virology – Page 2 – Drug Discovery Opinion

Antibody Recruiting Molecules Target HIV, Cancer

hiv virus cartoon Both HIV and cancer cells have found ways to evade the body’s immune system but researchers at Yale University have now found a way to boost the body’s ability to fight HIV and cancer. The team have identified bifunctional small molecules, termed “antibody-recruiting molecule targeting HIV” (ARM-H) and “antibody-recruiting molecule targeting prostate cancer” (ARM-P), which bind simultaneously to antibodies and to proteins on HIV, HIV-infected cells or cancer cells.

ARM-H molecules bind to gp120, a component of the Env glycoprotein on the surface of HIV and virus-infected cells and to anti-2,4-dinitrophenyl antibodies already present in the bloodstream. The ternary complex formed between the antibody, ARM-H, and gp120 is immunologically active, and leads to complement-mediated destruction of Env-expressing cells. ARM-H also prevents virus entry into human T-cells and so has the potential to inhibit viral replication by two mutually reinforcing mechanisms.

ARM-P molecules bind with high affinity to prostate-specific membrane antigen (PSMA) and, by inducing complexes of anti-2,4-dinitrophenyl antibodies with prostate cancer cells, mediate antibody-dependent killing of the cancer cells.

The team has begun to evaluate the ARM molecules in mice, and hope that the strategy of using antibody-recruiting small molecules to boost the immune response will prove useful for treating HIV, cancer, and other diseases.

Both studies are published in the Journal of the American Chemical Society (ARM-H and ARM-P).

Retrovirus Linked to Chronic Fatigue Syndrome

Tired Man by József Somogyi Image: Wikimedia - Burrows — "Tired Man" by József Somogyi

Image: Wikimedia - Burrows

Xenotropic murine leukemia virus-related virus (XMRV) was in the news a month ago as researchers provided new evidence that the virus could contribute to the development of prostate cancer. Now, scientists have linked XMRV to Chronic Fatigue Syndrome (CFS).

Knowing that patients with CFS can share an immune system defect with prostate cancer sufferers, the team looked for XMRV in blood samples from patients with CSF. In the study, which is published in the journal Science, XMRV was detected in blood samples from 67% of CFS patients and in only 4% of samples from healthy individuals. The infected blood samples not only contained viral DNA but were shown to produce viral proteins and infectious viral particles. Retroviral particles were also detected in patient samples using transmission electron microscopy. The virus, which is transmitted through body fluids and is not airborne, was found to be transmitted by both cell-to-cell and cell-free mechanisms. Secondary viral infections could be established in uninfected primary lymphocytes by exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients.

The data demonstrate the first direct isolation of infectious XMRV from humans and raise the possibility that XMRV may be a contributing factor in CFS. Although the research provides a strong association between XMRV and CFS, it does not prove that XMRV causes CFS. If cause-and-effect is established, antiretroviral drugs could potentially be developed to treat CFS. Since the Science paper was submitted, the team have shown that 95% of plasma samples from CFS patients tested positive for XMRV antibodies. A clinically validated test to detect XMRV antibodies in plasma is currently under development, and the team plan to collect more data on the incidence of XMRV in the general population. The team are also investigating whether approved antiretroviral drugs are effective against XMRV and could benefit CFS patients.

Blocking Host Target Stops Flu

Nexbio have recently presented an update on their new, broad-spectrum candidate for the prophylaxis and treatment of flu, DAS181 (Fludase®) [If this link is not working, please click here]. DAS181 is a recombinant sialidase fusion protein comprising the active domain of Actinomyces viscosus sialidase and a heparin binding sequence derived from the human protein, amphiregulin, to anchor the fusion protein to epithelial surfaces. As well as delivering DAS181 to airway epithelial cells, the anchoring domain may reduce the potential for systemic toxicity by limiting systemic absorption. DAS181 has been shown to be effective both prophylactically and therapeutically against seasonal influenza virus, the highly pathogenic H5N1 avian influenza virus, and multiple strains of parainfluenza viruses. The new study showed that DAS181 is also effective against strains of influenza virus that show resistance to the neuraminidase inhibitor, oseltamivir (Tamiflu®), both in vitro and in a mouse challenge study.

