Robert Huber (2010) - Basic Science and Co-entrepreneurship, my Experience (Lecture + Discussion)

The design and development of inhibiting (or occasionally activating) ligands of target proteins in medicine and crop protection guided by molecular structures and functions has become an established technology in academia and industry recently

Good afternoon, it’s a pleasure to see you here. I compete with Werner Arber, Brian Josephson, Walter Kohn, Harry Korto and Carlo Rubbia, Gerard t’Hooft and so on. So I’m glad that you found the way to the forum C to listen to what I am going to say. Now the title is basic science and business. I first have to tell you what I mean by basic science, it’s a small section actually of basic science, it’s structural biology. So the research on bio structures and their use in generating business. So let me first tell you in a very broad sense of why we analyse and look at protein structures at a time when it has become rather simple to analyse the genomes, the DNA, so why do we need that information on proteins. We need it because the phenomena of life are determined by the protein repertoire. So what you here see as an example is a mature butterfly and its larva and they do have exactly, except for some epigenomic changes, the same genome. But they are as different as you can imagine in any respect in how they look, how they move, how they feed. And this is a consequence of the fact that the protein repertoire, the proteome in the larva is relative from that in the adult insect. So in order to understand we have to know more about proteins. Now how do we study them. It’s certainly not sufficient to study sequences of proteins that would not help much in our understanding of protein function. A bit too much light in here, so we have to, can you see it, it’s a rather dark slide, I’m sorry but. So we have to know about protein structures and the main tool to determine 3 dimensional protein structures is x-ray crystallography. So this is my tool which I help to develop from the late ‘60’s on, you need crystals and you need x-rays to determine protein structures. But there are other tools available, there’s electron microscopy which has the advantage that it is a technique that can help to visualise individual molecules, all be it at low resolution. So usually we get shapes by electron microscopy, but we get atomically dissolved structures by crystallography. Now there is NMR, which was also developed in the last 30 or 40 years to become a tool of structural analysis. With problems, great problems in analysing large proteins. And all of the proteins that you will see today are large in terms of what NMR can do. But NMR has its virtues as well. For instance it is able to, by NMR one is able to determine dynamic properties, this you cannot do usually with crystallography. All of these are details which I will not dwell on this afternoon. Now there was, since the first analysis of protein structures by crystallography, the father of protein crystallography is Max Perutz who worked in Cambridge, England together with his student John Kendrew, they got a Nobel Prize for finding a way to decipher the x-ray diffraction pattern of protein crystals and to derive molecular structures from it. Well there is a lot of progress in these 30 or 40 years and let me just very briefly tell you where this progress comes from. The first one is that we now do have recombinant proteins available. When I started crystallography in the protein crystallography in the late ‘60’s we had to work, to isolate the protein material from the animal. So from the plants now we do have bacteria, bacteria working for us, so big progress allowing us to analyse the rare, to make and to analyse the rare proteins. The other important progress came in by using instead of a classical x-ray generator, this is Röntgen’s x-ray generator which you see, again still see in museums, Röntgen was the first physics Nobel laureate in 1901. So we now use synchrotrons as x-ray sources which have, provide us with x-ray densities which are millions of times stronger than what you get with a conventional x-ray generator. So even the modern in-house generators, they of course look very much different from Röntgen’s apparatus, use, work on the same physical principle and produce relatively weak x-rays. Now here the physics behind the x-ray generation is very different and the x-rays are much stronger, as I said millions of times stronger. What you see in the next movie is an actual experiment as we do it now with a protein crystal on a synchrotron. We have very fast and sensitive detectors so we rotate the crystal in front of the x-ray beam and record the diffraction pattern and this is on line evaluated in terms of the intensity of these many hundred thousand of spots that appear when we rotate the crystal. So there is the development of fast and sensitive detectors. And of course computers then are able to process these millions of pixels on this detector plane. So all of this progress did not come from the crystallographers, it came from the molecular