Similarities between pandemic influenza viruses

Scientists from the Mount Sinai School Medicine (New York) recently publish an interesting paper about the similarities between two viruses, namely the Spanish influenza virus and H1N1. The Spanish influenza virus caused a incredible number of death in 1918 during the first world war, worldwide. This virus is formerly extinct and for this reason it could be used as a bioterrorist agent. The comparison between the Spanish influenza virus and H1N1 demonstrates an homology of antigenic sites of haemagglutinin A. This observation induced scientists to investigate whether mice and humans previously vaccinated against H1N1 were also immunized against the Spanish influenza virus. Human sera containing antibodies anti-HA were collected and passively transferred into mice infected with the Spanish influenza virus and protected them from lethality. Thus, immunization against H1N1 protects also from the Spanish influenza virus infection. In case of bioterrorist attack or accidental virus escape from scientific laboratories the emergency could be easily managed. This study is pionieristic and other researches could be done to identify similarities between viruses. Such as approach, it could be useful to validate current therapies against other viral target, diminishing the cost of clinical trial required to market new anti-viral drugs.

Novel molecules to fight Pseudomonas

Infections are the most dangerous and common problems in hospital wards. Patients are weak and ill and their bodies are easily infected by opportunistic parasites. Pseudomonas Aeruginosa is a Gram negative bacterium that is responsible for the major part of nosocomial infections. In US at least 160000 patients are contaminated by Pseudomonas every year. Usual treatment is a course of antibiotics, but the presence of resistant strains often makes useless the therapy. For this reason, physicians prescribe polymyxin antibiotics like colistin to care resistant infections, but these compounds are really toxic and cannot be used for long lasting treatment. The need of novel molecules and drugs is urgent and at the same time challenging.

Researchers at the University of Zurich (Swiss) have identified a group of molecules that seem to block outer membrane generation. Differentially from other class of antibiotics that causes hole into the membrane, POL compounds are able to interact and compete with proteins responsible for the correct assembling of the membrane because of their secondary structure containing beta- hairpin and alpha helix motifs. The result is high specificity, selectivity and potency.
In mouse septicaemia model, this novel class of antibiotics shows an effective dose 10 times lower than generic aminoglycoside gentamycin. Giving the promising conclusion, Phase I testing is planning for the next months. Other favourable properties characterize POL compounds: the lack of activity versus other strains, for instance, reduces the risk of general resistance. A common mechanism of resistance is the selection of adaptive mutations into genome that make possible survival in presence of weak antibiotics; if selective pressure is not present, none resistance will be generated. Furthermore, POL compounds self have the same resistance frequency of other antibiotics, even if they target only a limited class of proteins. Indeed, some critical concern due to this high specificity has been already risen: it’s more likely that bacteria evolve resistance versus an antibiotic that selectively target one protein, rather than one that interfere with a general mechanism of survival. Finally, another advantage of POL compounds is the synergistic effect of other drugs. Indeed, Pseudomonas strains treated with POL compounds seem more sensitive also to further antibiotics. This makes possible to reduce dosage, better manage the therapy and decrease the risk of resistance. In conclusion, there are several evidences that POL compounds are promising molecules to control and block Pseudomonas infections in hospital. Novel research has been already started to identify other compounds that should be able to kill Gram negative bacteria strains in order to dispose on numerous tool to fight infections. Financial advantage of these new therapies is to shorten the hospital staying and better save patients’ health.

Nuocytes, a novel cell type

A recent work published in Nature demonstrates the existence of a new class of cells, namely noucytes. This cells seem really important for the type II immuninity. The type II immunity protects our body from parasitic helminth infections, by promoting the release of type II cytokines, activating eosinophiles and goblet cells and secreting mucus and IgE.
The same system is also responsible for asthma and other allergic disfunctions. Nuocytes are a novel component of innate immune system and seem involved in IL13 release during the first steps of helminth infection. Nuocytes primarily respond to IL25 and IL33 stimuli either in vitro or in vivo, then start the immune response. Lack of IL25 ad IL33 cytokines determines nuocytes expansion failure and lastly severe defect in worm expulsion. Thus, nuocytes play a crucial role in body defense and this and subsequent studies will help us to understand better the mechanism of action of innate immune system. Nuocytes will represent a new possible target for immune stimulating molecules in order to increase the protection versus infection, as well as it could be possible that new anti asthma drugs will be directly act on this class of cells.

