Accelerated drug development by real time kinetic studies

The development of new drugs for the treatment of e.g. cancer or infectious diseases like HIV is one of the most challenging topics in medical sciences. More frequently the design of new drugs is based on knowledge about specific molecular interactions e.g. the reaction of a hormone with its membrane receptor at the target cell. Following the molecular design and the production of the drugs, the biological activity of the candidates has to be tested in vitro. Screening assays often serve as a tool to obtain a kind of ranking by excluding molecules with only low activity in the selected assay set-up. Due to the high costs incident to this research area, methods speeding up the process of development by accelerating and increasing the information content of screening assays are of utmost interest. Therefore methods which allow real time monitoring of biorecognitive reactions are superior to commonly used endpoint assays (e.g. ELISA) because of. more reliable data than one-point measurement short readout times no sample pre-treatment measurement of very fast reactions information content of kinetic data (e.g. affinity) The most widespread technique for real time monitoring at the moment is surface plasmon resonance. Although it doubtless benefits from the advantage to enable label free investigation of the compounds of interest, the high costs of the measurement device often limit its application. In this project we would like to demonstrate the value of a simple and inexpensive fluorescence enhancement technique for real time monitoring of biomolecular interactions. Silver colloids bound to the surface of microplates enhance the fluorescence of molecules close to them, thus making a discrimination between molecules binding to the surface and molecules in solution possible in a one-step reaction without washing steps. This technique is fully compatible to standard microplate procedures and ordinary fluorescence microplate readers can be used as measurement device. In previous experiments using this technique we have achieved detection limits comparable to ELISA techniques in less than 10 minutes readout time. In this project, we want to demonstrate the use of the assay design described above for accelerated drug development in cancer research by optimisation of the molecule design via improved information about binding kinetics and by reducing the number of experiments in cell culture. The work will be focused on site directed drugs which bind to a pathological target cell via a specific molecular interaction thus preventing harmful side effects to normal tissue. In general production of such drugs in cancer therapy includes the combination of a biomolecule (most often an antibody) which provides cellular specificity and a cytotoxic compound. Since two major groups of drugs, one which`s cytotoxic activity depends upon uptake of the drug into the cell and one who`s cytotoxic activity depends upon the binding to a receptor on the cell surface exist, we are intending to investigate exponents of both groups using real time kinetic monitoring with the described colloid fluorescence enhancement technique: lectin-doxorubicin conjugates targeted at colon-carcinomas (endocytosis dependent). Aim: Separate the influence of binding kinetics from the influence of drug endocytosis and release at the other side of the cell layer, which is considered an important mechanism involved in systemic distribution of the drug after transport across the gut wall. tumor necrosis flictor-immunotoxin targeted at breast carcinomas (non endocytotic system). Aim: covalent conjugation of TNF and antibody fusion proteins with different spacer length with special focus on TNF-motility, which is important for TNF-receptor binding. Testing of the different drugs constructs will be done using a competitive assay set-up, thus avoiding the need to fluorescence label each component to be tested. We intent to use two different surfaces to determine the affinity of the site directed drugs: target receptors directly bound (coated) to the colloid coated surfaces and artificial tumour-cell surfaces applied to colloid coated surfaces. These "life-like" surface will be created by the fusion of cell-membrane vesicles to self assembled monolayers (SAMs) bound to the colloid-surfaces.

The phenomenon of silver nanoparticles is that they enhance only the fluorescence intensity of molecules close to their surface. This unique feature is of utmost importance for high throughput screening of drug candidates as removal of unbound molecules is not required and interactions can be monitored in real time. Upon coating the surface of the silver nanoparticles with antibodies and isolated receptors, key parameters about biorecognitive interactions are collected which allow identification of leads. Furthermore, the set-up of biomimetic surfaces such as mucus layer or artificial cell membranes offers rapid information about biorecognitive processes at barriers to successful absorption, not only of drug molecules but also of prodrugs. As compared to expensive commercially available devices, the silver nanoparticle enhanced flourescence-technique represents a simple, cheap and versatile tool to characterize biological interactions and probably helps to reduce the number of animal trials required. To date, the development of new drugs for treatment of cancer and infectious diseases is one of the most challenging topics in medical sciences. As high costs are inherent to this research area, methods speeding up the process of drug discovery and development are of utmost interest. Most frequently, the design of new drugs is based on knowledge of specific receptor-ligand interactions and the biological activity of such drug candidates has to be tested in time consuming assays and animal experiments. Thus, real time monitoring of biorecognitive interactions provides features to establish a kind of ranking and to exclude less efficient substances at early stages of drug discovery and development. The silver nanoparticle enhanced fluorescence is introduced as a cheap alternative to established surface plasmon resonance techniques. Silver colloid nanoparticles enhance the radiative rates of fluorescent molecules localized within a distance of 5-50nm from the surface of the noble metal particles. In order to take advantage of this fluorescence enhancement zone for biorecognitive interactions, the surface of the nanoparticles was modified by attachment of different biomimetic ligands: The adsorption of antibodies via an intermediate layer is useful for immunoassays. As biologically active proteins are not fluorescent, a single step competitive assay for insulin was investigated which revealed a working range of 10-200nM in serum. Although receptor-ligand studies usually require radiolabelling, silver nanoparticle bound receptors and flourescent labelled ligands featured detection limits sufficient to follow their interaction. Despite of immobilization, the native structure of the receptor was maintained. As exemplified by wheat germ agglutinin, which represents the targeting moiety in lectin-mediated drug delivery systems, the mean velocity, the affinity and specificity of interaction was estimated. Not only key parameters of drugs, but also biopharmaceutical characteristics can be investigated by this technique. The anchoring of biomimetic cell membranes prepared from cancer cell lines or cell lines similar to absorptive enterocytes offers a test system which allows elucidation of events at the cell surface. Not only the membrane of intestinal cells but also the mucus layer represents a barrier to drug absorption after oral administration. Experiments with mucin-coated silver nanoparticles extend the area of applicability of the test system to formulation development of drugs. All in all, the silver nanoparticle enhanced fluorescence technique allows real time monitoring of biological interactions within 3-5min with a detection limit comparable to ELISA-techniques. The single step assay design and the compatibility to the microplate format makes this technique useful for high throughput screening.