New Ways of Vision: Protein Structures in Translational Medicine and Business Development, my Experience
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00:00
ChemistryNobeliumProteinMolecular biologyWeinfehlerElectronic cigaretteCrystallographic defectNeoteny
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NobeliumProteinProtein domainEmission spectrumAtomChemical structureCell (biology)FluorescenceCrystallographyProteinOrganische ChemieChemical structureMultiprotein complexArzneimittelforschungWasserwelle <Haarbehandlung>ChemistryMoleculeOrders of magnitude (radiation)Protein domainNuclear magnetic resonanceNanoparticleNitrogen fixationElectronMolecular biologyMicroarrayComputer animation
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CrystallographyNobeliumCrystalGolgi apparatusChemical structureAtomProteinMarlChemical structureQuartzChemistryMolecular biologyNobeliumLactitolIce frontFrictionChlorideComplication (medicine)WaterfallAusgangsgesteinExciter (effect)SodiumProteinOrigin of replicationLecture/ConferenceMeeting/Interview
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NobeliumCell growthNucleolusProteinChemical structureAcidCrystallographyMoleculeBiotechnologyX-ray crystallographyProteinChemical structureGolgi apparatusProteinPharmaceutical drugCell membraneMoleculeKristallkörperComputer animation
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ChemistryGenePharmacyProteaseExtracellularCell (biology)ProteinGenregulationActivity (UML)Reaction mechanismActivity (UML)ProteaseChemistPharmaceutical drugProteinActive siteEnzyme inhibitorSubstrat <Chemie>Organische ChemieSurvival skillsBlock (periodic table)PeptideProteolyseHydrolysatMoleculeProtein biosynthesisProteinCell (biology)InhibitorAcidBinding energyBase (chemistry)Chemical reactionRegulatorgenMachinabilityElectronic cigaretteComputer animation
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Wine tasting descriptorsThoriumProteaseNobeliumChemical structureStructural steelMoleculeMultiprotein complexNuclear magnetic resonanceTrypsinDigestive enzymeAprotinin
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Recreational drug useElectronic cigaretteAnticoagulantActive siteDeterrence (legal)Substrat <Chemie>Binding energyBlock (periodic table)Recreational drug usePhenylalanineProteinArzneimittelforschungThrombinSolutionWursthülleBis (band)
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Chemical structureEnzyme inhibitorMoleculeActive sitePsychopharmakonLeaching (chemistry)Blue cheeseAnticoagulantActive siteSolutionThrombinC-terminusPeptideChemical structureBlock (periodic table)Enzyme inhibitorProteinLigandMoleculeAbbruchreaktionTandem-Reaktion
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SunscreenRecreational drug useThrombinLactitolFactor XChemical structurePharmaceutical drugWeizenbier-Brauerei J. BayerTandem-ReaktionProteinFoamSystemic therapyClaus process
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Enzyme inhibitorChemical compoundNational Rifle AssociationSerine proteaseBinding energyThiolproteaseMetalloproteinasenFactor XMultiprotein complexStem cellThrombinEnzyme inhibitorSubstrat <Chemie>Computer animation
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PathologyProcess (computing)Multiprotein complexProteinInhibitorAgricultureAngiotensin-converting enzymeZincPhysiologyMetalloproteinasenN-terminusPathologyActive sitePharmacologyProtease
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PeptideMetalloproteinasenAcidInhibitorLigandPeptideDeterrence (legal)ZincN-terminusProtein
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BagelProteaseProtein domainDemocratic Progressive PartyStarvation responseNobeliumInsulinDelicatessenBinding energyActivity (UML)GenregulationVerschlackungCell (biology)PeptideCell membranePressureAntigenSubstrat <Chemie>ProteinProteinProteolyseGrammatical conjugationExtracellularMoleculeSoft matterPharmacologyChemical structureProteinDietary supplementCell (biology)Systemic therapySetzen <Verfahrenstechnik>RegulatorgenBase (chemistry)HashishMan pageGrowth mediumChemical compoundActivity (UML)InsulinProtein domainIsotopenmarkierungFunctional groupPitting corrosionReaction mechanismWine tasting descriptorsProteaseN-terminusChemische SyntheseLigandT cellBarrel (unit)UbiquitinDenaturation (biochemistry)Binding energyActive siteDipeptidylpeptidase IVDipeptideDeterrence (legal)SecretionComputer animation