Unlike neuraminidase inhibitors that inhibit the viral neuraminidase, DAS181 prevents viral entry into cells by blocking binding of viral HA glycoproteins to sialic acids on cells in the respiratory tract. Cell-surface sialic acids act as entry receptors for all influenza virus A and B strains as well as for parainfluenza viruses. Antiviral drugs that target viral proteins lead to selection pressures that favour the emergence of resistant strains and it is hoped that targeting a host receptor may reduce the likelihood of resistance. Extensive, prolonged, nonclinical studies are reported not to show the development of any meaningful resistance.

A study carried out in collaboration with researchers at the University of Hong Kong and Imperial College, London and published in the September issue of Antimicrobial Agents and Chemotherapy has shown that DAS181 strongly inhibits infection of human lung tissue and cells by H5N1 virus. This study was also able to resolve mechanistic details of the action of DAS181. Human lung tissue and cells have two receptors that allow influenza virus to enter cells; α2-3-linked sialic acids, to which the H5N1 strain preferentially binds; and α2-6-linked sialic acids, to which the currently circulating pandemic H1N1 strain preferentially binds. DAS181 was shown to remove both α2-6-linked and α2-3-linked sialic acids found in human respiratory tissue from adjacent glycans, thereby suppressing viral entry.

DAS181 is administered by oral inhalation, using a device called ‘Cyclohaler’ and is currently undergoing phase I clinical studies.

Retrovirus Linked to Prostate Cancer

A number of human cancers are linked to viral infection including cervical cancer (human papilloma virus), liver cancer (hepatitis B and C viruses), and lymphoma (Epstein Barr virus). The human retrovirus, XMRV (xenotropic murine leukemia virus-related virus) has previously been found in prostate cancers – most often in men with a defect in the antiviral defence protein, ribonuclease L – but, since only malignant tissues were analysed, a direct association with prostate cancer was not established. In a study published in the September 7^th online edition of PNAS, researchers at Columbia University and the University of Utah have now analysed specimens from 233 cases of prostate cancer for the presence of XMRV and compared them with 101 benign control specimens. XMRV infection was found to be associated with prostate cancer, especially with more aggressive tumours, and infection was also found to be independent of reduced activity of ribonuclease L. The virus was detected in 27% of prostate cancer specimens compared with 6% of benign tissue samples and was found almost exclusively in malignant cells.

XMRV is a member of the gammaretrovirus family, other members of which are known to cause cancers in rodents, cats and primates. The study suggests that XMRV infection could contribute to the development of human prostate cancer, although the mechanism by which the virus transforms cells remains to be fully elucidated. As with other retroviruses, XMRV integrates into host DNA and it may be that it inserts close to a gene that regulates cell growth. XMRV was found to replicate efficiently in a cell line derived from human prostate cancer but not in other human cell lines, suggesting a viral tropism that needs to be investigated further. If further studies provide a causal link between XMRV and the development of prostate cancer, opportunities for diagnostic tests, antiretroviral therapy, or ultimately a vaccine against prostate cancer, could follow.

Not Channelling for Success

Globally, hepatitis C virus (HCV) infects almost 200 million people and is a leading cause of cirrhosis and hepatocellular carcinoma – albeit several decades after initial infection. In a majority of cases, the virus is able to establish a persistent infection and, even with current gold standard treatments, sustained cure rates once the infection has become established are only around 50%. Researchers at the University of Leeds have now uncovered a previously unrecognized mechanism that allows the virus to evade the immune system and establish a chronic infection.

Establishment of persistent infection means that HCV-infected cells must be resistant to pro-apoptotic stimuli and the team found that one of the viral proteins, NS5A, is able to block apoptosis in human hepatoma cells either infected with HCV or harbouring an HCV subgenomic replicon. Amplification of an outward K⁺ current mediated by voltage-gated K_v2.1 channels normally precedes apoptosis triggered by oxidative stress and NS5A was found to block this process by inhibiting phosphorylation of K_v2.1 by p38 MAP kinase. Inhibition of a host cell ion channel by a viral protein as a means of preventing apoptosis has not previously been described, and the researchers hope that their findings could lead to new strategies for antiviral therapy. The study is published in the August 26^th online edition of PNAS.

