the autoimmune disease, but also inhibit the anti-tumor immunity. Recently, many studies have demonstrated that cytotoxicity T cells (CTL) can control the growth of EBV-positive tumor cells in vitro, including Hodgkin's lymphoma (HL), nasopharyngeal carcinoma, posttransplantation lymphoproliferative disorders (PTLD), depending on the large mount of EBV antigens presented by MHC molecules on the surface of these malignant cells. However, limited benefit of CTL adoptive immunotherapy has been reported in the treatment of EBV positive HL and NPC, and Tregs are regarded as a critical hurdle in this issue. In the present review, we discuss the correlation of EBV antigens expression in the tumor cells and the induction of Tregs in tumor microenvironment. Treg subsets and its possible mechanism to attenuate the anti-tumor immunity in EBV associated malignancies are also discussed, following by the possible strategies of targeting Tregs in the future immunotherapy for EBV positive cancers.     

Drug delivery technologies for autoimmune disease

Importance of the field: Targeting autoimmune disease poses two main challenges. The first is to identify unique targets to suppress directly or indirectly autoreactive cells exclusively. The second is to penetrate target tissues to deliver specifically drugs to desired cells that can achieve a therapeutic outcome.

Areas covered in this review: Herein, the range of drug delivery methods available and under development and how they can be useful to treat autoimmune diseases are discussed. Polymer delivery methods, as well as biological methods that include fusion proteins, targeted antibodies, recombinant viruses and cell products are compared.

What the reader will gain: Readers will gain insight into the progression of clinical trials for different technologies and drug delivery methods useful for targeting and modulating the function of autoreactive immune cells.

Take home message: Several tissue-specific polymer-based and biologic drug delivery systems are now in Phase II/III clinical trials. Although these trials are focused mainly on cancer treatment, lessons from these trials can guide the use of the same agents for autoimmunity therapeutics.     

Recent developments in the prevention and treatment of Epstein-Barr virus-associated lymphoproliferative diseases

Epstein-Barr virus (EBV) infection and its relevance in post-transplant lymphoproliferative diseases (PTLDs) is an increasing area of concern. PTLD can differ clinically from a mononucleosis-like syndrome to malignant lymphoma. The incidence varies between < 1 and > 20% depending on different risk factors and the kind of transplant. Despite several treatment regimens, including reduction of immunosuppression, antiviral drugs, adoptive immunotherapy and administration of anti-CD20 monoclonal antibodies, the mortality rate is still high. Novel therapeutic strategies for managing PTLD use pharmacological induction of the viral thymidine kinase gene in tumour cells, the target of antivirals based on nucleoside analogues, followed by treatment with ganciclovir. Further treatment modalities include the development of vaccines and targeting of the latent EBV episomes. Prevention of PTLD by pre-emptive therapy based on molecular monitoring of EBV load will represent the main aim to reduce occurrence. This review examines patents and literature for the treatment of EBV-associated lymphoproliferative diseases.     

Recent developments in the prevention and treatment of Epstein–Barr virus-associated lymphoproliferative diseases

Epstein–Barr virus (EBV) infection and its relevance in post-transplant lymphoproliferative diseases (PTLDs) is an increasing area of concern. PTLD can differ clinically from a mononucleosis-like syndrome to malignant lymphoma. The incidence varies between < 1 and > 20% depending on different risk factors and the kind of transplant. Despite several treatment regimens, including reduction of immunosuppression, antiviral drugs, adoptive immunotherapy and administration of anti-CD20 monoclonal antibodies, the mortality rate is still high. Novel therapeutic strategies for managing PTLD use pharmacological induction of the viral thymidine kinase gene in tumour cells, the target of antivirals based on nucleoside analogues, followed by treatment with ganciclovir. Further treatment modalities include the development of vaccines and targeting of the latent EBV episomes. Prevention of PTLD by pre-emptive therapy based on molecular monitoring of EBV load will represent the main aim to reduce occurrence. This review examines patents and literature for the treatment of EBV-associated lymphoproliferative diseases.     

Pharmacophore-based discovery of ligands for drug transporters

The ability to identify ligands for drug transporters is an important step in drug discovery and development. It can both improve accurate profiling of lead pharmacokinetic properties and assist in the discovery of new chemical entities targeting transporters. In silico approaches, especially pharmacophore-based database screening methods have great potential in improving the throughput of current transporter ligand identification assays, leading to a higher hit rate by focusing in vitro testing to the most promising hits. In this review, the potential of different in silico methods in transporter ligand identification studies are compared and summarized with an emphasis on pharmacophore modeling. Various implementations of pharmacophore model generation, database compilation and flexible screening algorithms are also introduced. Recent successful utilization of database searching with pharmacophores to identify novel ligands for the pharmaceutically significant transporters hPepT1, P-gp, BCRP, MRP1 and DAT are reviewed and the challenges encountered with current approaches are discussed.     