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StockfishBaker's yeastAlpha particleProtein subunitBaker's yeastSpeciesHexamereWine tasting descriptorsComputer animation
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VinylverbindungenPhenyl groupArzneistoffträgerCancerMultiple myelomaSynthetic oilCovalent bondEnzyme inhibitorSymptomAbundance of the chemical elementsSeedlingReaction mechanismNobeliumSunscreenChemistryHumusMajor histocompatibility complexAntigen-ProzessierungWasserbeständigkeitAcidBinding energyConstitutive equationCell (biology)Angular milDielectric spectroscopyPolypropyleneInterferon <gamma->PsychopharmakonTiermodellMoleculeMolekulardynamikLigandNanoparticleMultiprotein complexElectron donorDexamethasoneLithiumdeuteridProteinResearch Institute for Mathematical SciencesStereoselectivityDiseaseAntibacterialMultiple chemical sensitivityNaturstoffPathogenicityWine tasting descriptorsEnzyme inhibitorChemical structureChemical plantLigandPharmacologyLeadChemical formulaPeptideAntibacterialMultiprotein complexActivity (UML)CyclineBoronMoleculeToxicityQuartzPolymorphism (biology)Gene clusterProtein subunitLibrary (computing)Recreational drug useProtein domainSingulettzustandHydrogen bondGesundheitsstörungCancerCollectingChemical compoundWhite blood cellChemistryActive siteAddition reactionProcess (computing)VirulenzfaktorTargeted drug deliveryRiver sourceResidue (chemistry)Hope, ArkansasTiermodellFunctional groupWursthülleOrganische ChemieChemical elementNecking (engineering)WaterfallDietary supplementTeilentrahmte MilchChain (unit)Electronic cigaretteDeterrence (legal)PlasmodiumMolecularitySmoking (cooking)Growth mediumWaterAreaHydrogenPotato starchProteinCell (biology)DiseaseController (control theory)Boyle-Mariotte-GesetzComputer animation
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Binding energyGene clusterChemical structureTiermodellChemical compoundChemical propertyLeadActive siteCrystallizationRecreational drug usePerm (hairstyle)SemioticsAnomalie <Medizin>NobeliumSerum (blood)ProteinAlpha-1-RezeptorDiseaseCrystalCell (biology)SecretionAntibodyEnzyme inhibitorTyrosinActivation energyAntigenMultiprotein complexAntibodies (film)AlumBenzodiazepinePeriodic acid-Schiff stainClaySclerosis (medicine)AvidityRheumatismCollagenHydroxybuttersäure <gamma->HydroglimmerAngular milAntibodies (film)Chemical structureAlpha-1-RezeptorProteinAntibodyAreaActivity (UML)White blood cellIce shelfProteinKohlenhydratchemieQuartzLigandEnzyme inhibitorProcess (computing)PathogenicityReaktionsgleichungX-ray crystallographyMultiprotein complexStoichiometryProteaseGolgi apparatusPharmacologyBinding energyFaserplatteMoleculeWine tasting descriptorsSolubilityThermoformingStop codonFunctional groupIonenbindungButcherElectronBlue cheeseComputer animation
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NobeliumAageOcean currentPhysical chemistryX-ray crystallographyComputer animationLecture/Conference
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NobeliumLecture/ConferenceComputer animation
Transcript: English(auto-generated)
00:14
Good morning, it's a pleasure to be here with the colleagues and the young students.
00:24
Now this is Max Peritz, the father of molecular biology together with others in Cambridge, UK. And this is what he wrote to his family when he first saw a protein molecule revealed by X-ray diffraction.
00:43
Now 50 years later, we have many more reasons to join him or join his words because of the discoveries made to see organs, cells, large protein complexes and protein domains.
