Proteases for Drug Design and Development, My Experience
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Transcript: English(auto-generated)
00:14
Welcome to the Agora Talk,
00:23
where young physicists, you, get to meet structural biology. My name is Tobias Meyer. I'm the deputy director of the National Institute for Science Communication here in Karlsruhe in Germany. And the laureate who is going to speak to you now is Robert Huber.
00:45
He was awarded the Nobel Prize in 1998 for structural biology, crystallography, discovering what proteins actually look like and how their function can be understood.
01:01
Robert is still active. He's in Martin's lead, close to München, running a research group there still. After a very long career, he founded two pharma companies, two biotech companies also, and we might get into that aspect too. With no further ado, I give the stage to Robert for his talk.
01:33
Well, thank you so much. Do you understand me? It's a pleasure for me to be here and to see so many young faces,
01:45
most of you from the field of physics, although my lecture is biochemistry, pharmacology, medicine. But most of the experiments are based on physics, as you will see.
02:12
Well, this is Max Perutz, the father of protein crystallography, Cambridge Ingen, and this is what he wrote to his...
02:21
Can you see it? A little too much lighter, I think, maybe. So this is what he wrote to his family in the late 50s, early 60s, when he was excited about seeing the first protein structure.
02:40
What a time I'm having, he wrote. Now this is the century of vision that we have now, because we learned in the last few decades to use not only X-rays, as Max Perutz did, but to use a very large spectrum
03:01
of electromagnetic radiation from X-rays to radio waves. And you were lucky to hear during this Lindau Conference on Physics, many of the founders of these different techniques speaking.
03:24
So we learned to see small organic molecules, and we learned to see eukaryotic large cells by applying these various new technologies.
03:41
This is a different view of the same story, the century of vision, I would like to call, adding chemistry for tract design. So that was not right.
04:04
Now what I would like to do, just to remind you on what you have heard during these days of the conference, just show a few slides. For instance, the super resolution fluorescence microscopy, and this is the statement of the Stockholm Committee
04:26
about the Nobel Prize to Betsy, Helen and Myrna, breaking our base diffraction limit. Now our base diffraction limit means that with a conventional optical microscope, you cannot resolve details closer together
04:43
than half of the wavelengths, which is about 250 nanometer. And of course, not sufficient to resolve atomic details. And what these three researchers discovered, that there is a way navigating around our base diffraction
05:03
limit by finding a way that only one of these chromophores, these are eight chromophores in this object, is emitting light and then collecting thousands and hundred thousands of images
05:22
and then combining them, and so get a super resolution image. This is electron microscopy again. You heard from Joachim Frank about the development there. These are huge machines which, of course,
05:42
were developed because of new technologies, detector technologies, for instance. Now Frank was the person who took care of the mathematical background that led,
06:02
as the Stockholm Committee stated, developing cryo-electron microscopy for the high resolution structure determination of biomolecules in solution. Now this is one of the examples that we see now of cryo-EM images
06:22
of a large protein complex. And I show this. This is a structure by my colleague Wolfgang Baumeister in Martin's read. I show this because it shows the interplay between X-ray crystallography and cryo-EM.
06:41
Now we had determined in the middle 90s the structure of the core component of this large particle consisting of 28 subunits at high resolution, at two angstrom resolution by X-ray crystallography. But we were unable to crystallize the whole complex.
07:01
But by EM, you can visualize it. And at moderate resolution, so the combination of high resolution structures of fragments and the hollow complex then at low resolution by electron microscopy is, in my mind, the way to go.
07:23
NMR and radio waves and modeling. Now the structure that I show you is related to the topic of my talk about proteases and their regulation. It is the basic pancreatic trypsin inhibitor, which was the model for developing high resolution
07:46
nuclear magnetic resonance spectroscopy. Kurt Wüthrich, you heard yesterday. And it also served as the model for molecular dynamics. Again, a Nobel Prize to Karpus, Leavitt, and Warshall.
08:02
And they used the BPTI as their model to do the initial calculations. Michael Cole did the lab red of theoretical chemistry. Well, we did the high resolution crystallography,
08:23
developed refinement at that time. And this is the model as we see it. The advantage of this material was that it forms very big crystals, almost half a centimeter. So we could not only do X-ray crystallography,
08:42
but we could do NMR. We could do neutron crystallography. And this was actually the first neutron crystallography structure of BPTI of any protein, by the way. And this allows one to determine the proton deuterium
09:06
exchange rate in crystals. And this then we could compare with the exchange rate seen in solution by NMR, and one finds complete correspondence.