Influenza Infection Could Lead to Neurological Complications Later in Life

Neurological complications such as encephalitis have been associated with influenza outbreaks since the middle ages but links with neurodegenerative diseases are more controversial. The association was strengthened after the 1918 Spanish influenza pandemic when some patients who developed von Economo’s encephalopathy – an atypical form of encephalitis – went on to display symptoms of Parkinson’s disease. Although a recent study showed that a reconstituted virus is not directly neurotropic, the engineered virus did strongly induce a variety of cytokines, some of which have been implicated in the pathophysiology of Parkinson’s disease.

Providing new evidence for an association between influenza infection and neurodegenerative diseases, researchers at St. Jude Children’s Research Hospital, have now shown that mice that survive infection with a virulent H5N1 strain of avian influenza are more likely to show changes in the brain associated with neurological disorders such as Parkinson’s disease and Alzheimer’s disease. Using an antibody to the influenza virus nucleoprotein, the team were able to track the progress of the virus into the CNS: 2-3 days after infection, the virus appeared in the peripheral nervous system; by day 3, the virus had invaded the brain stem and, by day 7, the virus was found in areas of the midbrain including the substantia nigra pars compacta (SNpc), and the mice now showed Parkinson’s disease-like symptoms such as tremor and movement problems.

Although after three weeks there was no evidence of the virus in the nervous systems of the surviving mice, inflammation in the brain triggered by the infection persisted for the entire three month course of the study. The Parkinson’s disease-like symptoms disappeared as the flu symptoms eased but, 60 days later, the mice had lost almost 20% of dopamine-producing neurones in the SNpc. α-Synuclein, a protein which forms insoluble plaques in the brains of Alzheimer’s disease and Parkinson’s disease patients, was also found to accumulate in H5N1-infected cells, including those in the midbrain where key dopamine-producing cells are located. The authors propose that a significant loss of dopaminergic neurons and long lasting immune response caused by influenza infection may worsen the effect of a second trigger, leading to increased risk of neurological disorders, such as Parkinson’s disease, later in life.

The H5N1 strain used in this study is so virulent that 61% of the 433 people who have been infected to date have died and, for the survivors, it is too early to say whether they will develop neurological problems. The influenza pandemic now engulfing the world is caused by an H1N1 strain rather than an H5N1 strain and, although the neurological threat posed by this virus is still being examined, early indications are that the H1N1 pandemic strain carries a low neurologic risk.

The study is published in the August 10^th early edition of the Proceedings of the National Academy of Sciences.

Small Molecule Inhibitors of Kaposi’s Sarcoma Virus Protease

Kaposi’s sarcoma (KS) is caused by Kaposi sarcoma herpes virus (KSHV) which is also known as human herpes virus 8 (HHV8). HHV8 infection rates vary widely amongst different populations but KS rarely develops unless the immune system is compromised, by AIDS, transplant drugs, or ageing. There is currently no specific treatment for KS but researchers at UCSF have now identified small molecules that target the viral protease. Along with other members of the herpes family of viruses – including herpes simplex viruses I and II, varicella zoster virus, cytomegalovirus and Epstein-Barr virus – HHV8 encodes a serine protease that is essential for viral capsid formation and viral replication. Many previous attempts to discover inhibitors of herpes virus proteases targeting the active site of the enzymes met with limited success, perhaps because of difficulty in finding molecules that bind tightly to the shallow substrate-binding cleft. The UCSF team have chosen instead to inhibit catalytic activity by disrupting dimerisation of the enzyme.

A number of earlier studies have shown that dimerisation is a common mechanism for activation of herpes virus proteases, and the UCSF team have previously identified a helical peptide that prevents dimerisation of herpes virus proteases. In the new study, published online in the journal Nature Chemical Biology, the team describe small molecules, DD2 structure including DD2, which inhibit dimerisation of both HHV8 and CMV proteases with IC₅₀s in the low micromolar range.

HIV protease also acts as an obligate dimer but, in this case, dimerisation inhibitors have been less successful than compounds that directly target the active site, many of which are now in clinical use. The difficulties experienced in trying to identify active site inhibitors of the herpes virus serine proteases may mean that disruption of dimerisation presents a more attractive target for these challenging enzymes.

Host Kinases Linked to HCV Infection

Globally, around 200 million people are infected with Hepatitis C virus (HCV). Infection generally leads to chronic liver disease, albeit over a period of decades, which can lead to cirrhosis, hepatocellular carcinoma and liver failure. Current treatment, a combination of pegylated interferon-α and ribavirin, is only effective in 50-85% of patients, dependent on viral genotype. Not surprisingly, much effort is being expended to identify new therapeutic interventions.

