Robert Huber (2015) - Structural Aspects of Protease Control in Health and Disease and my Experience with Translation into Practice and Business

If one speaks of structural aspects, then of course, the question is, how do we determine structure? So let me show a few slides on the history of Structural Chemistry, and Structural Biology, and how we learn to see atoms, and molecules. It goes back almost exactly 100 years, with the discovery of x-ray diffraction, by crystals, by Max von Laue, who worked in Munich. We commemorated this event worldwide, the Laue Centennial and then in 2014, the year of crystallography. This is so to say the birth document of Structural Chemistry and Structural Biology. A diffuse, I would say even ugly, diffraction photo of a crystal. This is Laue's publication in 1912, in the Berichte der Bayerischen Akademie der Wissenschaften, a journal few would read, I would say. But the Nobel Committees, their members read it and they awarded Laue the Nobel Prize two years later. This is Laue, at one of the first meetings in Lindau, sharing to Count Bernadotte a crystal lattice. Foundation of x-ray diffraction. This is Roentgen, this is Laue, they both worked in the Munich University, side by side. Laue a professor of Theoretical Physics, Roentgen professor of Experimental Physics. These are their instruments that you can see in Munich in the Deutsches Museum. So instruments that I would say, changed the world of natural sciences. The experiment of Laue was, became known quickly, to the, physical and chemical community. And became known to father and son Bragg who worked in Cambridge, England. And they immediately grasped the importance of Laue's discovery as a structure determination tool of first simple crystals, and then more complex ones, in determining the structure of the molecules making up those crystals. The Braggs founded a school, which became the birthplace of Molecular Biology. Max Perutz worked there, he's the father of protein crystallography, because he found a way to decipher the complex diffraction patterns of proteins. Founding fathers of Molecular Biology and Structural Biology. Growth of biological crystallography. A slow start in the early 60s, by the first structures, myoglobin and haemoglobin, Kendrew and Perutz in Cambridge, England. And then from 1990 on, we saw an exponential growth simply because of the technological advances of fast detectors, x-ray detectors, powerful x-ray sources, fast computers, and of course then, the applications in medicine and biotechnology, in which I would like to focus right now. Hartmut Michel spoke about a similar exponential increase in our knowledge in membrane protein structures. The first one was determined in 1985, the photosynthetic reaction center, which led to a Nobel Prize to Deisenhofer, Hartmut Michel and myself. This is a picture of my institute, when it was inaugurated. And I moved in in 1972. An institute in the forest, and some called it, not Martinsried but Martinsruh, it was a peaceful place, these are the outskirts of Munich. It was scientifically not peaceful. There was a group of prominent scientists there, two Nobel Laureates, this is Adolf Butenandt who was chemistry Nobel Laureate for his work on human sexual hormones. This is Feodor Lynen, physiology Laureate for his work on fatty acid synthesis, and there is a very young guy sitting on the edge, this is Pehr Edman, by the way. All of you know about Edman degradation. He unfortunately died prematurely. Now this is the campus in Martinsried today. This was the first institute building that I showed to you. A second Max Planck Institute was added, and here is the number three, which is the Innovation Center. I come back to that, it's an incubator for small companies, and many university institutions around. Now, how do we use, for biotechnology and drug design, ligand design, protein structure information? It's very simple in principle. I have chosen thrombin, which is still an important target for cardiovascular diseases. So we first look at the apoprotein. We look at the substrate binding site, and then we perhaps let our chemical fantasy work, that is, we look at the stereochemistry, the shape of the binding site, the electrostatics, hydrogen bonding. And we design a small molecule which has to be synthesized and then tested of course. This is a circular process, improving, changing and improving, the ligand and testing. Well we need, and do have programs that help us in screening the target protein surface, according to the finding of binding pockets, their shape and their electrostatic hydrogen bonding properties. And this is actually very much necessary, if we search for small molecules, for a protein target. The chemical space is incredibly large. There is no way to synthesize all these molecules, or to screen them against a certain target. So we have to use modeling, to design, and perhaps synthesize focused libraries. This is an important aspect of drug design, right now. We can use nature to help us. Again, I'm focusing on one target, that is thrombin, which you see here in blue. We analyze the structures of some extremely powerful natural anticoagulants in the tick. In the tick here, in the leach here, and in a blood-sucking bug. They produce, they are bags full of coagulant, protein inhibitors, very powerful ones. Some of them are actually used in the clinic, hiridun, for instance, with some problems, because it is so powerful, that it shuts coagulation completely down, which is unwanted. This can be used to make a chemical chimera just using the C-terminal tail, and adding a small side group that binds them to the active site. So we can use nature as a guide for designing our library. Now I wanted to tell you about structural, the protease control, as it has been found by structural and functional studies. Hundreds of such natural inhibitors have been discovered, analyzed, and I'll just briefly go through this, simplified, a cartoon to show you what regulatory mechanisms have been found. So, a very simple one is, we have the enzyme, and there is a proteinaceous inhibitor that has a shape that is exactly complimentary to the protease. It exactly fits, is very specific and, usually, and the sual of such proteinaceous inhibitor is, by the way, substantially larger than sual of proteases. So nature takes great care in regulating protease activity. Some