09:21
So what they see by NMR in solution, we see also by neutron crystallography in the crystalline state. Also, crystallography and NMR. Now, for a while in the 80s, I would say, people were skeptical about the potential of NMR
09:44
to determine unbiased protein structures. So Kurt and I then started a project where we began to work with our techniques, that is X-ray crystallography and NMR at the same time,
10:02
and without any communication, then publish. And what you see here is an overlay of the chain trace of these two. So quite clearly, there is complete correspondence. And it was quite important for the scientific community showing that by NMR, you can determine
10:22
the reliable protein structures. So we've seen these beautiful crystals that you've just seen, half a centimeter long. And then we've seen the overlaid structure of NMR results and crystallography results. How do you get from having such a crystal
10:42
to the actual drawing of the chain that we've just seen? Well, this is the next part of my story. I've now come to two X-ray crystallography. It's history. It's my field, and it is the field with which protein structure research started.
11:02
Not only protein structure research, but structural chemistry by itself. This is the person, Max von Laue, presenting or showing to Count Bernadotte, the founder of the Lindau meetings, at the third Nobel Laureate meeting in physics in 1953.
11:28
He showed him a crystal lattice. Now, Laue had discovered that X-rays are diffracted by crystals.
11:42
And what you see in the middle of this is the birth document of structural chemistry. Ugly, I would say. But it showed that first, X-rays are electromagnetic radiation, and second, that crystals are ordered lattices.
12:04
So really, the birth document, hand signed by Max von Laue, I should say, 1912. This is the publication that he wrote in the Berichte de Baerischen Akademie der Wissenschaften. I think nobody in the audience has ever seen that journal.
12:24
It still exists. Nobody reads. But anyway, it led, after the publication in 1912, two years later, 1914, to the Nobel Prize. So it's not the impact factor that counts.
12:43
Now, these are the fathers of crystallography, in protein crystallography, Roentgen and Laue. These are the original machines that you see on display in Munich. You see, in the Deutschen Museum, you're just 150 kilometers away.
13:00
If you find some free time, then go there. It's fascinating, because these people and with their instruments change, the revolutionized the science and technology. This is a son, Brecht, Lawrence Brecht, who the father and son, Brecht,
13:21
they worked in Cambridge, England. And they had heard of Laue's experiments, repeated it, and did the right thing. They found out that it can be used for structure determination, a simple crystal sodium chloride. And then they built up a laboratory
13:40
where Max Perutz were the father of protein crystallography. Now, there is an enormous progress in the field of protein crystallography, because we learned to make and to use recombinant proteins. And because we had available, since two decades or so,
14:01
the enormously powerful X-ray machines of the synchronous ones, which, of course, were built by the physicists for their purposes. And in a sense, they thought we, by making use of the X-rays that are generated during the operation, we misuse them.
14:23
So we were allowed to use them in parasitic times, when they were sleeping or having lunch or whatever. This is Roentgen's original X-ray generator, with which he discovered the X-rays.
14:45
Now, these were the models that we built of proteins in the 1960s and 1970s, from screws and wires, because there were
15:00
no graphic systems available. That came later. I had a postdoc, Alvin Johns, who developed the first interactive graphic system for protein crystallography. We learned to use the variable wavelengths
15:27
of the X-rays that are generated in a synchodron. And we use the anomalous dispersion at the anomalous absorption edge of heavy atoms
15:41
that might be in a protein, metalloproteins, for instance, but also artificially introduced heavy atoms like selenomethione and replacing the sulfur by selenium Z is possible with modern biochemistry gene technology.
16:03
We learned to improve the quality of the electron density. This is an initial electron density that we get by isomorphous replacement. This is the method that Max Perutz discovered to solve the phase problem.
16:20
And you see already polypeptide chains. And if you have some experience, you can build in a model. But it's not really nice. Now, what we learned is we made use of the fact that we do know how an amino acid looks
16:41
like in great detail by small molecule crystallography or other techniques. And this is information that we can put in, improve the phases, and get an electron density of that quality that is called refinement.
