Team:Freiburg/Interview Münch
“If we can make peptide therapeutics orally available, then peptide-based therapeutics will be the medicine of the future - I am convinced of that.”
To get a more in depth view on the present situation of peptides in the therapeutical applications we talked to various experts. A key influence was the visit at the Ulm Peptide Pharmaceuticals (U-PEP) which focuses on the development of safe and innovative peptide therapeutics. There, we learned a lot more about the advantages of peptide therapeutics but also about the obstacles that still have to be overcome in the development of peptide therapeutics.
Prof. Dr. Jan Münch is the co-founder of U-PEP and director of the Institute of Molecular Virology at University Hospital Ulm. In collaboration with Prof. Dr. Frank Kirchhoff and Prof. Wolf-Georg Forssmann they isolated a potent HIV peptide inhibitor from human hemofiltrate. This endogenous peptide, which has been named VIRIP, binds to the fusion peptide in the gp41 fusion protein, a key component in HIV entry mechanism. Further, they developed various derivatives to optimize stability and activity, from which one derivative has entered a phase 1/2 clinical trial in HIV-1 infected individuals. This derivative, termed VIR-576, suppressed viral loads by more than 98 % without causing severe side effects. During the interview Prof. Münch mentioned several advantages of peptide therapeutics “Peptides are highly specific in comparison to small molecule compounds. A lot of those small molecules have off-target effects and they cannot be modified easily. And this is one of the biggest advantages of peptide therapeutics: they are easily and quickly modified. Peptides can be modified in amino acids sequence, shortened, extended or conjugated with various fatty acids, this does not work for antibodies or small molecule compounds.” Despite their numerous advantages, poor oral availability is an obstacle, that still has to be overcome in order for peptides becoming a competitive drug class on the market. Prof. Münch emphasized that: “As soon as oral availability is achieved, peptide will play a major role in the pharmaceutical sector. Peptide pharmaceuticals have less side effects, are highly specific and cheap in production, which gives them major advantages towards any other therapeutic classes.”
To get a more in depth view on the present situation of peptides in the therapeutical applications we talked to various experts. A key influence was the visit at the Ulm Peptide Pharmaceuticals (U-PEP) which focuses on the development of safe and innovative peptide therapeutics. There, we learned a lot more about the advantages of peptide therapeutics but also about the obstacles that still have to be overcome in the development of peptide therapeutics.
Prof. Dr. Jan Münch is the co-founder of U-PEP and director of the Institute of Molecular Virology at University Hospital Ulm. In collaboration with Prof. Dr. Frank Kirchhoff and Prof. Wolf-Georg Forssmann they isolated a potent HIV peptide inhibitor from human hemofiltrate. This endogenous peptide, which has been named VIRIP, binds to the fusion peptide in the gp41 fusion protein, a key component in HIV entry mechanism. Further, they developed various derivatives to optimize stability and activity, from which one derivative has entered a phase 1/2 clinical trial in HIV-1 infected individuals. This derivative, termed VIR-576, suppressed viral loads by more than 98 % without causing severe side effects. During the interview Prof. Münch mentioned several advantages of peptide therapeutics “Peptides are highly specific in comparison to small molecule compounds. A lot of those small molecules have off-target effects and they cannot be modified easily. And this is one of the biggest advantages of peptide therapeutics: they are easily and quickly modified. Peptides can be modified in amino acids sequence, shortened, extended or conjugated with various fatty acids, this does not work for antibodies or small molecule compounds.” Despite their numerous advantages, poor oral availability is an obstacle, that still has to be overcome in order for peptides becoming a competitive drug class on the market. Prof. Münch emphasized that: “As soon as oral availability is achieved, peptide will play a major role in the pharmaceutical sector. Peptide pharmaceuticals have less side effects, are highly specific and cheap in production, which gives them major advantages towards any other therapeutic classes.”
iGEM Freiburg: What first incited your interest in peptide pharmaceutics and what makes peptides in the therapeutical application special?
