Dr Timothy Johanssen, Alzheimer's Research UK Drug Discovery Institute

Transcription

1 Turning Discoveries at the Bench in to Therapies for Dementia Dr Timothy Johanssen, Alzheimer's Research UK Drug Discovery Institute Hi, My name's Tim Johannsen. I'd like to thank Melanie Witt and Mark Dallas for the opportunity to come and to introduce you the drug discovery institute at Oxford. It's the Alzheimer's Research UK Drug Discovery Institute or as we like to call it the ODDI. This is housed within the Nuffield Department of Medical Research building. This is over at the Old Road campus. It's a very impressive new building over there on the on the site. What we are endeavouring to do there, is to take academic research, what we say is "at the bench". Then translate that into a therapeutic, that we can then hopefully progress, through collaboration with pharmaceutical industries, through to a therapy for Alzheimer's disease. I actually began working for a pharmaceutical company down in London some time ago now, about 15 years ago. At that time, there were only actually four drugs on the market. This company that I worked for, they make Donepezil or you might know it as Aracet. This is one of the major treatments. It's of a set of drugs called acetylcholine esterase inhibitors. At the time they were all of that same class of drug. What I want to emphasize is that, since then, there's only actually been one other approved drug to come to the market, and that's memantine. What I've put up here is a diagram of the clinical, preclinical, and the outer ring, and the clinical stages. This is a snapshot, back in 2010, of the drugs and where they were at. What we can see today, is only one of those have actually made progress all the way through to phase four. Which is being approved for therapy. What this has led to is an exodus of pharmaceutical companies from this really important preclinical research, because of the risks that's involved. Obviously the financial cost of this.

2 Fortunately for us, Alzheimer's Research UK has identified this major problem in this preclinical phase of drug discovery. In order to address this, they've established these 3 drug discovery institutes that Robin mentioned earlier. They're housed at University College London, at Cambridge, and here at the University of Oxford. The Oxford Drug Discovery Institute wants to combine academic excellence at the University of Oxford and the researchers here, with the expertise coming from the pharmaceutical industry in the hope of discovering new treatments for dementia. They're set up, somewhat like a pharmaceutical company, in the sense that they have a chief scientific officer. Then underneath there there's a lead biologist, lead chemist, and biology team and chemistry team. Then we collaborate directly with academics, here in Oxford, and further afield, on specific projects that they've actually validated in their own labs and they've shown to be efficacious in preclinical models at the very early stages. Where the Oxford Drug Discovery Institute steps in, as well as the other Institutes, is we want to take that basic, at the bench research and develop the high-throughput assays. We want to take this, say it's a protein, that they've found and come up with assays where we can study the effects of that assay and that protein in a high-throughput sense. That is so that we can study and we can test, tens of thousands, to hundreds of thousands, of compounds to find a compound, or a class of compounds, that actually are able to prevent the negative activities of these targets that have been discovered in the academic laboratories.

3 Today I'll just I'll just describe two projects that I work on. Both in collaboration with lead academic scientists at the University of Oxford. The first project I work on is a collaboration with Professor Richard Wade Martin's lab who's down in the Department of Physiology, Anatomy & Genetics. In their previous research they've actually taken one of the key mutations in Parkinson's disease and they've been able to express this in a transgenic rat model. They take a human protein that's abnormal, and express that in a rat model. Then they study this rat model to see what the changes are. One of the major changes they found is there's this normal process that occurs in the cell. It's a requirement for cell survival. It maintains a balance between the manufacture and the breakdown of cellular components. I've just got a little picture here that shows the cellular components. Then here, if these intracellular vesicles, they're called Autophagosomes, they're just vesicles. They come along and they engulf, this is all within a cell, they engulf these cellular components. Then they fuse with another intracellular vesicle, called a lysosome. This creates an acidic environment which leads to the degradation of these intracellular components, and then these can be recycled and used again. This is ongoing at all times and it's vital for cell survival. With this discovery, that when you have this mutation in these rats that were expressing this human protein, that autophagy is prevented. This means we have what we call a phenotype. We can study the prevention of autophagy. We can set up assays that screen, 10s of thousands or 100s of thousands of compounds. Then we'd look for compounds. Generally, about 1% of will show these will show an activity in preventing this phenotype. They're called hits. Then they can provide us with a starting point that we can then, with work with the medicinal chemists in the Institute, go about refining the properties of these drugs that have come out.

4 This project, at the moment, is in its early stages. It's just been developed from the research in Richard Wade Martin's lab. We've been able to take that from a very low throughput system and miniaturize it into high-throughput scenarios. Here, we actually take neurons from these animals that I referred to. Then we can grow them in these little microplate dishes they have 384 wells on them. On one plate we can screen 300 odd compounds on a plate. Then using state-of-the-art technology that's housed down at the Department of Physiology, Anatomy and Genetics with its high content imaging. This machine is called an Opera Phoenix It's definitely a state-of-the-art technology. It allows high resolution, as well as high magnification images of these neurons that we're growing from the brains of the animals or nerve cells. Here we can see them, and using antibodies we can we can stain and highlight the neuron in red, and it's little extensions (the neuritic extensions) that join up with other neurons. More importantly, I don't know how the imaging is for you? You can see all these little dots. Those dots are coloured yellow and green and they're stained with yellow and green antibodies and they detect those vesicles that I was referring to. With this image we can use sophisticated software to really extract this information about autophagy.