Herpesvirus vaccines: challenges and future prospects

Human herpes viruses are a family of large double stranded DNA viruses. Their life-cycle is characterized by an initial lytic cycle of infection in which large numbers of new virions are generated, followed by a pattern of lifelong latent infection with intermittent reactivation and viral shedding. Co-evolution over millennia has resulted in an interaction between host and pathogen which leads to the successful establishment of a lifelong latent infection with typically minimal impact upon the host, and subsequent maintenance and control of latently infected cells by the host immune system. Infection is usually asymptomatic or leads to an acute transient disease. However more severe disease can arise during primary infection, latency or upon reactivation; in both an immunocompromised and an immunocompetent setting, indicative of a need for the development of vaccine strategies which can either prevent infection or treat virally-associated diseases.     

The DNA helicase–primase complex as a target for herpes viral infection

Introduction: The Herpesviridae are responsible for debilitating acute and chronic infections, and some members of this family are associated with human cancers. Conventional anti-herpesviral therapy targets the viral DNA polymerase and has been extremely successful; however, the emergence of drug-resistant virus strains, especially in neonates and immunocompromised patients, underscores the need for continued development of anti-herpes drugs. In this article, we explore an alternative target for antiviral therapy, the HSV helicase/primase complex.

Areas covered: This review addresses the current state of knowledge of HSV DNA replication and the important roles played by the herpesvirus helicase– primase complex. In the last 10 years several helicase/primase inhibitors (HPIs) have been described, and in this article, we discuss and contrast these new agents with established inhibitors.

Expert opinion: The outstanding safety profile of existing nucleoside analogues for α-herpesvirus infection make the development of new therapeutic agents a challenge. Currently used nucleoside analogues exhibit few side effects and have low occurrence of clinically relevant resistance. For HCMV, however, existing drugs have significant toxicity issues and the frequency of drug resistance is high, and no antiviral therapies are available for EBV and KSHV. The development of new anti-herpesvirus drugs is thus well worth pursuing especially for immunocompromised patients and those who develop drug-resistant infections. Although the HPIs are promising, limitations to their development into a successful drug strategy remain.     

Contribution of Epstein-Barr virus infection to chemoresistance of gastric carcinoma cells to 5-fluorouracil

Although Epstein-Barr virus (EBV) is associated with 6–16% of the gastric carcinoma (GC) cases, the effect of EBV infection on the tumorigenesis process and the responsiveness to chemotherapy remain unclear. We compared chemosensitivity of the EBV-positive GC (AGSEBV) and EBV-negative GC (AGS) cells to 5-fluorouracil (5-FU). Although 5-FU inhibited the growth of both cell lines in a dose- and time-dependent manner, the sensitivity of EBV-positive GC cells to 5-FU was lower than that of EBV-negative GC cells. The cleavage of PARP and caspase-3 was also lower in AGS-EBV cells than in AGS cells following 5-FU treatment. Both the level of Bcl-2 expression and the ratio of Bcl-2/Bax were higher in AGS-EBV than in AGS cells not only at basal state but also following 5-FU treatment. Moreover, p53 and p21 expression was enhanced further by 5-FU in AGS than in AGS-EBV cells. Immunofluorescence assay and Western blot showed that 5-FU induced the expression of EBV-lytic genes including BZLF1, BRLF1, BMRF1 and BHRF1. Our results suggest that latent and lytic EBV infection contributes to the chemoresistance to 5-FU in gastric carcinoma by modulating apoptosis related cellular genes.     

Targeting Mcl-1 for the therapy of cancer

INTRODUCTION: Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers. AREAS COVERED: Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments. EXPERT OPINION: We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.     

Avidin-biotin technology in targeted therapy

Importance of the field: The goal of drug targeting is to increase the concentration of the drug in the vicinity of the cells responsible for disease without affecting healthy cells. Many approaches in cancer treatment are limited because of their broad range of unwanted side effects on healthy cells. Targeting can reduce side effects and increase efficacy of drugs in the patient.

Areas covered in this review: Avidin, originally isolated from chicken eggs, and its bacterial analogue, streptavidin, from Streptomyces avidinii, have extremely high affinity for biotin. This unique feature is the basis of avidin-biotin technology. This article reviews the current status of avidin-biotin systems and their use for pretargeted drug delivery and vector targeting.

What the reader will gain: The reader will gain an understanding of the following approaches using the avidin-biotin system: i) targeting antibodies and therapeutic molecules are administered separately leading to a reduction of drug dose in normal tissues compared with conventional (radio)immunotherapies; ii) introducing avidin gene into specific tissues by local gene transfer, which subsequently can sequester and concentrate considerable amounts of therapeutic ligands; and iii) enabling transductional targeting of gene therapy vectors.

Take home message: Avidin and biotin technology has proved to be an extremely versatile tool with broad applications, such as pretargeting, delivering avidin gene into cells enabling targeting of biotinylated compounds and targeting of viral vectors.