prof. dr. W.J. Quax
Enzymatic Degradation of Bacterial Quorum Sensing
Pseudomonas aeruginosa , a Gram-negative bacterium, is an opportunistic pathogen able to infect animals, plants and humans. This bacterium has been associated with several types of infections causing a major threat to immunocompromised, severely burned and cystic fibrosis patients.
P. aeruginosa is able to secrete various signalling molecules, such as N-acyl-homoserine lactones (AHLs), which control the production of many virulence factors, including toxins and biofilms, making it highly pathogenic and resistant towards antibiotic treatment. The production of these virulence factors is under the control of a communication system known as quorum sensing. Quorum sensing is used by a number of bacteria to co-ordinate their behaviour, according to their local population density.At low cell density, the bacterium secretes very low levels of signalling molecules, however, when the cell density increases, higher amounts of the signalling molecules are produced, and when a threshold is reached then production of the virulence factors is initiated.It’s by no surprise, that quorum sensing became an interesting target since the disruption of this communication system (a process known as quorum quenching) can lead to a more effective antibiotic treatment. The discovery of two different types of enzymes able to break AHLs was a big step towards quorum quenching. The fist type of these enzymes, AHL-lactonases, is capable of cleaving the lactone ring, while the second type, AHL-acylases, is active against the acyl-chain of the signalling molecule.
In the search for novel antimicrobials, we have based our efforts in the development of potent quorum quenchers that allow us to effectively interfere with these communication systems to obtain an advantage in the fight against infection. We are actually studying the effects of these quenchers using pvdQ, an acylase from P. aeruginosa, as a model to create potent enzymatic variants with enhanced quorum quenching activity that will also be tested in a live model.
In parallel, we are studying the role of a multitude of genes involved in the complex communication network of P. aeruginosa. Understanding the function of its components will allow us to develop more specific tools to fight this important pathogen that is a major cause of mortality in cystic fibrosis patients.
Structure based design of rhTRAIL variants for increased induction of apoptosis in cancer cells
Computational protein design (CPD) methods have been successfully employed to redesign several protein - protein interactions, but have as yet hardly been applied to redesign target binding preferences of therapeutic proteins. CPD methods allow the rational design of tailor-made protein therapeutics by modifying the binding characteristics of the protein, for example in order to reduce target binding promiscuity or to design novel mechanisms of activity.
A member of the TNF-ligand family, Tumor necrosis factor (TNF) related apoptosis inducing-ligand (TRAIL, Apo2L) is attracting great interest as a potential anti-cancer therapeutic as it selectively kills various types of cancer cells, and unlike other apoptosis inducing TNF-ligand family members, appears to be inactive against normal cells.
TRAIL is a promiscuous ligand as it binds to five different cognate receptors of the TNF-receptor family: the death receptor 4 (DR4, TRAIL-R1), death receptor 5 (DR5, TRAIL-R2) containing a cytoplasmic death domain through which TRAIL can transmit an apoptotic signal and to the decoy receptor 1 (DcR1, TRAIL-R3, TRIDD), decoy receptor 2 (DcR2, TRAIL-R4, TRUNDD) and the soluble secreted receptor OPG, that may act as antagonist receptors, lacking an intact death domain. The occurrence of DR4 or DR5 responsive tumor cells indicates that a DR4 or a DR5-receptor specific TRAIL variant will permit new and selective tumor therapies.
Using protein design algoritms, we successfully generated DR5-selective TRAIL variants. These variants do not induce apoptosis in DR4-responsive cell lines but show a large increase in biological activity in DR5-responsive cancer cell lines. Even rhTRAIL wt insensitive ovarian cancer cell line could be brought into apoptosis.
The existence of certain cancer cells only responding to DR4-mediated apoptosis and favorable results obtained with agonistic anti-DR4 antibodies, prompted us to design a DR4 selective TRAIL variants. This variant has been showed to enhance apoptosis induction significantly.
Directed evolution of enantioselective biocatalysts.
The European Pharmacopoeia mentions around 1600 different synthetic pharmaceuticals of which around 40% display chirality. Despite the fact that the "wrong" stereo-isomer only contributes to unwanted side effects, the majority of chiral drugs are administered as a racemate. As a result of more stringent safety precautions there is now a strong need for efficient and clean enantioselective catalysts. Lipases are very promising candidates, however, most of them have evolved under natural conditions different from the industrial applications. This project aims to develop novel tools for directed evolution of enzymes in the laboratory and to provide novel lipase variants as enantioselective biocatalysts for important synthons. The model proteins are Bacillus subtilis and Pseudomonas aeruginosa lipase, whose 3D structures have recently been solved.
Podophyllotoxin (POD) is a lignan, which is used as educts for the semisynthesis of Etoposide®, Teniposide®, and Etopophos®. These antineoplastic drugs are of importance for the therapy today and also there will be a high demand for the commercial drug and its precursors for the treatment of leukemia. For the production of POD three different routes are known: extraction and isolation from Podophyllum hexandrum and other Linum species, production in plant cell cultures  and o rganic synthesis .
Extraction and isolation of POD from Indian Podophyllum hexandrum species is not economic and characterised by a high price for the final product. Another problem is that the cultivation of the plant is not easy because of the limited growth rate. Mostly POD is extracted from wild collected species. As a consequence from wild collection P. hexandrum is actually endangered in India, especially in the Himalaya region. Working with plant cell cultures as described in the past or obtaining the drug by organic synthesis is difficult, the yield is limited and the price can not compete with wild collection.
Anthriscus sylvestris (L.) Hoffm. (Apiaceae) is a common wild plant in Northwest Europe that accumulates considerable amounts of lignans. Deoxypodophyllotoxin (DOP) as the main attractive constituent can be used as a precursor for the production of podophyllotoxin but this is not yet applied on an industrial scale.
|Laatst gewijzigd:||06 november 2012 01:40|