Münch: Well to begin with, everything was a giant coincidence. I started as an intern in the group of Dr. Kirchhoff when the topic of HIV/AIDS was just getting more public attention. After two to three weeks I joined a talk by Wolf-Georg Forssmann, who at that time was the director of the Institute for Peptide Research in Hannover. In his talk he presented a method where they isolated peptide libraries from body fluids, especially hemofiltrate, which could have potential in antiviral drug development and they were searching for collaboration partners. Frank Kirchhoff then agreed to do further tests in his lab and gave me the duty to screen these hemofiltrate fractions for HIV inhibitors.
So I started the screening and was successful relatively fast. From 300 fractions I could find anti-HIV activity in 10-12 fractions, without showing toxic side effects.
And at this time this was a huge deal. As we were on cloud nine we started isolating our first peptide which was a RNAse, that we immediately patented together with partners from industry. And then one night I woke up, drenched in sweat, because I noticed that my detection system for HIV was a reverse transcriptase test. And what is the function of a RNAse? It digests RNA or single-stranded DNA.
So there was activity in those fractions that destroyed my detection system, which, after testing them on RNAse activity, were all false positive, except for one.
And from this fraction we found VIRIP and out of this, this whole peptide project developed.
iGEM Freiburg: What advantages do peptide therapeutics provide compared to small molecule compounds or therapeutic antibodies?
Münch: Well there are lots of them. Peptides are highly specific in comparison to small molecules. A lot of those small molecules have off-target effects and they cannot be easily modified. And this is one of the biggest advantages of peptide therapeutics: they are easily and quickly modified. Peptides can be modified in amino acids sequence, shortened, extended or conjugated with various fatty acids. This does not work for antibodies or small molecule compounds.
iGEM Freiburg: Are degradation products a relevant topic? If you look at small molecules, for example in chemotherapy, those are metabolized in the liver where their degraded products can cause severe side effects.
Münch: Yes of course. In general you can say that peptides have less side effects than small molecule compounds. Especially in our case we did not expect a lot of side effects because the ligands we isolated are derived from human peptides.
iGEM Freiburg: The prize for developing a peptide therapeutic is also an advantage, would you agree?
Münch: Yes for sure. And also with chemical synthesis you save a lot of time. If you had an antibody you would first have to clone it until it can be expressed recombinantly and this takes a lot of time. For peptides you just have to synthesize your sequence and it will be lab-ready within a week.
iGEM Freiburg: Could immunogenicity be a problem?
Münch: Yes this can be a problem. However, with the peptides we developed we did not have any problems concerning immunogenicity, but we are not sure what is the reason for that. But I think with certain software programs you can calculate the immunogenicity even though you still have to test this in vivo. Also it depends on the function of your peptide therapeutic. In stem cell mobilization, immunogenicity does not play that big of a role, because it is only administered once. In contrast, if you are developing a therapeutic for chronic therapies, you would have to pay attention to any immunogenic problems and do specific analysis to circumvent those.
iGEM Freiburg: Are there any other disadvantages or risks when working with peptide therapeutics?
Münch: Oral administration, respectively bioavailability is a major factor and there is a lot happening in this direction at the moment. A lot of companies are investing tons of money in developing orally available therapeutics. This is the so called „holy grail“, and if we can make peptide therapeutics orally available, then peptide pharmaceuticals will be the medicine of the future - I am convinced of that.
Another problem is the half-life in vivo. Extension of this can be achieved by incorporating D-amino acids, or with modifications in the protease cleavage sites within the peptide.
iGEM Freiburg: While developing VIRIP what were other hurdles that you had to overcome?
Münch: The first hurdle of course was: how do we get this peptide more active? Our original peptide had an IC50 of around 20 μM and if you project this, you would need grams of this peptide to achieve an effect in vivo. So our first goal was to optimize the anti-viral activity as well as stability. For this we did a systematic structure-activity relationship analysis in order to design optimized derivatives of VIRIP. This was 15 years ago when peptide synthesis was still expensive. For mg amounts you would pay around 5000-6000 €, whereas now you would get ten grams for that sum of money.
iGEM Freiburg: We already came across the topic of modifying peptides: what are the most common modifications for peptide optimization?