Monoclonal antibodies

Monoclonal antibodies are important tool in molecular biology, diagnostics and clinical studies. These protein are produced by a single cells isolated from immunized animals. Current protocol requires an immunization of an animal host, for instance rabbit or mouse; then spleen cells are selected in vitro and B cells are isolated. B cells from spleen are fused with tumoral mouse cells of mieloma in order to stabilize and make possible the B lymphocyte culture. Indeed, B cell culture is difficult to set up and maintain.
The hybridoma technology allows overcoming these difficulties because of genetic transformation of mieloma cells. Finally, hybridomas are serially diluted and the antibodies are obtained from cell hybridomas cultures derived from a single cell. Which are the advantages of monoclonal antibody in respect with polyclonal ones? Monoclonal antibodies are codified by the same gene and none point mutations are present to generate some difference into antibody population. Thus, the whole population is identical and specifically recognizes one antigen. Cross reactivity is reduced with monoclonal antibodies and the interaction between antigen and antibody is usually more stable. Furthermore, this technique is also really flexible because it’s virtually possible to create antibody versus each antigen, when it’s possible to immunize the host animal. Which are the applications for monoclonal antibody? Monoclonal antibodies are currently used in molecular biology and biochemistry laboratories for imaging, western blotting, immunoprecipitation and so on. A lot of protocols are based on antibody use. In diagnostics, monoclonal antibodies are used in ELISA dosage or in flow cytometric analyses, as well as infection detection or pregnancy diagnosis.

Clinical applications of monoclonal antibody are prevalently in oncology. In 1997, the first monoclonal antibody was approved for non- Hodgkin lymphoma treatment. Since this year, several antibodies have been optimized against breast cancer, leukaemia, colon cancer and recently lung cancer. Each antibody recognizes a tumoral antigen and specifically kills only the cells (tumoral) that present that molecule. Thus, adverse effects associated with the use of monoclonal antibodies are reduced if compared with traditional drugs. Based on their specificity, antibodies can be used to carry other useful drugs to cells. For instance, an antibody can be conjugated to radioactive compounds to be addressed to cancer cells. Furthermore, other drugs can be carried into the brain, giving the capability of monoclonal antibody to overcome the blood- brain barrier. Parkinson’s disease can be treated with this approach. Improvement of biochemical characteristics of monoclonal antibodies is one challenge for scientists for the next future. Indeed, it’s important to improve the delivery of monoclonal antibody into all districts of human body. The specificity will be a must if clinical or diagnostic applications are planned for the monoclonal antibody. Furthermore, cheaper technology must be optimized to allow large scale production. Research development in this field is really promising.