biologist, it came from the physicists, it came from the informatics people, from the engineers. But we were able to incorporate, to integrate this progress in very different fields for our process. So first actual determination. This is the last of these slides I would like to show, namely the use of graphics systems for interpreting electron density maps. This is, I have to get a very good electron density map and it’s easily interpreted. So easy that a computer can do it when we feed in the sequence. This is actually what is done but there was also, when I started in the ‘70’s we built our models from metal parts and we drew up the electron densities on plastic, on paper and plastic sheets. And then modelled the electron density with the help of metal parts. So the first interactive graphic systems were developed in my laboratory in the early 70’s. And they have progressed now in such a way that if the electron density is good it can be automatically interpreted. I just wanted to say as Astrid mentioned my Nobel Prize in 1988 together with Johann Deisenhofer and Hartmut Michel, I thought I should say a few words about the importance of their work. This is an area view on earth, on Europe during the seasons, the snowy winter, spring, becomes green and then full photosynthesis, chlorophyll colour that you see in summer. Now Sweden is very white in winter of course and gradually it becomes somewhat more friendly in spring but it’s also rather green in summer. Ireland is always green. Spain, Andalucía in particular is always brown which is not really true, it’s very beautiful in spring time. So it is a process that is very important, we live on it, our food, our oxygen comes from it and these are the basic components. There is the reaction centre, this is the biological photocell, these are the peripheral and the membrane as light harvesting complexes which feed in light into the reaction centre, into the photocell. And we started work first on a large peripheral light harvesting complex from some bacteria, these are huge organelles with hundreds of sub units which you can see in the electron microscope. They are these phycobilisomes as they are called, not attached to the photosynthetic membrane but they can be removed and looked at in the electron microscope to see this kind of structure. It’s not crystallisable and neither you can understand, you see how heterogeneous these individual objects are but you can do what we now make more and more frequently use of, use electron microscopy and x-ray crystallography, EM to get a general, an overview picture, shape and then dissect the material into its component and crystallise them. This is what you see here, so this is the inner component, the middle and the outer component. Beautiful blue and red colours. This we can crystallise and analyse the crystal structure and then model back into the EM structure, the atomic structure that we see by crystallography. Then we start to understand the function of this system which actually is a concentrator of light. Very simply because the outer components have more a grower force than the inner component. So it acts like a concentrator of light. But of course we understand more out of , we can calculate the spectra from the arrangement of these chromophores in the structure and so on and so forth. Now a few words about the Nobel awarded work on the photosynthetic reaction centre from bacterium rhodopseudomonas viridis. Now that was chosen because it showed already a regular arrangement of reaction centres in the membrane and this is the reason why we, that is in particular Hartmut Michel who was the protein chemical part in this team, made us believe that this might crystallise. And actually it did crystallise, relatively large crystals and they were well ordered. This was a big surprise for an integral membrane protein to form well ordered crystals. Now at that time it was a very large protein, the largest that had been worked on at that time and we didn’t quite know how to solve the phase problem we managed. But let me first show what was known before we started the structural work, what was known was a large, many data on, spectroscopic data on the kinetics and the function, the kinetics of electron transfer, the spectral properties, the energetics and so on and so forth. But this was so to say a matrix of numbers that was known. That there is electron transfer between different chromophores with certain rates, unable to lead to an understanding of the situation of the system. And this is when we produced in the first brief publication the arrangement of the chromophores. So it was, I think the general feeling that it was like switching on the light. So there was this system of numbers in literature and now we switched on the light and associated the numbers now with molecular entities. This was then the complete protein structure of the chromophores, the chlorophyll chromophores and the protein, it was a membrane protein also that was exciting for the scientific community. Showing that the part that sits in the membrane is in contact with