Münch: At first you want to find the minimal active sequence in order to identify the amino acids that are responsible for the function of the peptide. With specific changes you then try to optimize the activity of your peptide. For our CXCR4 antagonist we found the structure of the amino acids responsible for activity and are now in silico modeling our ligands into the receptor in order to make predictions for improvement. With modeling we could also predict shortened derivatives. So you typically start with empiric changes of amino acids in your sequence as well as playing with hydrophobicity of your peptide. How could your ligand interact with its receptor? For this you have to consider not only activity but also stability. We are trying to always test this in parallel, however stability assays always are more complicated.
iGEM Freiburg: In your paper ”Reduced Susceptibility to VIRIP-Based HIV-1 Entry Inhibitors has a High Genetic Barrier and Severe Fitness Costs” the term „bispecific peptide inhibitors“ occurred. Could you tell us a little more about this?
Münch: The term bispecific inhibitor describes a molecule that has two different mechanisms of action.
iGEM Freiburg: Could this also be an advantage in comparison to small molecule compounds, because they are not as easily merged together as peptides?
Münch: Of course, this is a major advantage! If you design a peptide, you can always look for unimportant amino acids and exchange them for a cysteine, which due to the free thiol, can be modified to multimers. And dimers or multimers are in most cases much more active than the monomer. Which again is an enormous advantage Peptides basically work like building blocks: You couple them to a fatty acid, which you then can bind to Albumin, this again enhances retention time.
Coincidently the fatty acids can interact with the membrane, which allows membrane targeting as well. Depending on the location of the fatty acid you can achieve various effects like coupling to gold-nanoparticles for drug delivery or to dendrimers to enhance multi valency effects.
iGEM Freiburg: Did you try gold nanoparticles as a system to get your peptide into the cells?
Münch: Yes, we tried this with our CXCR4 antagonist but the conjugates not as active as other modifications that we tried. That’s why we did not continue to work with this method or published anything in that direction.
But again: If you have a peptide and want to ameliorate it, chances are very high that there is a way to do so and that is truly astonishing.
iGEM Freiburg: For VIRIP you did a hemofiltrate screen, where you had a library of around one million peptides to screen against. If you compare this to a phage display, where you’d have a library of 1011 peptides, isn’t a smaller library a restriction? Or does that also have benefits?
Münch: Well, since they’re endogenous peptides, they are not immunogenic when we isolate them. But with modifications a gain of immunogenicity is definitely possible, so this is not really an argument. The most valuable argument is the biological function. As scientists we are also interested in basic research: How does the human body work? What are principles of immunity in context of virology? In this case some peptides become interesting, even if there is no possible therapeutic function. The first step is always finding the biological function. Only secondly there comes the consideration if it is applicable as a therapeutic.
And you don’t have that with phage display, where one out of the billion peptides might have an inhibiting function to your target but nothing more. Whereas we always have the physiological relevance that we can also work with.
iGEM Freiburg: With various ligands you tested the incorporation of D-amino acids, did this lead to any advantages?
Münch: With D-amino acids you expect higher stability within the organism because there are no specific proteases for them. What we noticed with one of our candidates that entered clinical trials is that the incorporation of a D-amino acid did not only lead to better stability, but also increased the activity of the peptide.
iGEM Freiburg: This sounds very interesting. Are there any other reasons why one should consider including D-amino acids into a peptide therapeutic?
Münch: Well of course there is the proteolytic resistance but if you want to know more about D-amino acids or even D-peptides you should contact Prof. Dr. Dieter Willbold. He really is a pioneer in this field and has a D-peptide in clinical trials that seems to be very promising.
iGEM Freiburg: Thank you for your advice we will take this into consideration. To conclude, how would you describe the future of peptide therapeutics?
I think as soon as oral availability is achieved, they will play a major role in the pharmaceutical sector. They have less side effects, are highly specific and cheap in production, which gives them major advantages towards any other therapeutic class.
iGEM Freiburg: Thank you for the interview!