proteases are extremely specific. By having a substrate binding site, with a large number of sub-pockets, so it recognizes a long peptide chain very specifically. We do have a regulation by proenzyme activation, so most of the proteases are made in an inactive form, and they need activation by often limited proteolysis. And what happens here is that a part is cleaved off, and then the active site, the substrate binding sites, becomes accessible, but there are also cases where cleavage of the pro part then leads to an allosteric change. Some proteases are membrane bound, membrane anchored, to limit their action on the vessel surface. There are cofactors, that change the activity of an enzyme profoundly, because they offer an additional binding site for the substrates. So without that, the enzyme would be inactive. And there are cofactors that cause an allosteric change of the enzyme, and make it active, or inactivate. And there is a rather new activation process that we found when we looked at an ubiquitously present protein family, that of the dysk, or HTRs, high-temperature requirement proteases. They do have an enzyme pod, trypsin-like enzyme pod, and a PDZ domain. And substrate binding then leads that binding to the PDZ domain, leads to an allosteric change in the enzyme, and activation. And that was very new and very exciting to an aggregation, to an oligomerization, though the enzyme forms a shell around the substrate. And that is the proteasome on which I would like to focus briefly. So the proteasome showed us a new regulatory mechanism in this sense, that it forms a cage, and the active sites are inside the cage. And the entry into the cage is closed usually. And by opening the entry ports, then the enzyme becomes active. Well, you will see in a minute, that I, we wanted to translate the structural information that we had on the proteasome, into application. And we needed help. We needed help, and the help was given by an institution that was founded by the Max Planck Society called Lead Discovery Center, which brings a project, at the beginning stage, to a stage where it becomes interesting for big pharma. So this is the proteasome which I had suggested, to the Lead Discovery Center for further development. First we analyzed an archaeal enzyme, which showed the basic architecture, these four rings, forming a cylindrical, barrel-like. The active sites are inside here, and then a few years later, the eukaryotic yeast, and then again, mouse and human. Now the major difference between the simple archaeal, and the developed and evolved eukaryotic one is that all the subunits here are different. They are related, but they are different, generating specificity and higher activity. So this was the project I suggested to the Lead Discovery Center, an explanatory slide for the proteasome function. I mean, you have heard, or you will hear from Aaron Ciechanover and from Hershko about the discovery of the ubiquitin conjugation system, which labels proteins that ought to be degraded with a polyubiquitin chain. Now the execution of this system is the proteasome that we stucturally analyzed. In addition, it is the waste cleaner. The proteasome removes unfolded, non-functional, polypeptides from the cell, and it is essentially in the immune response. Because if this is of a vital, pathogenic origin, then the resulting peptides, resulting from proteasomal cleavage, are antigenic and they trigger the T-cell immune response. So a quite essential molecule. The more surprising it was, that an American pharma company, Millennium, had found, it was a serendipitous finding, that this little, small peptide like boronic acid, offers a new strategy against blood cancer, multiple myeloma. Big business, it became in no time. And stimulated the search for other proteasome inhibitors. I forgot to say that the bortezomib, this is the product label. Then is an inhibitor of the proteasome, so an intense search started. And many of the compounds that were found worldwide were sent to us to analyze them, about their binding to the proteasome. And this is a very small collection. And what you see here in yellow is what has been found in natural compound libraries. What fantastic compounds. They all have a warhead, a head group, that covalently binds to the N-terminal threonine. And no time to discuss the enzymatic mechanism of the proteasome, but it needs an N-terminal threonine as the active site residue. They all label that in different ways. Well this is an example of a discovery of a new chemical entity, a plant pathogen, Pseudomonas syringae. Needs a virulence factor, which our colleagues, plant biological colleagues, had been isolated. And we had found that what it does, it inhibits the plant proteasome, leads to accumulation of polyubiquitinated cyclin B1. This is the, molecular structure bound to the proteasome, bound to the N-terminal threonine. Well, so, the question for us was, and for the Lead Discovery Center, whether we should continue, with the proteasome, ligand development in view of the enormous activity worldwide. Can we compete for them? We thought we cannot, but we can find a niche. And the niche was, provided by the fact that, immune cells, upon an immune stimulation, express a different kind of proteasome, which is called immunoproteasome. It's very, very similar, but it has a somewhat different specificity. It makes more of the MHC class I ligand. In addition, the inhibition of the constitutive proteasome, so I mentioned bortezomib, is, involving, serious neuropathic toxicity. So there was a need for something else, and we focused on the immune proteasome. Again, there was structural information of mouse and now it's in the human case, so we could apply the tools of Structural Biology, to develop an immunoproteasome-specific inhibitor. So it would not only avoid the neuropathicity of the constitutive proteasome inhibition, but it would also offer a new strategy against autoimmune diseases. Well this is a, a, busy slide. And I would like to, skip that, but it is a summary of 50 or more, structural studies on proteasome, ligand complexes, whereby we found, that, by using a tool that is called Principal Component Analysis, that these structures cluster into two different, make two different clusters, the red cluster and the black cluster. And the black cluster is the structures of the apoenzyme and non-peptidic