17:01
We can also use X-rays in order to look at molecules, at protein molecules, a solution that's small angle X-ray scattering. You do not need crystals in that case. Now, the problem that we had here was this is a tetramer of linked actins,
17:27
beautiful crystals. So we had a high resolution crystal structure. However, the linker between these four actins was invisible in the electron density
17:41
because it was flexible. So the question was, how does, in solution, that tetrameric molecule look like? And there were three alternatives. So a compact, a semi-compact, semi-extended state, and a fully extended state. And this we can then compare with,
18:01
we can calculate the solution scattering from these three models. And clearly, we find this is the solution structure now at high resolution because we have high resolution crystal structures. The growth of biological crystallography,
18:21
this slow growth from 1960 on up to 1990, so slow growth because the techniques were not available. And then the exponential growth because
18:41
of the technological advances, so these strong X-ray sources, the fast detectors, the computers. Also, it became clear that in order to understand biology, we have to see the molecules. There is no way around. And the applications in medicine and pharmacology,
19:02
which is a great driving force right now. Thousands, hundred thousands of protein structures are determined in industry and hidden in the drawers because they would not like to disclose what they have found
19:21
as a molecular structure. You mentioned the technological advances. And now I remember that Laue and other Max Perutz, I believe, were physicists by training. Now, you're a chemist originally. Yes, thank you. Thank you for mentioning that. Now, both Roentgen and Laue were physicists.
19:41
Now, the Brex, they had a chemical touch, I would say. I think they were chemists. And Max Perutz was a chemist. So the foundation is physics. But then was taken over by the users, by the chemists and the biologists.
20:01
But those technological advances, were they done by chemists? Or who were the people that invented those machines that drove the technology forward? Chemists just make use of it. Crystallographers make use of what the physicists had built initially for their purpose.
20:20
They needed for their particle physics experiments, they needed the detectors. But we made use of it. And now, synchrotrons are built for our purposes as machines to generate x-rays.
20:46
Well, now I come to what I have announced with my title, proteases. This is the life cycle of proteins. So it's protein synthesis. You heard Ada Yonath speaking about the ribosomes.
21:04
So proteins are made, and proteins are folded. This is work that our moderator did in Martin's read. So many proteins fold up from an extended polypeptide chain
21:24
that emanates from the ribosome spontaneously. But many others need help. This is called protein folding, and there are specialized proteins that help protein folding. And there is protein degradation.
21:42
So proteins are sensitive materials. They become damaged under some environmental conditions. And then they have to be removed. Otherwise, cells would die, waste removal. But there is more to it.
22:00
And it's a rather simple chemical reaction. There's a peptide bond, which is hydrolyzed. And you know if you boil proteins in acid or base, then the protein is cleaved into single amino acids,
22:23
a very simple chemical process. But we do have about 600 different proteolytic enzymes in the higher organisms. So does that make sense?
22:41
I mean, of course, we need proteases for digesting proteinaceous meals. It's quite good. But we have many, many more that has to do with the fact that proteolytic activity, limited proteolysis, is a major regulatory mechanism
23:03
in cells. And this was the story we worked on since 1970, starting with this basic pancreatic trypsin inhibitor, now to the very big proteases, which is the proteasome. So how did structural biology help
23:21
resolving those mechanistic details of how proteins get degraded? Well, it helps us to see the molecules. And without seeing, there is no way to understand it. So with photosynthesis, that was the work for which Hartmut and Hans and I got the Nobel Prize. It was given for the fact that we produced
23:45
a high resolution picture of the biological photocell. And so it is with all the proteins that we would like to understand in their function, but also in disease process, we have to see them.
24:02
And then in the case of protease regulation to develop inhibitors or activators. So by visualizing it, you can see where those inhibitors or activators would dock and how those protein particles would interact with each other.
24:21
Well, I do have a large number of slides. I could know how obvious with time. We can continue talking to each other maybe a little bit. Because you mentioned on a previous slide that there are applications, current applications of this now, that these compounds that you resolve by crystallography,
24:40
that they are used by pharma companies to do what, exactly? Well, to develop drugs. So many of, well, let me go through this scheme. This is a scheme that shows what kind of protease
25:03
regulation we found when we started in 1970 up to 2019. What did we find? So we looked, of course, first at the protease itself, which we isolated, crystallized.