the hydrophobic fatty acid side chains, is devoid of charged residues, there are charged residues on the periplasmic, in the cytoplasmic side. This is another somewhat later membrane protein that we determine shows perhaps even better this non-uniformed distribution of charged neutral and charged residues in such an integral membrane protein. I think I have to skip that. I would like to show that, because I think it was rather unusual that the Nobel Prize was awarded 3 years after the publication, showing that it was regarded as an important work. These are the 3 laureates, all of them you see the meeting. Well I wouldn't say that none of them looks very happy, if you look at the faces. But we were happy I should say. Now work went on in my group, it was already a very large group in 1988, we worked on many projects in parallel and many of them had a medical implication and this is the story I would like to tell you today which finally then led to the foundation of businesses. This was a collection that I got on my 65th birthday to show the various protein structures that we had analysed over the years. Now what is the basis of the application in medicine and drug design that goes back to inefficient paradigm of the lock, the key and lock principle of proteins, enzyme specificity. So the enzyme has a complex binding surface and the ligand is complementary to that. Now once we know the structure of the apo-protein then we can design and also develop a ligand. Now this is an example of work that we did on thrombin clotting enzyme in a major target for drug design. So the first hit with which we started work was this simple tripeptide. You see that it fits nicely. But it is not perfectly filling the subsites of these parts of the ligand. So the next step was then to make this a bit larger, to make this a bit larger and this was the next step then. So instead of the phenyl, we have a naphtyl group. It fills the hole perfectly, instead of the arginine side chain we have an amidino group which is also somewhat larger. And we immediately gain a factor of 100 in binding strengths and also we gain in specificity. So this is the thing that we do. And we do that with many of the proteins that we analyse. Let me show you one of our recent, more recent systems on which we focus, the proteasome. The proteasome is the executioner of the ubiquitin pathway and you have heard about it, Ciechanover spoke or will speak about it, Avram Hershko will speak about it, so it was honoured with a Nobel Prize in chemistry to Ciechanover, Hershko and Rose in 2004. So they discovered this poly ubiquitin labelling cascade. By which proteins that are to be degraded, removed let’s say, cell cycle proteins, should be in a cell only for a brief time, signalling molecules, they are labelled and then they are removed and the machine that removed it is the proteasome. Which is a huge intracellular protease whose core particle we analysed about 10 years ago. It has more functions, it’s the waste cleaner in the cells. So it recognises denatured proteins and our cells are full of them. Makes peptides out of them and if this substrate is from a virus, if it is virally infected cell for instance then the foreign nature of these peptides is recognised and they are transported to the cell surface in complex with MHC class 1 molecules and trigger the immune response. So there is no, I would say higher life without proteasome, extremely important. The more surprising, it’s a huge molecule as I said, consisting of 28 subunits. The surprising thing was the American company discovered that the proteasome is a cancer target for certain types of leukaemia. They found that this boronic acid compound, so a short tripeptide with a boronic acid is useful as treatment for some forms of leukaemia. Now it was clear and we did the crystallography of that and this binds to the proteasome, inhibits the proteasome. And of course here we did this, we helped in this structured based design and development by looking at the structure of this compound, how it binds and how it may be modified. Now this finding and the success on the market of this compound caused an enormously intense search of other proteasome inhibitors, but also by the simple fact that bortezomid, as important as it may be has terrible side effects. So neuropathies which are quite severe, so people now screened libraries, in particular natural compound libraries for other proteasome inhibitors which may be less toxic. Now in all cases, almost all cases seem to us to analyse the quite fantastic chemistries which we saw in these. Now what you see in yellow is natural compounds. You see their very diverse nature. What you see highlighted in red is the pharmacophore, no time to say that the essential catalytic group in the proteasome is an n-terminal threonine as we found by the structural studies. Now here you do have an aldehyde and aldehydes make hemiacetals with hydroxyl groups, this is what we found. Now this is a beta lactone, various forms of it, this is a beta epoxy ketone, nature is enormously full of fantasy in making