ligands, while the red cluster is the structure with peptidic ligands. With peptidic ligands, I mean, a peptide bound that exhibits four hydrogen bonds, exactly four. And if that is the case, then the constitutive proteasome, closes upon the bound peptitic ligand. And the exciting observation was that the immunoproteasome does not. The immunoproteasome is already in the apo state, in the preferred orientation, preferred structure, of the peptidic, peptide-bound complex. So that immediately explains why the immunoproteasome is faster, than the constitutive proteasome. That was then a point where big pharma became interested, which was actually the goal of the collaboration between me and the Lead Discovery Center. So, what I told you is, translation, either by approaching big pharma directly as an academic research institute, or by having a mediator, like this Lead Discovery Center, of the Max Planck Society, or, by becoming an entrepreneur yourself. Now, this is not what I wanted to do, but I found people, that wanted to go into this direction. And so, we founded in 1999, a company called Proteros. What does it do? It offers enabling technological services, that is the process which I just described, using structures, assays, protein production, to develop, to develop new drugs. And they work and integrate Lead Discovery. Of course at first it was a very small group. They had a home in the innovation center that I described before, in the middle of the campus. It has grown substantially. It has grown profitably now, to 70 people, and does not need an investor. This becomes important for what I am going to say, with the next company. Now this is what they offer, and what they sell. And they make a living on it. An interesting aspect is, because they have customers all around the world. And what this list shows to you, is, what they have is gallery structure. These are the structures, where the processes of protein preparation, crystallization, structure analysis, have been defined. They have crystals in the fridge, so if a customer comes and wants the structure of his compound for CDK2, he can get an answer in a few weeks. So these are shelf gallery structures. The interesting thing in that, it is interesting after, Eddie's talk, is that the majority of targets, are the kinases, many of them. The second large group is proteases, and some others. So the importance of kinases is very obvious. It's customer-driven, it's pharma, global pharma research driven. Well this I'll skip. And I come to the second company. Which we founded, again, in 2003, and again they were located, are still located, in this innovation center. So the importance of, with such incubators, cannot be overestimated. Well the text says here, was founded 2003, and it made investors big, lawyers and broker and founders happy, in 2015. So let me very briefly show to you the story, which goes back 40 years, actually. When we, in the middle 70s, worked out the first antibody, structures, you know this, Y-shaped molecule, antigens binding on the tips of these arms. There is a hinge area, these are the so-called FAB arms. There is a hinge area, connecting the stem part. And there is a glycopart, which is also very important. It was actually at that time the first structurally-defined glycoprotein. It was quite unexpected that the carbohydrate has structure, and has structural function. So, this is the mediator of the cellular immune response and the humoral immune response. And we were interested in the cellular immune response, but it took us almost 25 years until we came to a, a material that we could crystallize, and analyze. Now what are these FC receptors? So this would be a bacteria, a pathogen, covered with antibodies. And these antibodies with the FC part, bind to the FC receptors, and trigger the immune response. So, leading to inflammation, for instance. So it is a process that must be tightly controlled. Otherwise, there is, so it must be controlled by inhibitory receptors, and activating receptors. And the balance between these two, is essential. So what we did, was analyzing, the receptor itself, and then its complex, with the stem part. The FAB is unimportant for this, the arm part is unimportant for this kind of interaction. A novel structure, and here you see the, the interaction with the carbohydrate. So again we thought, when we saw this structure prominently published, can we make use of it? And we, considered several possibilities. So what we would like to do is to modulate the immune response, for instance in autoimmune diseases, by inhibiting the interaction of an opsonized antigen, with the FC receptor. And we thought a possibility would be to have a soluble receptor. So the picture is simple. This is the antigen, this one's the antibody, and there is in, there is the soluble receptor. Of course this then no longer can bind to the cell-bound receptor. So this is a scheme that shows what is happening. Well this was, then, the time already, and a project that is outside of what an academic institute can do, and we thought about founding a company, which we did. First a very small, small group of people that we could do, pre-clinical experiments with mouse and rat models on prominent autoimmune diseases, rheumatoid arthritis, multiple sclerosis, and lupus. And this was then advertised, and investors had been found, which began to finance, the more, extensive clinical studies, which are very expensive, I should say. So it went to phase one and phase two, which were successful. And then, of course, in different steps of financiation. There was a financiation around A, then came B, then C, then D, and the E. And with the E there was little or almost nothing left, for the initial founders. But that does not matter. What matters is this is, the company was sold to Baxter for an enormous sum, and for me, most important was, that Baxter continues to run the company's operation, so the team will stay together, the project will continue. So the happy investors are those major shareholders, a whole collection of funds, including the Max Planck Society, I'm happy about that. A happy end, for the company, I think, and I would like to thank you. But show this last slide, this is the old University of Munich. So this is where Laue and Roentgen worked a hundred years ago, and did the first experiments that led to, Structural Biology. Thank you.

