260 research outputs found
Characterization of the Hemagglutinin Cleaving Transmembrane Serine Proteases Matriptase and TMPRSS2
Influenza is one of the commonest infectious diseases affecting millions of people every year including 290,000 – 650,000 heavy casualties. Influenza viruses undergo constant genetic changes and every 10 – 50 years new influenza virus strains emerge that potentially cause a severe pandemic. In this modern interconnected world, experts believe the next influenza pandemic will be a “devastating global health event with far-reaching consequences” [1]. Novel effective anti-influenza drugs are in need. One strategy of influenza research is to focus on host-specific proteases that are essential for virus activation and spread. Trypsin-like serine proteases are crucial for influenza activation by mediating the cleavage of the viral surface glycoprotein HA and hence promoting the fusion potential of the virus. Therefore, their inhibition provides a promising therapeutic approach. The present work focused on the characterization of two relevant HA cleaving type-II transmembrane serine proteases matriptase and TMPRSS2.
Chapter 3 and chapter 4 of this thesis engaged with the recombinant production of matriptase (chapter 3) in order to obtain a pure functional enzyme of high quality for a SAR study with novel monobasic (hence potentially bioavailable) matriptase inhibitors of the 3-amidinophenylalanine type (chapter 4). Adequate amounts of high-quality matriptase enzymes were isolated using a new expression system and in total 5 matriptase crystals were available at the end of this thesis for structural analysis. The matriptase inhibitor design in this thesis focused on matriptase-affine compounds with a fair selectivity profile against the blood coagulation enzymes thrombin and fXa. In total, 18 new monobasic and potentially bioavailable, as well as four new dibasic compounds of the 3-amidinophenylalanine types were tested. Based on the last published crystal structure of this inhibitor type in complex with matriptase from 2006 (PDB code 2GV6) docking was used as a structure-based virtual screening method for lead optimization of the compounds N-terminus. Selected compounds were suggested to interact with the carbonyl side chain of Gln175 of matriptase to achieve a higher affinity of matriptase compared to fXa.
The 4-tert-butylureido-piperidine could be identified as suitable C-terminus in combination with 3-fluoro-4-hydroxymethyl biphenylsulphonyl N-terminally in order to obtain excellent selectivity over thrombin. The binding mode of this compound (compound 55) was crystallographically determined in complex with matriptase as well as trypsin. Trypsin proved as a suitable alternative to matriptase for detailed binding mode analysis of the compounds N-terminus. However, different preferences were detected for the C-terminus.
Dibasic compounds showed higher matriptase affinity and selectivity in comparison with the monobasic analogues. However, the tested monobasic compounds were still decent matriptase inhibitors that are additionally suitable for cell culture and animal studies in their benzamidine prodrug forms, which are well established from related inhibitors of thrombin. In addition, selected monobasic as well as dibasic compounds demonstrated strong suppression of the replication of certain H9N2 influenza viruses in a matriptase-expressing MDCK II cell model. These matriptase inhibitors could be potential lead structures for the development of new drugs against H9 strains for influenza.
TMPRSS2 is widely discussed for its role in influenza activation. With a TMPRSS2 dependancy of HA-activation of certain subtypes, the characterization of this protease is an important prerequisite for being available as a target for influenza drug design. However, only little is known about the physiological function of TMPRSS2 and no experimental structure data are available at the moment to enable a structure-based drug development. Therefore, chapter 5 of this thesis focused on the characterization of TMPRSS2 in order to develop a strategy for the isolation of proteolytically active TMPRSS2 from cell culture. Even though, no functional TMPRSS2 could be recovered at the end of this work some new structural characteristics of TMPRSS2 were identified as crucial for functionality insight the cell. In general, TMPRSS2 without the cytosolic part, the transmembrane domain and the LDLRA domain is able to undergo autocatalytically activation if an artificial signal peptide was added N-terminal to enable entry into the endoplasmic reticulum. The presence of the cysteine-rich SRCR domain and the presence of the disulfide chain that connects the SPD and the stem region after activation cleavage have been identified as crucial for activity. N-terminal truncation of TMPRSS2 did not result in obvious dislocation within the cell: as the full-length positive control truncated TMPRSS2 was exclusively found in cell compartments surrounding the nucleus in immunofluorescence experiments. However, a reduced proteolytic cleavage activity towards H3-HA in co-expression experiments has been observed and might be a result of dislocation, since truncated TMPRSS2 is not bound to the biomembrane anymore. In addition, TMPRSS2 has been identified as a potential substrate of matriptase in vitro, which suggests possible participation in several zymogen cascades
A transient syntaxin/SNAP-25 interaction serves as ready-available binding site for synaptobrevin
Insect expression systems : improving intracellular and heterologous gene expression
Expression systems (ES) in a wide variety of biological systems are used to provide
foreign protein. Protein production in mammalian cells is a labour-intensive and expensive
process and insect cells have been used as cheaper alternatives. Insect ES are based on
two types of vector; viral- and plasmid-based. The baculovirus ES (BES) offers high-level
transient production of intracellular proteins. Yields of secreted and membrane-targeted
proteins have until recently been relatively low, however, the development of a novel
vector, lacking chitinase, has overcome these limitations. Plasmid-based vectors can be
used for transient and stable expression in insect cells but most utilise the relatively weak
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate early-1 (ie-1)
promoter, producing low-levels of intracellular recombinant protein. More recent vectors
have used stronger promoters, however, compared with mammalian stable ES these are
limited in use and most notable is the lack of an efficient inducible ES in insect cells. The
work in this thesis describes methods, with which intracellular levels of foreign protein may
be increased, while also developing the grounding work for the development of a fully
functional insect tetracycline regulatory system.
