Drug Discovery Opinion

Antiviral drugs that exclusively target viral factors rather than host cell processes offer the possibility of fewer side effects but at the cost of limiting possible targets and of providing selective pressure for the generation of drug-resistant viral variants. Recent advances in siRNA gene-targeting technologies provide a way of identifying host cell mechanisms that are essential for viral replication and researchers at the University of Texas Medical Branch at Galveston have used a siRNA screening approach to identify host gene products that play critical roles in Zaire Ebola virus infection. The team screened a siRNA library targeting cell kinases and phosphatases for inhibitors of cell penetration using a high-throughput assay based on pseudotyped virus which can be handled under biosafety level 2 conditions. Viral pseudotypes carry the glycoproteins of the virus of interest over the core of another virus and typically display the receptor specificity, cell tropism, and entry characteristics of the foreign glycoproteins. Ranking of siRNA efficacy followed by gene cluster analysis identified proteins in phosphatidylinositol-3-kinase and calcium/calmodulin kinase related pathways as important for Zaire Ebola virus infection and compounds targeting these pathways were prioritised for testing against both pseudotyped virus and wild type Ebola virus.

LY294002 was used to target the phosphatidylinositol-3-kinase (PI3K) pathway and KN-93 was chosen as an inhibitor of the calmodulin kinase 2 (CAMK2) pathway. LY294002, which directly inhibits the kinase activity of PI3K, had an IC₅₀ of 7µM and KN-93, which prevents association of CAMK2 with calmodulin (required for kinase activity), had an IC₅₀ of 21µM against infection by the pseudotyped virus. It was further shown that both KN-93 and LY294002 are able to reduce infection by wild type Ebola virus: at a concentration of 50µM, LY294002 reduced viral titres in cell culture experiments by 65% and KN-93 reduced viral titres by > 95%.

This is the first example of siRNA profiling coupled with network analysis being used to identify critical pathways for viral entry, together with lead compounds with confirmed antiviral activity. The study supports the use of pseudotyped viruses to facilitate lower containment level high-throughput screening and may lead to effective treatments for Ebola virus.

The study is published in the journal Drug Discovery Research.

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Neurotrophins – a family of proteins essential for the development, survival and function of neurons – exert their actions through two classes of receptor, Trk tyrosine kinase receptors and p75NTR. There has been considerable interest in using neurotrophins such as nerve growth factor (NGF) for the treatment of neuronal damage as well as for conditions such as stress and depression, but the use of NGF itself has been limited by poor CNS penetration and side effects such as hyperalgesia. This has led to a search for stable small molecules with neurotrophic activity and specificity for TrkA or TrkB receptors although, so far, none of the mimetics can fully reproduce the effects of NGF in animals.

Writing in the journal Chemistry & Biology, researchers at Emory University School of Medicine have now shown that the tricyclic antidepressant, amitriptyline, which had been thought to act predominantly by blocking serotonin and noradrenaline transporters, interacts directly with the extracellular domain of both TrkA and TrkB receptors. Amitriptyline induced TrkA and TrkB homo- and heterodimerization and activation in mouse brain, but heterodimerization was found not to be required for Trk receptor activation. Truncation of the amitriptyline binding motif on TrkA, but not the corresponding region on TrkB, abolished the receptor homo- and heterodimerization. Amitriptyline, but not other tricyclic antidepressants or selective serotonin reuptake inhibitors, promoted TrkA autophosphorylation in primary neurons and induced neurite outgrowth in PC12 cells. In mice, amitriptyline was further shown to suppress neuronal apoptosis caused by the neuroexitotoxin, kainic acid, in a TrkA-dependent manner. Inhibition of TrkA, but not TrkB, abolished the neuroprotective effect of amitriptyline without affecting its antidepressant activity.

Amitriptyline was found to bind to a motif in the first leucine-rich motif of the extracellular domain of the TrkA receptor with a Kd of 3 µM, which approximates to the brain concentration achieved when used to treat depression or neuropathic pain. Although significantly lower than the affinity of NGF for TrkA, it suggests that amitriptyline affinity for TrkA might be sufficient to explain at least some of its biological activity.

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Schistosomiasis is caused by infection with one of several species of parasitic worms and, although not usually fatal, is considered to be the second only to malaria in terms of human impact. Infection with the parasite typically occurs when wading, swimming or washing in water containing fresh water snails, the intermediate host for the parasite.

Although many countries are working to eradicate the disease by drug treatment, snail control, education and improved sanitation, the disease remains endemic in more than 70 countries and is estimated to affect around 200 million people worldwide. Schistosomiasis is readily treated with praziquantel but, despite being the mainstay of treatment for several decades, the mechanism of action of praziquantel is still debated. Writing in the journal PLoS Neglected Tropical Diseases, researchers at University of Minnesota Medical School have now shown how praziquantel kills a species of free-living flatworm that is often used as a model organism. These worms have remarkable regenerative properties and are able to reform a complete body from even a small fragment. Praziquantel was found to cause aberrant regeneration, producing two-headed organisms with duplicated, integrated central nervous systems and organs. The team further showed that voltage-operated calcium channel (VOCC) β subunits are important for the activity of praziquantel, supporting an earlier hypothesis about its mechanism of action.

The authors hope that elucidation of the mechanism of praziquantel toxicity – albeit in a free-living flatworm species – will help in the rational design of new antischistosomal drugs.

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