01:01
We learned to use the electromagnetic spectrum 12 orders of magnitude from radio waves to gamma rays. We learned to use particles, electrons, neutrons to visualize molecules.
01:23
The development of X-ray diffraction of NMR of super resolution optical microscopy and many others helped enormously and then combined with the chemistry helped in the development of new drugs.
01:44
This is the story I would like to tell you today. Now there is no doubt that the beginning of structural chemistry was with the discovery of X-ray diffraction by Max von Laue in 1912.
02:07
And this is the birth document of structural chemistry and then later structural biology and molecular biology. Here is an ugly photo of the X-ray diffraction of a crystal hand signed by Laue.
02:28
His publication in the journal, Few or Nobody Reads, I would say. And this shows Laue at the third Lindau Nobel Laureate meeting where he showed to Count Bernadotte a crystal lattice.
02:48
These are the fathers of X-ray diffraction, Roentgen, Laue. Laue's experiment was very quickly communicated to the world
03:06
and the Bragg's father and son, Bragg, who worked in Cambridge, England, grasped immediately the importance of Laue's experiment to determine crystal structures,
03:23
very small ones, simple ones, sodium chloride, founded a school where Max Perutz worked and he found a way then to decipher the complex and complicated diffraction pattern of large proteins.
03:41
By the way, the original instruments of Roentgen and Laue are on display in the Deutsche Museum in Munich. It's just 170 kilometers away. So if you find time to travel to Munich, don't forget to visit the Deutsche Museum. It's great to see the instruments that changed, revolutionized science and technology.
04:08
Now this is the development of then protein crystallography which began around here. There is no progress, just a handful of protein structures were known when I entered as a young postdoc
04:26
and then director at the Max Planck Institute of Biochemistry, less than a handful. Slow progress, as I said, but then the technological advances got their important role.
04:49
The fact that it was recognized that understanding biology requires to see the molecules and the application in medicine. This is what I would like to focus on later on.
05:03
This insert shows the development of membrane protein crystallography that Hans and Hartmut discussed which began with the publication in 1985. Again, relatively slow progress for over 10 years, but now we do see membrane protein crystal structures in the top journals published quite frequently.
05:32
Now this is my own institute which was inaugurated in 1972 where I moved in an institute in the forest.
05:41
And some called it Martin's Reed, it's a small village at the periphery of Munich. Some called it Martin's Roo because it was such a quiet place. Now this is how the campus Martin's Reed looks right now.
06:06
Many university institutes have been added. A second Max Planck Institute was also built. And in the center of this is the Innovation and Founding Center to which I come then later.
06:22
There is like a spider in the web trying to take up ideas and people from the academic campus and develop their ideas further.
06:41
Now I would like to focus on concerning application in medicine on a class of proteins. I have been studying early on from the late 60s on which are the proteases which degrade other proteins.
07:01
So you had about the life cycle of proteins, protein synthesis, you had or will hear about the protein synthesis machine, but the protein degradation is quite essential for our survival. Very simple chemical reaction, hydrolysis of a peptide bond, boiling acid or base can do it,
07:27
but in nature we do have in higher organisms 600 protein as is why. The reason for that is that proteolysis, in particular limited proteolysis, is an essential physiological regulatory mechanism.
07:48
Now what we began to study from the early 70s on is to find out how protease activity is regulated. It must be carefully regulated because otherwise proteins that are required in cells and outside cells would be degraded.
08:17
So we studied a large number of different proteases and in particular their natural inhibitors
08:24
and found different mechanisms of how protease activity is regulated. A very simple one is you have a proteinaceous inhibitor that binds in a substrate like manner
08:40
and blocks access to the active site. Well that was actually the very first one of these complex molecules that we studied. This is the simple digestive enzyme trypsin and this is the basic pancreatic trypsin inhibitor
09:02
which then later played a role in the development of nuclear magnetic resonance. I'm quite sure that Kurt Wüthrich will mention that in his talk. Now what we saw by looking at the individual components and in the complex there is no structure change.