25:23
And then often, these proteases do have natural inhibitors to control the activity. For instance, in our blood, I mean, there's a lot of hemoglobin that makes the red color. But the next material that we have in blood is antitrypsin.
25:53
So it's a trypsin inhibitor. So we are full of proteases. Now, I like to say if there were not so,
26:03
if I would stand here without protection by our natural protease inhibitors, I simply would melt away and remain as a skeleton in a short period of time. So there are natural inhibitors.
26:22
And this we studied. And there are complexes with their proteases. How does a natural inhibitor work? And that information also allows us to design and then synthesize synthetic inhibitors.
26:45
This is pharma research and pharma development. And there we helped because we produced and published freely available the structures. So they simply had to load a PDP file
27:01
and then start the design process. Well, what this cartoon shows is these various kind of inhibition mechanisms that we found. There is a simple one. Now, the green object is the protease. This is the substrate binding pocket.
27:23
Now, substrate must bind close to what is called the active site so that chemistry can go on. And this red object is a natural inhibitor. So it's perfectly designed for the protease.
27:40
It binds very, very precisely, specifically to the enzyme which no longer can process any substrate. There's no space for it. So this is the simplest regulatory mechanism that we found.
28:01
Another one is that many proteases are extremely specific. They just cleave a peptide bond in a long polypeptide chain. They recognize this long peptide chain which docks and is then precisely cleaved.
28:21
So that is specificity. And I marked it by this complex binding surface. A third regulatory mechanism is that many proteases are synthesized as proenzymes.
28:40
Now, imagine in a cell, a cell makes a protease which would be active. It would simply kill the cell by digesting the protein components there. So proteases are often made as inactive precursors,
29:01
proenzymes, which are then in a second, they are secreted then, and in a second step, then activated by limited proteolysis. So this is an inhibitory pro part which does not allow access to the active site.
29:22
It's cleaved off, the enzyme becomes free and becomes active. That we often see in all the digestive enzymes, for instance, in troops, in chymotryps, in carboxypeptidase. They are made in the pancreas. And if they would be active already
29:40
in the pancreatic cells, then impossible to think of. Which simply chew up the organ. We have colocalization. That is, there is the enzyme which has an anchor
30:00
to attach it to the membrane. So all the coagulation enzymes, thrombin, for instance, factor X and all the other factors, who have this architecture, to limit the activity to the surface of the vessel.
30:21
So they should not diffuse around and do harm to substrate that they should not attack. So localization. Then there is cofactor binding.
30:40
There are some proteases, which need this blue cofactor. Without that, they are not able to bind the substrate stably. Simply the affinity is insufficient to bind the substrate.
31:00
It's not turned over. But if there is a cofactor which binds to the enzyme and binds to the substrate, then this is a stable ternary complex and the substrate can be processed. There are other ways that the cofactor
31:22
then causes an allosteric structure change to the enzyme. It changes its shape such that it can bind substrate. And there is the protein I'm working on since the last 15 years or more, that is the proteasome I mentioned already,
31:42
which showed a new regulatory mechanism in that sense that the active sites, this is a huge protein, the active sites are buried inside. And there are entry doors which are closed.
32:02
So this molecule is actually latent. It's inactive. And there are ways to open the door and then the protein becomes active. So this is what we had found 15 years ago and are continuing. Now, all of these representatives
32:22
of all of these proteases and their regulation mechanism have importance for medicine and pharma. For instance, the proteasome, their structure led to discovery of bortezomib, which is a billion-dollar pharmacon
32:43
for some kind of blood cancer and many others. So you've showed wonderful examples now how structural biology helped to really understand the function of this class of proteases, of these different types of proteases.
33:00
I think that's just one example for how structural biology played an important role in understanding biology and understanding how we also work. Maybe we can even stop there. So only a few minutes left on the clock. And I would really invite you to ask some questions maybe while you make some questions up, while you think of some
33:21
and make your way to the microphones. I have an additional question for you. And that is, you've been obviously doing research now for quite a number of years and a lot of successful people also came out of your lab who are now professors, leading labs by themselves. Now we have here a room full of young
33:42
and upcoming scientists. What would be your sort of take on what are maybe the important characteristics that a young researcher should bring to the table in order to succeed in science? Well, I should say I was lucky to have excellent students for a long period of time.