5 Here's a very complex image with different types of cells in there in red. You see the neurons, and then there's all these little spots which are the intracellular vesicles that we want to measure. Using this really extensive pipeline on the software. I'll just skip through it. We can find the nuclei, so that's the centre of a cell, the heart of a cell, but they're not necessarily all neurons. Then we can just detect ones that are neurons. Then we further isolate the region of the neuron and the extensions that we want to look for intracellular vesicles. Then we can detect all those little vesicles within there, and then what we're looking for is when the vesicles overlap, that's when they fuse. That's our readout of autophagy. We want to look at drugs in this screen, this is still in its early development. We haven't actually screened libraries yet, but we will hope to do that in the near future, and look for compounds or drugs however you want to refer to them, that increase the number of those dots. Basically the overlapping vesicles. Then that's a sign that it's overcoming this prevention of autophagy.

6 In a second project that I work on at the Oxford Drug Discovery Institute, we collaborate with Professor Sir Simon Lovestone's group. They're in the Department of Psychiatry and also have laboratories down at department of Physiology, Anatomy & Genetics. In their previous work they've made a discovery that shows how important a certain protein called Dickkopf 1 is in Alzheimer s Disease. Along with other laboratories down at UCL, and in Italy, there's been a wealth of research that's validated this protein as a target, a therapeutic target for Alzheimer's Disease. We've collaborated with the Lovestone laboratory to further investigate this. Some of the further background, the reason this is so important. It might have been referred to before, and you might know about it. In Alzheimer s there is the amyloid plaques. This is made up of this abeta peptide. What they've shown is that when you increase the levels of beta amyloid you actually increase the amount of this protein DKK1 soup. This doesn't happen, in the cases where there's not Alzhiemer's present. This offers itself is as a really appealing target for us. It's also been shown in animal models, that if you block this protein, when it binds it has a toxic activity. If you block that in animal models you can rescue the health of these cells. That's what we wanted to do we wanted to set up a similar system like that, to screen for drugs that block this DKK1 protein. This project is a little bit more developed, we've actually been able to set up primary high-throughput screening. I wanted to demonstrate some of the equipment that we use because, I for one am quite impressed with it. Here's just a little cartoon, you have this protein, in this case it's called a donor, It's our DKK1 protein. When it's in close proximity to the binding protein where it conveys its toxicity, what we can do is use tags would transfer energy that we can measure. When they're close together, we get an increase in fluorescence. We can measure that on a spectrophotometer pictured here, in many plates screening tens of thousands of compounds. When we find a drug that blocks this proteinprotein binding, the fluorescence will decrease. We'll be able to measure that and then we will be able to find out which drug is doing that.

7 The way we're able to actually handle the logistics of tens of thousands of compounds is made possible by using such technologies shown here. This is an echo liquid transfer system. It's a roboticized way of dispensing the compounds from a plate into the experimental plates. This doesn't actually sound that interesting, but it's more of the concept behind it. What it does, is it actually uses sound waves to send nanolitre droplets, 2.5 nanolitre droplets, miniscule droplets, through the air to a plate that you want to do your experiment in, that's upside down above it. I find it quite amazing. We use this to to aliquot out our libraries across all these many, many plates, as pictured here. Then we can then perform these experiments. From these experiments, we've screened a library of more than ten thousand compounds now, this was at Christmas, and we found 52 drugs that are hits, that show the block the DKK1 protein from binding. So now we've taken these on. At the DDI we've developed secondary assays. These assays are in cell lines now. What we want to do with those, is to validate these hits that come from the previous assay. Because these experiments prior were just done, basically in a test tube. Now we're putting them on cells that are actually growing. We want to see that they're able to do the same thing.

8 In order to do that we've developed assays, again using this fluorescence spectrophotometer. We can measure when the protein is no longer binding, by the reduction in fluorescence. We can also image it, using this high content imaging that I introduced before. Again we've got a couple of assays we can see and we can show that the drug is actually stopping the binding of DKK1. From screening the hits that have come through to this stage of the project we currently have 4 drugs that have been effective, but we have many more to test. This is where the project is up to.

9 Just recently, in January, we've been successful in achieving one of the goals of the drug discovery institute, and that is to form a collaboration with a pharmaceutical company in order for them to inject funding into the pipeline, so into that first the high-throughput screening and then the secondary validation screening in cells. Then with the aim of taking hit compounds through to animal models, so that by 2021, we can have selective and potent drugs that can enter the brain. Get across into the central nervous system. This is a key problem with a lot of pharmaceutical products. Then actually show that they can treat the symptoms of Alzheimer s in models of that, normally in mouse models. At that stage, so this would be the end of the pre-clinical stage. The pharmaceutical company, then would have the right to acquire these drugs. Then they would fund the incredibly expensive process of clinical testing with the goal of getting it to market. With that, I'll just finish, but I'd like just introduce the people that I work with. John Davis is the CSO, the chief scientific officer. Over at Cambridge is John Skidmore and Paul Riley. Elena Di Daniel and Paul Brennan are the heads of chemistry and biology. There's a group of us working at the Institute over at Old Road. and we have our key collaborators. There's Simon and Chas and Richard Wade-Martins who I have just talked about and our other academic collaborators we work closely with. Thank You

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