09:27
So the two molecules are made for each other. Now this allows me to mention one of the first structure-based drug design endeavors.
09:46
Now here it's not trypsin but it is thrombin which is the major target for anticoagulant drugs and what you see here on this side is a simple lead compound which nicely fits into the substrate binding site and of course blocks access of substrate
10:05
but you also see that it does not, this phenylalanine, actually it's a D phi, does not fill its pocket so we thought at that time now by making the phenylalanine ring bigger, changing to enough gel,
10:21
we can gain specificity and proteins in effect. This was the case, 100-fold increase. It was one of the first examples of structure-based design. Now we can look, and this is what we did over the years,
10:42
at nature's solution of designing natural thrombin inhibitors. These sparks are blood-sucking and they keep the blood they eat liquid in order to process it. This is the leach which is actually, as the others, bags full of coagulation inhibitors.
11:08
Now we studied the three-dimensional structures of thrombin in blue and the various small protein inhibitors. Now the hirodine is actually an approved anticoagulant drug
11:23
and on the basis of the structure that we had determined, a chimera was made which has a peptide corresponding to the C terminus of hirodine and attaching a small synthetic ligand that then approaches,
11:44
blocks the active site. Now that story goes on, blood coagulation. There are a number of components in the blood coagulation cascade and upstream of thrombin is factor Xa.
12:04
Again, the structure that you see here was used then by Bayer to develop Xarelto which after introduction into the market about three years ago has become a blockbuster. So again, the importance of structure-based design and development is very clearly seen here.
12:28
We looked at many other proteinases, the major classes. So I talked about trypsin, the serine proteases and thrombin and factor Xa. Now there are the cysteine proteases and the metalloproteinases
12:44
and they all do have their natural inhibitors which we started in isolation and in complexes. They all bind in a substrate-like manner but they have exocytes to generate specificity and increase the potency.
13:05
This is an example of the metalloproteinases, the blue proteinase and the red natural inhibitor which sticks its N-terminus into the substrate binding site coordinating the zinc.
13:26
Now what you see here is the role the metalloproteinases play in pathology and physiology. So obviously the metalloproteinases are major targets for pharma research.
13:45
However, there is a problem. You can simulate what we saw in the natural metalloproteinase inhibitor with a small peptide which instead of an N-terminus has a hydroxamid acid
14:04
which is a better coordinating ligand to the zinc but there are about more than 20 different but closely related metalloproteinases and you would like, you must generate specificity but they all look the same.
14:23
They all look very much alike. So the problem here is very obvious generating specificity by further design and development. Well, this is one other example out of many that we looked at
14:42
in the course of these 35 years of protease research. This is D-peptidyl peptidase 4 which has become a diabetes target. It's a large protease which does have different domains.
15:01
We know where the substrate binds so it cleaves off dipeptides from the N-termini of their substrates. We know how the substrate enters the protein either through this barrel domain or a side entry.
15:25
Now what does it do? How did it become a diabetes target? Very successful one because D-peptidyl peptidase 2 inactivates the incretins which are gut hormones that stimulate after a meal insulin secretion.
15:46
And what you see here, this list of different synthetic compounds that inhibited D-peptidyl peptidase. You see many of the prominent big pharma companies and in fact it's a billion dollar business.
16:05
Again, by making use of the published structure of D-peptidyl peptidase 4. I still do have a very small research group, an emeritus group
16:21
consisting of three or four people. And we started to continue with the DPP family beyond the DPP4 which we had determined going to DPP8 and DPP9 which have a very different physiological function and they are cytoplasmic, involved in immune response and apoptosis
16:43
and again we continue with structure determination in order to help design of specific ligands. Now, well, what you see here is on this cartoon
17:01
the different mechanisms generating or helping to regulate protease activity. I have no time at all to go through this but we determined the structures and functions of many of them. And I now will focus on the last one
17:23
which showed a new way of protease regulation. These are the large intracellular proteases called proteasome that has the active sites buried inside and regulation is by closing or opening the entry ports.