34:05
Often students applying for a PhD work came with their project. They said, now this I'm interested in, you have the tools or the department has the tools. Can I work with you and do my PhD with you?
34:26
So I was lucky. Did I help them? I think I helped them by leaving them a lot of freedom, first even choosing their project.
34:43
Now, just an example. This is the Proteusome. Now, there were two PhD studies involved with that. One was in 1995.
35:02
This was the PhD work of Michael Grohl, who then became professor of biochemistry at the Technical University. The other one, also working on the Proteusome,
35:20
was Jan Loewy, who is now a professor and director of the LMB at the Medical Research Council in Cambridge. So obviously the Proteusome is a way for successful research and then a final career.
35:43
And so many others I had. I think if I tried to count them, there are 10 professors in Germany. In Switzerland and Austria, and 10 more abroad that did their PhD in micro.
36:03
I'm very happy about that. This is some kind of legacy that I have. So all of them did work in structural biology of,
36:21
in most cases, pharmacologically important molecules. Thank you. Yeah, I think there's a question over there. That microphone on the left. It's a more general question. So considering that we are at a physics meeting and we have several talks that combine biology,
36:40
chemistry, informatics, do you still think it's up to date to separate these topics? Or are we going to more uniform natural science in general that lives from the interconnection of different sciences? Well, this separation of the Lindau meetings in chemistry, biology, and physics
37:00
is something historical. But there are joint meetings as well, which are huge. Now, I am a chemist, but I am invited being a local. Just 150 kilometers away, I come to all of them.
37:21
Well, it's wonderful to be here, a wonderful place, wonderful to meet colleagues, and wonderful, in particular, to meet students and tell them about the beauty and usefulness of protein structures for application.
37:44
Now, you had mentioned, or I wrote in my CV, that I co-founded two companies. So I never, well, these two companies where all are based on academic work in my group.
38:03
That was structural biology and pharmacology and biochemistry. And one of them offered then, what was inaugurated 20 years ago, offered service in that field.
38:21
So big pharma needs that information. They usually do have their own structural biology groups, but these are expensive and slow. So the company, the dedicated company that I had in mind was fast and actually cheaper.
38:43
And this is on what I live. They have as clients almost all big pharma companies. The other company worked on antibodies. So one of my projects in the 70s
39:01
were antibody structures. We determined the first antibody structure. I mean, these are this Y-shaped molecule that you see in textbooks. And then we continued research and structure determination going to antibody receptors.
39:21
So when antibodies bind an antigen, they trigger the immune response, which for instance may be inflammation. They trigger also an autoimmune response. This is a disastrous disease where the immune system attacks its own, the components of its own body.
39:47
So this, we also defined structural and then thought that can be, can lead to a new strategy against autoimmune diseases. So that company was founded 15 years ago,
40:06
needed investment, substantial investment, but that turned out to be a big business for the investors because it was sold for an enormous amount of money, of which very little goes to the founders
40:21
because you have this stepwise financing. And at the end, there was financing around E, nothing or very little left for the founders, but a lot left for the investors. Investors, Robert, we're running out of time.
40:41
There's one more question. Is there one more question? Over there, let's hear it. Okay, I have a question to the state of structural biology right now. To what degree are you, or is structural biology able to really watch, for example, protein substrate
41:01
or protein substrate cofactor interaction in a real, let's say, biological environment? Well, there are some hopes now with these extremely strong X-ray sources, the X-ray lasers and microcrystals.
41:21
Exciting. Let us see what comes out from these experiments, where we really can see dynamics. Now, what we do see dynamics by analyzing a enzyme molecule
41:40
in different states, that is the apoenzyme, the substrate-bound enzyme, the product-bound enzyme, and perhaps some intermediate. These are often separate, different crystals, which we separately analyze, and this is not dynamic in the sense you probably mean that we see rates
42:03
that we usually, in that way, can't, we have hopes with the X-ray lasers that this may be possible, but this is, still, it gives a lot of information. If we combine these static structures with molecular dynamics calculations that we can do
42:22
and often do, that is, we determine the structure of a protein ligand complex, and then we remove the ligand and see how, in a molecular dynamic simulation, the structure reacts, and it often makes great sense.
42:43
So thank you for that. We have to cut short here. I think you'll be around for today. I will be around today. So you can approach Robert, just ask him any questions you might have. Thank you for attending and for your attention. Thank you. Thank you.