EH 2_2016

LABORATORY Early warning of future brain disease – up to 20 years before onset A new photo-switchable molecule for oxygen-independent PDT therapy Blood test detects early Alzheimer’s Advanced photo- dynamic therapy The cause of Alzheimer’s disease remains unknown. Early diagnosis is currently not possible as clinical symptoms only occur once a large number of neurones in the brain have already been destroyed. There is no treatment available either. Imaging procedures such as PET-MRI are expensive and not covered by rou- tine care. This situation hinders the early confirmation of a diagnosis. A new blood test now makes it possi- ble to diagnose Alzheimer’s up to 20 years before the onset of the first clinical symptoms. Amyloid-beta pep- tides, i.e. proteins, and an infrared sensor, play key parts in this process. In our interview, Professor Klaus Gerwert, Head of the Chair for Biophysics at the Ruhr University Bochum, Germany, discuses the procedure Researchers develop an oxygen-independent, photoswitchable molecule and successfully test this in the lab to observe effects on tumours. Photoswitchable agents may reduce chemotherapy effects Report: Melanie Günther ‘There are different hypotheses that describe how Alzheimer’s develops, but none of these has been conclu- sively proven,’ explained biophys- ics expert Professor Klaus Gerwert, when asked about the role of amy- loid-beta peptides in Alzheimer’s disease. ‘We assume that, at the beginning of the disease, there’s a change in the amyloid-beta peptide. This means that the peptide changes from a healthy alpha-helical form into a diseased, beta-pleated sheet form; the secondary structure of the protein changes. The pleated sheet structures can attach to one another and form oligomers. We believe that the formation of soluble oligomers is the critical point at the beginning of Alzheimer’s disease. The oligom- ers develop into fibrils and finally into visible plaques.’ Along with other scientists, Gerwert has developed a test to detect change in amyloid-beta pep- tides and thus diagnose Alzheimer’s earlier. How does the test, or procedure, work? ‘We have developed an infrared sensor. This detects the second- ary structures and differentiates between alpha-helix and beta pleat- ed sheet forms. However, various different types of proteins are found in the blood or spinal fluid. The dif- ficulty is being able to look only at the amyloid-beta. Other substances impair this and interfere with the actual signal. We utilise antibodies as ‘interceptors’ to specifically bind amyloid-beta peptides. These are covalently bonded to the surface of the infrared sensor, meaning that we have a type of ELISA-test (Enzyme- Linked-Immunosorbent Assay). ‘We then measure the bonded amyloid-beta peptide with the infra- red sensor and analyse whether it is present in its healthy or diseased form. We utilise structure-sensitive infrared bands, i.e. so-called amide 1 bands. When these bands are below a certain threshold we know that Alzheimer’s disease is present. ‘We determined the threshold level based on experimental inves- tigations.’ What does the importance of amyloid-beta peptides distribu- tion mean? ‘This is the key part of the test: There are antibodies that can only selectively bind one form of amyloid-beta peptides – either the healthy or diseased form. The blood of a healthy human contains a large number of healthy amyloid-beta peptides, but naturally also some diseased ones. An antibody that only selectively looks for the beta pleated sheet form would therefore also find diseased forms in a healthy person. Therefore the test is initially confusing. ‘The advantage of our technol- ogy is that we can determine the secondary structure distribution of all amyloid-beta peptides extracted from body fluids label-free. When the peptides are healthy we can see alpha-helical dominated bands above the threshold level. When the majority of peptides are of the beta pleated sheet form, the band goes below the threshold level. Unlike the ELISA procedures we measure the distribution of all amyloid-beta peptides and not only specific con- formations. Early detection timescale ‘Our primary objective was to detect an early stage of Alzheimer’s, i.e. before the onset of clinical symp- toms. Therefore, we selected the amyloid-beta peptide, because the change occurs around 15 – 20 years before the beginning of clinical symptoms. ‘We’ve already carried out a mini- study which, although not yet sta- tistically significant, has delivered some promising results. We ana- lysed samples from a cohort from the year 2000, at the Heidelberg- based German Cancer Research Centre (DKFZ). We could analyse, conclusively, which of the initially healthy study subjects developed Alzheimer’s disease within the fol- lowing 8-15 years. To substantiate our analysis with statistical accuracy, we are currently examining 1,000 samples from study participants. Once we can make this prediction for all 1,000 samples we’ll have achieved the necessary statistical significance. So far we have tested 300 out of the 1,000 samples.’ Other neurodegenerative disease ‘The sensor is also potentially suit- able for the detection of Parkinson’s disease. In the case of Parkinson’s the so-called alpha-synuclein plays an important role. We assume that this protein also converts from an alpha shape to a beta shape. We are currently looking for a specific anti- body for alpha-synuclein, allowing us to use the sensor for the detec- tion of Parkinson’s disease.’ When might the test enter clinical routine? ‘The procedure is very robust in the laboratory. It is suitable for the detection of Alzheimer’s. It is a good, additional clinical-chemical test to confirm an Alzheimer’s diag- nosis. ‘We need to await the results of the current study to assess its use for early detection. We are currently optimising the sensor and are trying to increase the sample throughput so that large collectives can be measured in short periods of time.’ So far, photodynamic therapies have been dependent on oxygen in the tissue. But hardly any oxygen exists in malignant, rapidly grow- ing tumours. A group of researchers at the KIT Institute for Biological Interfaces and the University of Kiev has now developed a photo- switchable molecule as a basis of an oxygen-independent method. The researchers’ successful labo- ratory tests on tumours have been reported in the journal ‘Angewandte Chemie’ (Applied Chemistry – DOI: 10.1002/ange.201600506). Photodynamic therapy (PDT) in medicine usually uses a substance that reacts to light and converts the oxygen in the tissue into aggressive radicals. These reactive substances are toxic and damage neighbouring cells, in such a way that tumours, for example, are decomposed. As a result of their quicker growth, however, many tumours have a high oxygen consumption. This reduces the concentration of oxygen avail- able in tissue, which may aggravate conventional PDT. What the researchers at the KIT Institute and the University of Kiev have developed is a new photo- switchable molecule for oxygen- independent PDT. The effect of the GS-DProSw molecule can be ‘switched off’ by ultraviolet light prior to therapy. Only upon applica- tion is it ‘switched on’ in the tumour tissue by visible light to damage the tumour tissue there. ‘The sur- rounding organs remain in the dark and are not affected by the active substance,’ Anne S Ulrich, Professor of Biochemistry and Director of the KIT Institute for Biological Interfaces, explains. ‘As a result, side effects are reduced significantly.’ Animal testing Now, For the first time, this new concept has been tested on animal models. Once per day, the photo switch- able GS-DProSw molecule was administered. Then, the tumours were irradiated locally with visible light for a period of 20 minutes. After ten days of PDT treatment, the tumors were found to be far smaller than comparative groups not treated with light. To initiate an oxygen-independ- ent reaction in PDT, the molecule applied has to be of a cytotoxic nature. This means it has to directly attack the tumour tissue irrespective of other reaction partners. A suitable molecule with cytotoxic properties against tumours is the biomolecule gramicidin S (GS), which is a natural antibiotic. To prevent it from damaging healthy tissue, the research team inserted a photo-switchable diaryl ethene segment into the ring struc- ture. As a result, the GS-DProSw molecule can be switched between two states with the help of light: The agent can be administered in the inactive state and is activated at the desired location by specific irra- diation with light. There, it attacks the surrounding tumour tissue and, contrary to conventional PDT, it does not require any oxygen for this purpose. Professor Klaus Gerwert is Head of the Chair for Biophysics at the Ruhr University Bochum. As Chair of Biophysics, the professor. and his collegues, investigate structure, function and interaction of proteins at the atomic level, using a wide variety of interdisciplinary methods from biology, biophysics, biochemistry and computing. In the post-genome-era the focus of research has moved more towards proteins, which are, besides DNA, key players on a molecular level. The GS-DProSw molecule in its inactive form (blue) can be activated by visible light (red) and ‘switched off’ again by UV light. (Figure: KIT) 20 EUROPEAN HOSPITAL  Vol 25 Issue 2/16

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