17:41
That was very new and very exciting. These are large proteins consisting of 28 subunits. Well, the role of the proteasome is extremely important. It is involved in the ubiquitin system. You heard Hershka speaking about it.
18:00
It is the executioner of the ubiquitin communication system by degrading labeled proteins but it is also the garbage cleaner in cells by recognizing denatured proteins and it is essential in the immune response
18:20
triggering the T cell immune response. So the surprise, so we looked at the structure first at a simple version of an Arhael proteasome and then of course continuing to the eukaryotic, first yeast and then mouse and human proteasomes.
18:45
So they have the same architecture as you can see but while the Arhael proteasome has just two different subunits which are arranged in this 28 oligomer
19:01
using 7-fold symmetry for instance, we do have 14 different subunits in the mammalian species. Well, now the big surprise concerning drug design came by the finding by serendipity
19:24
and the lack of an American company that this boronate which we know inhibits the proteasome was developed and approved against some blood conscious multiple.
19:44
That was a big surprise. You have an essential molecule as a drug target. Big money again here, exciting and stimulating research all around the world, screening libraries
20:02
and the successful candidates then have been sent to us, to Michael Grohl who is professor at the technical university and myself to find out how these compounds are found. Now it's a very small collection that you see here.
20:21
In yellow you see what has been found in natural compound libraries. Now very different chemistry that you can see but they all do have a head group, a warhead that attacks the active site residue which is an N-terminal trion. I have no time to go into that
20:42
but this is the example of such a discovery process because a plant pathogen in Zurich had found that this Pseudomonas syringae which kills bean plants needs a virulence factor with this formula
21:01
and we found out what it does, namely binding to the proteasome and causing then apoptosis because the polyubiquitinated cyclin B1 is no longer degraded. Now of course this has been synthesized and modified and is now used for further development.
21:26
Well the problem with the constitutive proteasome inhibition is that it offered a new strategy for cancer but it is very toxic, in particular neuropathic toxicity.
21:46
Now what we thought then is that we look at a variant of the proteasome which occurs in hematopoietic cells in usable during an immune response.
22:01
Now this immune proteasome is extremely similar, identical, almost all of the subunits, but there are some differences in the active site and we thought that this should be sufficient to generate immune proteasome specific inhibitors
22:22
and thereby avoiding the toxicity and offering additional strategy against autoimmune disorders because we would hit immune cells specifically.
22:40
Now this is far outside of what an academic group can do so I approached the lead discovery center of the Max Planck Society which is an organization of the Max Planck Society to help further development of ideas coming from academic institutes in the Max Planck Society.
23:04
So they accepted that project. We still continue of course to work on it and put in new ideas, for instance in this way by finding out
23:21
that when we reanalyze the about 50 different proteasome complex crystal structures, then we found that the ligands that have a peptidic nature cluster in a certain area, this we did by principle component analysis
23:43
different from the non-peptidic one. Now what do the peptidic ligands do? They cause a domain closure of one of the active subunits and it depends exactly on these four hydrogen bonds which are required. The non-peptidic ligands cannot do that
24:02
so the structure closes and the important finding was that the immune proteasome does not undergo this change, does not require this change. It is already in the close confirmation, immediately explained the enhanced activity of the immune proteasome. We have pursued that further then
24:24
with molecular dynamic simulation. Well, also the Lead Discovery Center is not big enough in order to pursue a drug development program so they teamed up or we teamed up with big pharma Merck
24:43
and had quite successful further development. For instance, developing a way of treating rheumatoid arthritis in a red model and their work is continuing. Well, the other quite important development
25:03
was that the proteasome turns out to be a target for novel antibiotics. This was work of others that had looked at the plasmodium proteasome or a mycobacterial proteasome and developed specific ligands. Now here the need is to inhibit
25:26
the bacterial proteasome and not touching the human proteasome but with the structures available you can do that. So there are great hopes in very novel antibiotics.