Robert Huber (2015)

Structural Aspects of Protease Control in Health and Disease and my Experience with Translation into Practice and Business

Robert Huber (2015)

Structural Aspects of Protease Control in Health and Disease and my Experience with Translation into Practice and Business

Abstract

As a student in the early nineteen sixties, I had the privilege to attend winter seminars organized by my mentor, W. Hoppe, and by M. Perutz, which took place in a small guesthouse in the Bavarian-Austrian Alps. The entire community of a handful of protein crystallographers assembled in a room which served as living and dining room and as auditorium for the lectures.
Today structural biologists organize large congresses with thousands of attendants and there exist many hundreds of laboratories specialized in this field. It appears to dominate biology and biochemistry very visibly.
Structural biology was successful, because it was recognized that understanding biological phenomena at the molecular and atomic level requires seeing those molecules.
Structural biology revealed the structure of genes and their basic mechanism of regulation, the mechanism of enzymes’ function, the structural basis of immune diversity, the mechanisms of energy production in cells by photosynthesis and its conversion into energy-rich chemical compounds and organic material, the mechanism that makes muscle work, the architecture of viruses and multi-enzyme complexes, and many more.
New methods had an essential impact on the development of structural biology. Methods seemed to become available in cadence with the growing complexity of the problems and newly discovered methods brought biological problems within reach for researchers, a co-evolutionary process of the development of methods and answerable problems.
An important additional incentive for structural biology came from its potential application for drug design and development by the use of knowledge of drug receptors at the atomic level. The commercial interest in application spurred this direction of research enormously.

My lecture will start out with a very brief review of the history of protein crystallography and continue with our studies since 1970 on proteolytic enzymes and their control. Proteolytic enzymes catalyse a very simple chemical reaction, the hydrolytic cleavage of a peptide bond. Nevertheless they constitute a most diverse and numerous lineage of proteins. The reason lies in their role as components of many regulatory physiological cascades in all organisms. To serve this purpose and to avoid unwanted destructive action, proteolytic activity must be strictly controlled.
Control is based on different mechanisms which I will discuss and illustrate with examples of systems and structures determined in my laboratory:

a) by specific inhibition with natural and synthetic inhibitors
b) by enzymatic specificity
c) by activation from inactive precursors accompanied or not by allosteric changes
d) by co-localization of enzyme and substrate
e) by cofactor binding accompanied or not by allosteric changes
f) by controlled access to the active site
g) by substrate-induced allosteric changes of the active site associated with oligomerization

The regulatory principles offer new opportunities of intervention for therapeutic purposes and use in crop science.

I then will let you share my experience with the foundation and development of two biotech companies with different business models, but both based on basic academic research in structural biology:
Proteros (www.Proteros.com) offers enabling technology services for Pharma- and Crop science companies imbedding all steps of the workflow molecular and structural biology can provide and commands and uses its platform for the generation of leads from identified targets to in vivo Proof of Concept (PoC).
Suppremol (www.Suppremol.com) specializes in the development of novel immune-regulatory therapeutics for the treatment of autoimmune diseases on the basis of a recombinant, soluble, non-glycosylated version of the human Fcg receptor IIB and of receptor binding antibodies.
Suppremol was recently acquired by Baxter International Inc. (NYSE:BAX) offering an ideal setting for its therapeutic projects.

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