From a protein to pharmaceutical target

Current pharmacological approaches to identify target have strong molecular basis. Giving the important results of genome and proteome projects in understanding cell biology and biochemistry, also pharmaceutical research changes its approach and obtains great advantages from this new knowledge. Indeed, once identifying mutations in genes involved in particular diseases in the genome project, the role of proteins have been defined by the proteome project. Numerous proteins responsible of cancer, neurodegenerative disease, immune disease and so on have been studied in this way, obtaining important results in disease understanding.
The first step to treat one disease is knowing the pathological mechanism on its basis. Altered proteins could have a high activity, in this case we talk about gain of function mutations, or lower activity than normal, and so we talk about loos of function. In both cases, genetic alterations cause an impaired protein activity with deleterious consequences on cellular homeostasis. One protein acquires pharmacological significance when has a crucial role in disease arising. The common way to demonstrate the importance of one protein in pathogenesis is silencing or restoring it, in the case of gain or loss of function respectively. SiRNA approach is often used to silence proteins and analyse the effect of this lack in cellular system. The big advantage of siRNA is the selectivity: indeed, if siRNA is well designed, it’s possible to obtain complete depletion of the protein of interest without interfering with other cellular functions. So, this approach is safer than the use of chemical inhibitor because of the lack of collateral events. Furthermore, siRNA is applicable to every kind of protein even if their function has not been clarified yet. In the case of loss of function, it’s possible to restore protein function by transfecting cells with wild type version of the protein. Unfortunately, this system is not so theoretically universal like siRNA, because in the case of dominant negative mutation the normal protein function cannot be restored. Numerous animal models are available to confirm results obtained in cellular assays: knock out and knock in animals have been developed to simulate loss or gain of function, respectively. In summary, in cellular system we can define the role of a protein in the disease of interest and we can understand how it’s possible to modulate its functionality in order to restore normal homeostasis. Then, in animal model we can verify the consequences of proposed treatment in a whole organism. This is the way to transform a simple protein into pharmaceutical target. Basic studies on protein function are usually performed in public institution or universities, in some cases with the contribution of pharmaceutical sponsor that would have major benefits from research results. Otherwise, the presence of spin off or start up companies inside the university allows preserving the economic value of research for the institute. In this manner, research independency is guaranteed and also diseases which pharmaceutical companies are not strictly interested in, could be studied.

Improving fluorescent probes

Cell imaging is one important tool to verify protein expression, localization and interaction in living cells. Several probes are now available to specifically colour cells, but intracellular retention is a problem for an high number of them. It’s great challenge to improve intracellular retention and also a great deal because improving intracellular retention means also improve the sensitivity of fluorescent detection.

Calcein, a fluorescein derivative, contains two imino- diacetic acid groups which are responsible for the optimal intracellular retention. Researchers of the University of Tokyo synthesized membrane- permeant derivatives of fluorescein containing either one or two imino- diacetic acid group and decided that two groups, as are present in calcein, are optimal for improving intracellular retention.

They designed novel fluorescent probes for imaging highly reactive oxygen species and nitric oxide. With these new probes they visualized low levels of target proteins in living cells over a relatively long period of time, which it’s not possible to monitor with traditional probes. This chemical approach to prevent cellular leakage should be broadly applied for all probes fluorescein- based as well as on other kind of scaffolds and could be considered a general strategy to increase the sensitivity in living cells.

Novel small molecule inhibitor has immunosuppression properties

Immunosuppression is one approach to care some autoimmune diseases, such as rheumatoid arthritis. Etarnercept and cortisone are currently used to care these disorders. In Nature Medicine has been recently published a work that describes a novel possible therapy for this kind of disease. Scientists focussed their attention on small molecule inhibitors of immunoproteasome. This cellular compartment has been identified in monocytes and lymphocytes and is specialized in generating MCH I peptides.

The selective inhibitor of LMP7, a specific protein belonged to immunoproteasome, down-regulated the expression of MCH I complex and reduced the level of some cytokines, for instance IL23, IL6 and TNF. All these molecules are implicated in the autoimmune response in rheumatoid arthritis. The expression of other cytokines were tuned in following inhibitor exposure. The small molecule inhibitor could have pleiotropic effects of various cell types, by ameliorating drug efficacy. In animal models, indeed, this molecules gained better results than Etanercept in TNF blockade and consequently showed faster resolution of symptoms of immune disorders. This study represents a great hope for all people that are affected by rheumatoid arthritis, but a lot of work still to be done before entering in clinical phase.

Novel approaches to interfere with HIV infection

Several studies are currently ongoing to identify some strategy to care HIV infection: in this post we would like to propose you two novel approaches, based on glycerol monolaurate (GML) and griffithsin. GML is a mild detergent that in high concentration can prevent inflammation and recruitment of CD4 T cells, usually cell- host of HIV virus.

