h2g2 The Hitchhiker's Guide to the Galaxy: Earth Edition

Project Title: Clinical application of Automation of phage display and SELEX technology, the basis for the development of targeted therapeutics
A-number of Project Title Page: N/A
Description of Project: The main goal of the present project is definition of a ligand/receptor-based molecular map of human vasculature, which have been useful in functional genomics and proteomics, to translate the phage display technologies into clinical applications and turn out to be the basis for the development of targeted medicine.

Within the framework of this project, the phage display technologies will be used to develop targeted medicine . Mainly we will focus on phage-library-selected peptides and proteins with anticancer and immune system address.For this reason, novel ligands for human tumor cells and immune system from diverse libraries of targeted phage particles, random peptides(aptamers) that inhibit essential intracellular processes, phage libraries from CD lymphocytes and HIV positive will be studied with implementation the new stradegy;targeting proteins in the host, rather than in the virus itself (20)..

Simultaneously with the above mentioned, the various aspects of the fictionalization process of low-molecular-weight ligands specific for the surface of cancer cells that use to detect and treatment cancer will be investigated.

In order to look for new antibiotic targets and lead compounds were developed a method for isolation of random peptides that inhibit essential intracellular processes in bacteria. A library of random peptides expressed as fusions to E. coli thioredoxin (aptamers) was expressed under the tight control of the arabinose-inducible PBAD promoter. Characterization and biological properties of aptameters from lymphocyte subpopulations celiac disease (CD) patients will be investigated.

It should be mentioned that CD patients often develop other autoimmune disease such as diabetes, thyroiditis, alopecia, autoimmune hepatitis, and cerebellar ataxia. Phage libraries from CD lymphocytes proved to be a valuable source of antibodies to auto antigens. Characterization and biological properties of these antibodies and random peptide libraries (RPL) that behaved as antigenic mimics of conformational B-cell epitopes generated in vivo in the course of the natural HIV-1 infection will be studied. Shotgun scanning uses combinatorial libraries of alanine-substituted proteins to rapidly map the functional epitopes of receptor-ligand interactions.

It should be mentioned that Phage display technology is used to select and affinity mature peptides, human antibodies, and single domain antibody molecules with exquisite cancer specificity. For these three different types of molecules, phage libraries have been designed, made, and used, and different affinity maturation strategies were applied. Examples, will include the isolation of anti-CEA peptides, anti-MUC-1 and MHC-peptide antibodies, and various single domain variable domain-based antibodies. We suppose and hope that the obtained results will allow to, such phage-library-selected peptides and proteins with anticancer address are expected, form the basis for more specific cancer diagnosis and treatments what will be the most significant result.

To achieve the goals of the present reseacrh the biophysical methods (hydrodynamic, thermodynamic and spectroscopic) will be used.. The necessity of biophysical approach is to get extensive knowledge about the structural and functional properties of proteins and peptides, how they interact with nucleic acids and proteins, form complexes, and ultimately impact pathways involved in disease. As well known, capsid protein of AIDS, acting like a key, binds to Nup358, a protein on the nuclear pore complex, unlocking the gateway and granting the virus access to the DNA To access the DNA, the HIV must pass through the nuclear pore complex, a gateway into the nucleus. So that, the mechanism that allows the virus to pass through this gateway was well be studied(20).

For realization of the intended work it is necessary to use entire phage particle, i.e. with undamaged structure. For example, if the conditions of phage environment will be changed even insignificantly (which can be caused by pH, ionic strength, ions), this can induce the modification of phage structure (even insignificant) so it won’t be able to infect bacteria. It should be also that the cause of negative results of biological studies on bacterial infection by phage consists not in the incorrect choice of phage to the corresponding bacteria, but the above discussed considerations are not taken into account. Therefore, in the present project when studying the infection process all the above-mentioned considerations will be taken into account. That is why before starting infection experiments, the phage native state in those environmental conditions, in which phages have to “attack” bacterial cells, should be checked. The study of all phages to be investigated in the framework of the project by means of biophysical methods will be carried out in different environmental conditions (influence of pH, ionic strength, ions), to have a clear idea of phage ability to infect bacteria.

Also, it well known that functional display of diverse genomic mammalian proteins usually requires native machinery for correct folding and post-transnational modification, not possible with current technologies such as prokaryotic phage display and in vitro RNA-protein fusions. We have overcome this limitation, by developing a display technology platform that creates protein display libraries using native expression systems, including human cells, where each expressed protein is covalently linked to its corresponding cDNA. Therefore, in the present project ,all expressed proteins in a cell can be screened for interaction with a compound in a single experiment using this display technology.


Proposed Completion Date: N/A

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