To improve stable and regulated expression of heterologous genes in insect cells, work
was undertaken to compare and characterise the transcriptional activity of a range of
promoters successfully used in insect and mammalian systems. Of the promoters tested,
Orgyia pseudotsugata MNPV (Op) ie-2 and Bombyx morl actin 3 (BmA3+E) were found to
be transcriptionally stronger than AcMNPV ie-1. Drosophila melanogaster metallothionein
and actin 5.1 did not produce any detectable activity, and the Cytomegalovirus (CMV) ie
promoter, although active, was weaker than AcMNPV ;e-1. Attempts to develop a new
inducible ES for use in stable cell lines was based on modifying a mammalian tetracyclineinducible
ES. The original CMVie promoters were replaced by the Bm A3+E and Op ie-2
promoters that had been shown to be optimal in insect cells. The Bm A3+E promoter was
successfully used to express the tetracycline transcriptional activator protein. Studies also
demonstrated that the constitutive transcriptional activity of Op ie-2, used to drive
expression of a reporter gene, was successfully suppressed in normal medium. However,
in the presence of the inducer, doxycycline, transcriptional activity of Op ie-2 was not
activated. Work to elucidate why Op ie-2 remain repressed in the presence of the inducer,
indicated that other, uncharacterised vector sequences may have interfered with the
activation process.
To determine whether stable insect cell lines could be used as an effective alternative to
the BES for producing large quantities of intracellular foreign proteins, protein production
from stable cell lines, using the Bm A3+E promoter, were compared to the BES using a
range of reporter proteins. It was concluded that with vectors currently available, stable
cell lines would not normally provide an effective alternative to the BES. However, a stable
insect cell line expressing Discosoma red was used to develop methods for scaling-up
continuous cultivation of cells in an open fermenter system. This stable cell line was
successfully maintained without contamination or total loss of cell viability for 4 weeks.
Improving intracellular expression of foreign genes using the BES, initially focused on
investigating whether a baculovirus vector (BV) lacking chitinase could produce larger
yields than normal BV. It was concluded that recombinant BV lackingchitinase could be
used to improve intracellular levels of foreign protein, but demonstrated the importance of
optimiSing production conditions for each recombinant protein (e.g., vector and cell line).
Intracellular levels of foreign protein were further improved from the BES using different
culturing methodologies (fermentation and shaker flask) and concluded that fermenters
produced optimal conditions for intracellular protein production, probably due to
maintaining a constant level of dissolved oxygen concentration during virus infection
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The Engineering of Chinese Hamster Ovary Cells to Achieve More Efficient Gene Amplification for Improving Biopharmaceutical Development
This dissertation addresses the issue of the long development times of obtaining Chinese hamster ovary (CHO) cells capable of producing high quantities of therapeutic proteins. It addresses the specific time bottlenecks associated with developing high producing CHO cell lines and reviews various methods that are employed to alleviate these bottlenecks. The specific time consuming process of gene amplification is the focus of this work. Gene amplification is the process of selecting CHO cells which have been genetically modified to contain many copies of a therapeutic transgene, and therefore has the ability to produce a high amount of therapeutic protein. Two separate projects are described which decrease the time necessary to obtain a high producing cell. The first project describes a novel process developed which can measure and quantify the amplification rate of a transgene in CHO cells. This process was used to successfully isolate a CHO cell clone with the capability of amplifying a transgene targeted to a specific location in the genome and thus produce higher quantities of protein in a shorter time period. Site-specific recombination (SSR) technology was utilized to target the transgene to this location which was deemed capable of amplifying a transgene at a high rate. The second project also utilizes SSR technology to integrate many copies of a transgene into many recombination sites in the CHO genome. A cell line containing several thousand integration sites was isolated, however only about twenty of these sites successfully integrated a transgene after optimizing cell transfection conditions. Efforts towards engineering an improved recombinase for this purpose has led to the result that DNA sequences flanking recombination sites have the ability to greatly improve this integration process. Potential future experiments are described which may isolate such sequences and ultimately increase the number of transgenes integrated into the CHO cell genome. Overall, these improvements to CHO cells have the ability to ultimately isolate a higher producing cell line faster, thus decreasing the time to get a potential drug candidate to market
Viral Gene Therapy
The development of technologies that allow targeting of specific cells has progressed substantially in recent years for several types of vectors, particularly viral vectors, which have been used in 70% of gene therapy clinical trials. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. This book is designed to present the most recent advances in viral gene therap
Oncogene and Cancer
This book describes a course of cancer growth starting from normal cells to cancerous form and the genomic instability, the cancer treatment as well as its prevention in form of the invention of a vaccine. Some diseases are also discussed in detail, such as breast cancer, leucaemia, cervical cancer, and glioma. Understanding cancer through its molecular mechanism is needed to reduce the cancer incidence. How to treat cancer more effectively and the problems like drug resistance and metastasis are very clearly illustrated in this publication as well as some research result that could be used to treat the cancer patients in the very near future. The book was divided into six main sections: 1. HER2 Carcinogenesis: Etiology, Treatment and Prevention; 2. DNA Repair Mechanism and Cancer; 3. New Approach to Cancer Mechanism; 4. New Role of Oncogenes and Tumor Suppressor Genes; 5. Non Coding RNA and Micro RNA in Tumorigenesis; 6. Oncogenes for Transcription Factor
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