Study of the viral lifecycle has been difficult in the past since serum-derived HCV (sHCV) replicates poorly in primary human hepatocytes and hepatoma cells in vitro. Surrogate systems have been developed, however, that have enabled reproduction of all steps of the HCV replication cycle, including cell entry (recently reviewed).

Viral cell entry is still incompletely understood, but a new study from researchers at the University of Rennes 1 has identified host kinases involved in HCV infectivity. Using a small-interfering RNA (siRNA) library, the scientists have shown that knock-down of phosphatidylinositol 4-kinase type III-α (PI4KIIIα) prevents infection by either HCV pseudoparticles (HCVpp) or by cell-culture grown JFH-1-based HCV (HCVcc). A second PI4K-family member, PI4KIIIβ, also influenced cellular susceptibility to HCVpp infection and the ability of the cells to sustain HCV replication. These kinases may therefore represent new targets for HCV therapeutics.

The study is published in the FASEB Journal.

Renin Links CMV Infection to High Blood Pressure

Human cytomegalovirus (hCMV) is a widespread member of the herpes family of viruses, with well over half of the world’s adult population thought to be infected. Most individuals are infected in early childhood and remain infected with latent virus for the rest of their lives. For the majority of people, infection is asymptomatic and usually undiagnosed but immunosuppression, caused by HIV-1 infection or drug therapy after transplant surgery, can lead to reactivation of the virus and overt infection. A number of studies have linked hCMV infection to cardiovascular disease, but the mechanisms underlying the pathology are not well understood. Writing in the May 15^th edition of PLoS Pathogens, a team led by researchers at Beth Israel Deaconess Medical Center have now shown that infection with murine CMV (mCMV) leads to a significant increase in arterial pressure in mice. mCMV infection alone was sufficient to cause the observed increase in blood pressure. mCMV infection alone did not cause atherosclerosis in the aorta but, when combined with a high cholesterol diet, did cause classic plaque formation. The team went on to show that mCMV stimulated production of pro-inflammatory cytokines, IL6, TNF-α and MCP-1 which have previously been linked to high blood pressure. They also showed that mCMV infection induced renin expression in an infection dose-related manner in mouse renal cells and that hCMV induced a similar increase in human vascular endothelial cells. In mice, mCMV infection was additionally shown to lead to an increase in angiotensin II levels in serum and in tissue from the aorta.

Since the roles of renin and angiotensin II in hypertension are well established, the study provides a mechanism by which persistent CMV infection might increase blood pressure and also suggests that vaccination or antiviral therapy could have the potential to provide new treatments for cardiovascular disease.

Nuclear Attack on HSV-1

Cold sores are fluid-filled blisters caused mainly by the highly contagious herpes simplex virus type 1 (HSV-1), which is passed on by close skin contact with someone with a cold sore. Most children come into contact with the virus by the age of five but many show no symptoms until puberty. HSV-1 is not cleared from the body by the immune system and, following initial infection of cells of the epidermis, the virus travels to the nerve root ganglia where it becomes dormant and causes no symptoms. As a result of the primary infection, the body produces antibodies against the virus – the majority of adults have antibodies against HSV-1 and 20 per cent of these individuals will have recurrent attacks of cold sores throughout their lives. The causes of reactivation are uncertain but are thought to include physical or psychological stress as well as changes to immune function.

HSV-1 Virions Writing in the journal Nature Immunology, scientists from Canada and the US have now described a novel cellular process that helps the body to fight HSV-1 infection. In mouse cells infected with HSV-1, the researchers discovered a previously unknown type of autophagosome originating from the nuclear membrane. When conditions of low-grade fever were replicated by hyperthermia, or the cells were exposed to the pyrogenic cytokine, interleukin 1β, autophagy and vacuolar processing of viral peptides was observed. Viral peptides in autophagosomes were further processed by the proteasome, suggesting a complex interaction between the vacuolar and previously described MHC class I presentation pathways.

The research team hope that their discovery might eventually lead to novel therapies for HSV-1 infections, as well as for infections caused by other pathogens that are able to evade the immune system.