this exotic chemical entities. Now this also, it’s not on this slide, now this reacts with the n-terminal threonine with both the hydroxyl group which adds to the carbonyl carbon and the amino group which opens the epoxide ring and forms a morpholino adduct. So making this pharmacophore absolutely essential for an n-terminal threonine. Nature is wonderful to do that. But these are the kind of experiments that we did, showing the electron density of these ligands and the mechanistic interpretation of that. This is one of the last examples which, work that we did together with a plant biologist, Dudler in Zürich. Now showing the, again the fantasy of nature. So there is a plant pathogen which kills in plants, pseudomonas syringae. And as these biologists found kills in plants by secreting virulence factor, without the virulence factor the pathogen is harmless. The virulence factor is this and we found out that it inhibits the proteasome. It’s a complicated cyclic compound with a side group and as expected from the chemistry so the n-terminal threonine is added to a Michael-Addition to this double bond. This is the corresponding electron density of it. This is the functional interpretation. So what this virulence factor does, it inhibits the cell cycle by the accumulation of cyclin B, polyubiquitin aggregates. And so drives these plant cells into apoptosis. Well the last example and well there is more behind the proteasome so I spoke about the proteasome cancer target and perhaps other diseases but it is also a target for developing antibiotical and there are some quite exciting, recent publications of the role of the proteasome micro bacteria as a possible drug target or here another inhibitor against micro bacteria. Now the point here is that one can design and make ligands that inhibit the micro bacteria proteasome and are specific for that and do not touch the human proteasome. So this is a prerequisite of course for an antibiotic, to kill the pathogens and not the patient. Well this is another example of how we use structure information for drug design. This is an enzyme called furin and it is essential for human physiology because it is necessary for activating prohormones. Now what this enzyme does is it is a protease, it cleaves a polybasic site and only the cleaved product is then an active hormone. Now this physiological reaction is misused by pathogenic bacteria and viruses. They make use of this human enzyme to activate toxins, so anthrax toxin need a cleavage by furin at a polybasic site to become active. And so you have diphtheria toxin needs that cleavage. There are viral proteins, co-proteins which need processing of (00.31.10 inaudible). So there is of course the immediate idea of using furin as an antibiotica target but on the other hand there is the danger of toxicity because furin is also necessary for activating essential hormones. So we didn’t care much about that. We looked at the structure, designed and then helped to make a ligand. Now the ligand is polybasic as you can imagine, it cleaves a polybasic site so we colour the binding site according to the electrostatic potential, it’s deeply red. It’s full of aspartates and glutamates. And then our American colleagues started some clinical, well animal experiments to find out that mice are protected from pseudomonas aeruginosa exotoxin. They are rescued, they die after 2 days and they survive with this furin inhibitor. So there is some hope although obviously not much progress, this is work from 2002 and obviously there were problems with the human and clinical tests. A further example from relatively recent work which I would like to tell you, this is work on enzymes that are responsible for heme and chlorophyll biosynthesis. Heme and chlorophyll biosynthesis do have a common pathway. Up to this step the synthesis of this protoporphyrinogen IX and then comes an oxidation to make protoporphyrin. And then 2 branches leading to heme and to chlorophyll are separate. Now this enzyme, the protoporphyrinogen oxidase, both very interesting for us. For 2 reasons, there is a hereditary disease in some patients causing these skin lesions. So there is an accumulation of protoporphyrinogen and protoporphyrin and makes the skin very light sensitive. And the reason for that is that protoporphyrinogen oxidase is not working properly because of a sequence change in the molecule. More important is the fact that in the plant protoporphyrinogen oxidase is a target for a rather important class of herbicides. Now what these herbicides do is quite similar to what we see in human skin of these patients. Photosensitivity and the dying of these wheat plants. So we looked at the structure of, in this case of the plant enzyme as well, the binding of the cofactors and the binding of herbicide. We understood the function from the structure. Now of course on that basis we can help further development of the herbicide, so perhaps making it even better binding so that we need a lesser, smaller amount of herbicide in the field. So this is one avenue that companies are following. And a