In particular, some experiments have been done on vaginal epithelium –this site is frequently involved in the first phases of HIV infection- and decrease of activation of dendritic cells and, thus, of CD4 T cells, was observed. Infection couldn’t spread and systemic infection was blocked. Some authors noted that really high concentration of GML has to be used to obtain inflammation bock, while lower concentration could have the opposite effect. Griffithsin is a protein derived from algae and has been recently cloned into tobacco plant, thus recombinant variant is now available. This protein impairs the binding of viral particles to glycoprotein at the cell surface and determines HIV death. Interesting results showed a block of infection on human cervical explant by adding recombinant griffithsin protein. These two approach could be considered promising prophylactic treatment because both interfere with the first phases of infection and have quickly gained a great interest of some important pharmaceutical companies.

Microinjection of yeast

Yeast is a commonly used genetic model, budding and fission have been largely studied, but few methods to introduce DNA, protein or other compounds, have been described until now. Indeed, yeast presents the rigid cell wall that avoids injection and all techniques currently used to manipulate cells. It has been described that piezo-impact micromanipulator worked well to overcome biological barriers; thus, this tool was employed also for yeast injection with good results.

The procedure, reported in the last volume of Nature Methods, allows to introduce material during fission yeast. At the microscope it’s possible to immobilize and buckle one cell, through mechanical stress, while sorbitol in the surrounding medium avoids cell disruption. Material that has to be injected enter into the cell is released from pipette and passed through the hole into the wall. The major difference between usual microinjection technique is that the pipette doesn’t touch cell surface. Why is this method so innovative? For the first time yeast has been manipulated and now inhibitors, RNA and proteins could be introduced in. Thus, new experiments could be thought and performed, keeping in mind that yeast is really diffused, as a tool for genetic studies and recombinant protein expression and other applications could be now imaged.

The importance of QSAR studies in pre-clinical phase of drug discovery

QSAR is the acronym of quantitative structure activity relationship, is a modern tool to discover new molecules that could become drug. Indeed, a crucial step in the drug discovery process is the identification of lead compound, the first molecule that has an activity on the target and could be modified to improve its pharmaceutical features. Molecular modeling allows to show how this lead fits into the target, for instance a protein, and defines what are the important bonds that guarantee the interaction. The three-dimensional structure of the target is essential to perform this kind of studies and obtain results pretty sure.

It’s not a case that the major pharmaceutical companies have large structural and biochemical group in their Research & Development department. Anyway, an hypothetical model, based on structures of other protein similar to those of interest, can be build and tested: is the robustness of the model that determine the quality of the prediction. Indeed, in both cases virtual model can be used to screen libraries and identify the most promising compounds, named lead. Then, these molecules are tested in biochemical or biological assays to validate the activity. In this way, only few molecules of thousands that compose a library, can reach the bench and can be screened, by effectively reducing costs. Modifications of an active lead compound are crucial to improve its pharmaceutical properties and increase the likelihood to successfully enter in clinical phases. QSAR identifies all the positions that are important for the interaction between compound and target and suggests some chemical groups that could be inserted to further stabilize this binding. Thus, other compounds derive from this virtual study that have to be validated in experimental conditions. By analyzing the changes in the activities, it’s possible to define a quantitative relationship between structure modifications and activity itself.

Modifications that show an improvement in the activity are selected and further compounds are synthesized, by combining these changes. Again, a virtual screening is firstly run to check the new binding modalities and, then, experiments determine the activity. By repeating this process two or three times it’s possible to dispose on leads of second and third generation that have more chances to become drug. This pre-clinical phase could take some years, but efficiently reduces the cost of drug discovery. Indeed, the high rate of failure in the clinical phases, the most expensive part of the process, in part depends on the lack of accurate pre-clinical studies. QSAR is one of the tool now available to reduce the failure rate and identify only the compounds that could potentially have success. However, the results from clinical studies are always the limiting step to the entrance on the market of novel drugs.