25:43
Now the last few minutes then I would like to talk about my experience with the Foundation of Business. The first one is a company called Proteros that was founded in 1999
26:00
by a former postdoctoral student, it was Neufein and myself. And I should say I never was involved neither in this company nor in the one I'm discussing in a minute in the operational business. I feel as a scientist but I was lucky to find
26:23
former members of my department of being willing to pursue an idea. Now the idea in this case was to provide service for big pharma. So enabling technologies and integrated discovery
26:41
and offering this to big pharma. Now the company grew from two or three or four people to about 70. They have their own laboratory building in the Munich area and this is what they offer. It goes from protein production, assays, screening
27:02
and then of course the specialty which is structured determination. And this is then which they give to their clients. This I skip. And these are the targets which they have on the shelf.
27:25
They have established protein production, crystallization. So if a client comes with his ligand and then he will get an answer let's say in perhaps a month because all the processes for structure determination are already available.
27:42
Now as they have clients from all over the world it's quite interesting to look at the list of these shelf targets. These are the kinases. These are the proteases and these are others. Now it is all proteins with a molecular weight between about 20,000 to 60,000 or so.
28:03
So by the way as there was a discussion about this, this is a range of size that cannot so far be determined with electron microscopy. You need X-ray crystallography and you will need it forever.
28:22
That's my impression. But I mean there are wonderful, wonderful progress in cryo-EM in particular with the very large complexes. But concerning pharma design and pharma development you will need the structure, you will need the X-ray crystallography.
28:42
These are their clients from all around the world. So there is a tendency of big pharma to outsource these services and reduce their own in-house activities. Of course this is good for proteases.
29:02
Now a few minutes if I have for the second company which again was based on academic expertise and work founded in 2003. And what this title page says that it made in 2015
29:21
investors very happy, lawyers and broker happy, and founders also somewhat happy. So let me go through the story. It goes back to our work very early, that is when Hans was in my department,
29:42
very early work on antibody structures was actually the first antibody structure which showed in parts the FC portion and the FAB arms, antibodies central mediators by binding to FC receptors binding to complement
30:02
and this was what we wanted to study later then. Now what happens when a pathogen is optionized, it is covered with specific antibodies. So this complex then binds to FC receptors
30:22
and triggers the immune response and the immune response may be either activatory or inhibitory. There is a careful balance between activation and inhibition. So now what we did was looking at the complex
30:44
between the FC portion which is sufficient for making the FC receptor complex. You see it was an unusual stoichiometry because the FC is twofold symmetric while only one receptor binds to it and there is some structural change in both in the FC part.
31:07
Well what was the idea then that we had? Now first you can design small molecules that inhibit the receptor FC interaction.
31:20
We did not achieve anything in that direction so we thought that we should use the receptor in soluble form itself to block the binding to the immune cell and then stop immune response for instance in autoimmunity.
31:43
So this was the business idea. We followed another way too because we found that a specific antibody against the inhibitory receptor then also blocks the immune response but this is a separate story.
32:02
But I would like to mention great work done by Roche again on the basis of the structures I have shown where they have found that by modifying the antibody and eliminating this few cause
32:21
in the standard carbohydrate of the antibody enhances the binding to the FC receptor by 100 fold so an enormous effect.
32:42
And this is of course just the opposite of what we want if we want to stop or to reduce the immune response but for therapeutic antibodies you would like to enhance the immune response and this is exactly what they found
33:02
and they studied the structure also showing for the first time that there is carbohydrate-carbohydrate interaction between the receptor and the antibody and the receptor and the focus is in the way of this interaction.
33:23
So great work, wonderful and has enormous consequences in making therapeutic antibodies. Well we of course continued with the inhibition with our soluble receptor successful
33:43
so the investors stayed on board they invested a lot of money but they had great profit as you can see from this press release including my own institution the Max Planck Society made a lot of money
34:03
by investing in supremol. Now this is Munich, my hometown which as I said you should visit and visit the Deutschen Museum but I show that because this is the old part of university
34:24
and this is where Roentgen and Max von Laue worked Roentgen was professor of experimental physics Laue was with Arnold Sommerfeld professor of theoretical physics this is where 100 years ago all started
34:42
all of what I told you about protein crystallography began here. Thank you.