particularly interesting thing was a relatively novel finding of resistance developing against this herbicide. And the resistance, I’m sorry some of you maybe able to see, this is one of the cofactors, this is a herbicide and these herbicide resistant plants do have, weak plants do have a deletion of this glycine 178, which is part of the binding site of the herbicide. So when this is deleted there is a re-arrangement of this finding, a substantial re-arrangement of that, so that the herbicide can no longer bind. But as we can model the structure of this deletion, this glycine 178 deletion, we can give the chemist some advice, how to change the herbicide. So this is use of structural information in the crop science field. Well now I come, I have no time left I think. Well I make that very short and then on this company foundation thing, that you might wish to hear. This is just a few slides to have a summery and then we have time afterwards to elaborate if you are interested, a little more in detail. So the first company that I helped to found, more than 10 years ago was a company that is providing a platform, using the techniques that I have described, of structural biology and which I have helped to develop since the late ‘60’s, early ‘70’s. So they offer this to customers. So a customer comes and Bayer, Pfizer, Johnson and Johnson, these are their customers and they have discovered an interesting drug target and they often have found a ligand which needs development and in order to do that they need structural information. And they buy this structural information from Proteros, Proteros gets paid for it and then makes a living on it and developed substantially in these last 10 years. So what they do is they provide the gene protein, protein structure, protein ligand information. They have the technologies, protein production, structural analysis and ligand structure information. They usually, they have gained enormous expertise in this, are known for that, can live on this system. Interesting work, the scientists that are doing, and its often novel structures. The only problem is that they are not allowed to publish. You see they sell the structure information and that’s it. Well there are some arrangements can be made with the customers because sometimes they are all also interested in having a good publication as a kind of advertisement. But usually they are not allowed to publish. So this, I have much more to say about this company but this we can do after. But this is the second company which is more typical of biotech companies, suggesting a new avenue of, new strategy for autoimmune diseases. And it goes back to academic work, my own work which was on antibody structures in the middle ‘70’s, so we analysed at that time the first antibody structure, the FC part, the FAB part. And followed that work by looking at, I should go back. Now I have to tell you that when an antibody binds to an antigen, a bacterial, a virus, then the immune response is triggered by binding off this complex to FC receptors. And what we wanted to know and we started work on that about 8 years ago, on FC receptors and the FC receptor antigen or FC complex. So we were successful in crystallising that, doing the crystal structure of it. This is the FC part, part of the antibody, this is the soluble part of the receptor. You see it’s a very asymmetric finding that we find here. And then we thought we should make use of that, that is the people who worked on these structures, thought this is a possibility to interfere with the cellular immune response. And that was then the basis of the foundation of the company. The idea is very simple, so we use, they use the soluble FC receptor as an antagonist. Which then interferes of course if an antigen antibody complex has soluble FC receptors bound, it’s no longer able to interact with the cellular FC receptors. And this then was developed with animal models, quite successfully in various autoimmune diseases. And then the problem started to find money for the first clinical tests and further development. This is also a topic I would like to tell you, if you’re interested, but that we can do after the formal lecture. Thank you.

Robert Huber (2010)

Basic Science and Co-entrepreneurship, my Experience (Lecture + Discussion)

Robert Huber (2010)

Basic Science and Co-entrepreneurship, my Experience (Lecture + Discussion)

Abstract

The design and development of inhibiting (or occasionally activating) ligands of target proteins in medicine and crop protection guided by molecular structures and functions has become an established technology in academia and industry recently.
In my lecture I will present examples from the protein groups of proteolytic enzymes and antibodies describing research background gathered in academia (http://www.biochem.mpg.de/eg/huber/publications ) and translation into industry, a process which I attended as co-founder and scientific advisor with the companies Proteros (http://www.proteros.de) and Suppremol (http://www.suppremol.com).

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