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With printable biotech through the entire sequence space

A peephole is not enough for Frank Rosenau. He wants to see everything. The insatiable scientist is a biotechnologist at Ulm University and his aim is to exploit everything the emerging field of printable biotechnology can offer to miniaturization in pharmaceutical research. Working with chemists, physicists and medical doctors, Rosenau focuses on the cell-free production of all theoretically possible peptides in the human body.

Dr. Frank Rosenau, managing director of the U-PEP in Ulm, wants to produce all theoretically possible peptide compounds. © Pytlik/BioRegionUlm

Rather than focussing on the investigation of what are known as peptide hormones, Rosenau and his team of researchers are interested in the fragments produced by the enzymatic degradation of proteins. The complete repertoire of these degradation products is referred to as degradome and consists of a huge arsenal of compounds that have the potential to be used as pharmaceutically active agents. This potential has already been substantiated by the findings of a group of researchers led by Wolf-Georg Forssmann and Frank Kirchhoff. The two scientists and their teams have identified in human body fluids three compounds that are active against HIV-1. “This discovery is all the more amazing in that the technique, which is in fact a standard laboratory method for producing peptide libraries from human sources, only revealed a tiny section of the sequences the genome stores on the proteome level,” says Rosenau.

Rosenau’s goal is to produce a complete library of human peptides using cell-free mini-reactors. Rosenau and his colleagues from Ulm have applied for funding totalling six million euros for a period of three years from the German Federal Ministry of Education and Research (“Next Generation of Biotechnological Procedures – Biotechnology 2020+” funding programme).

Huge majority remains invisible

In body fluids such as blood we can “only isolate stable biomolecules, i.e. proteins that are present in the blood at a certain time and under certain conditions and therefore accessible to scientists.” However, many natural peptides have a very short half-life before they are broken down for the amino acids to be reused as building blocks. Peptides can be highly important to drug developers, but inevitably a large number of peptides escape the attention of scientists due to their instability. What the scientists from Ulm are therefore doing is to “try and make visible what would otherwise go undetected”. There is a good chance of success because Ulm University has the skills required to increase the stability of unstable peptides and optimize their pharmacological effect.

The pharmaceutical industry’s compound libraries have a limited yield

Researchers look for potential peptide drugs in all sorts of creatures, including in the venom of cone snails, in potatoes and in bacteria, but rarely in humans. However, searches in human material are very successfully carried out by scientists from the Universities of Hannover and Ulm. Some might consider this to be rather unorthodox. However, Rosenau strongly believes in the new approach and points to the limited yield of the pharmaceutical industry’s gigantic compound libraries. When Forssmann began his pioneering work on the development of peptide libraries from human body fluids around 20 years ago, he was only able to get hold of stable peptides. Since then, biotechnologists like Rosenau have been able to access methods and techniques with which they can create any peptide they wish.

The toolbox for such a systematic search is already available: the proteome is relatively well known, as are the sequences of around 500 protein scissors (proteases). These tools enable everything subject to cleavage (proteome) along with the cleaving tools (proteases) to be produced using laboratory methods. 

Frank Rosenau explains: “The technology we have in mind is something I like to call combinatorial degradation of proteins using different proteases that successively act on one and the same substrate protein. The resulting protein fragments are subsequently cleaved by other protease enzymes, and so on. If you imagine a pyramid with the protein under investigation at the top, this protein is broken down by different proteases and the number of peptides increases from one row to the next as you go down. Moreover, the order of the proteases can also be changed. This leads to an incredibly large number of peptide compounds.”

In vitro experiments give better control

Left: microcolumns 10 micrometres in diameter and 5 micrometres high. The photo shows how functionalization leads to simple reaction zones. Right: fluorescence image of the microcolumns with fluorescence labelled proteins. © Rosenau/Kay Gottschalk/Uni Ulm

Of course it makes a difference where the protease enzymes cleave a protein. The way a protein is broken down into smaller fragments depends on the protein cleavage site to which a protease is specific and also on the proteolytic product that results from the action of any preceding protease. The use of many different enzymes leads to a broad range of different degradation products, which can then be analyzed and tested for a specific effect. The Ulm scientists take advantage of in vitro conditions, which enable them to precisely control the production of peptides. “With our technique, we know exactly what we are doing. We simplify the system, select specific substrate proteins and break them down with specific proteases at defined times and in a defined order. At a later stage we will perhaps also use tissue.” 

“Reactors” in which the proteins can be continuously digested with different combinations of proteases are required for the in vitro generation of the theoretically possible peptidome. The Ulm scientists plan to use a microfluidic chip and “printable biotechnology”, a method that has been developed in a time-consuming process by Helmholtz (mainly at the KIT in Karlsruhe) researchers. Printable biotechnology refers to the transfer of biological mechanisms (e.g. cells, organs) to printable systems, especially the manufacturability of three-dimensional structures (e.g. microfluidic channels) and their equipping with specific catalysts/enzymes. 

Cell-free reactors using nano-3D printers

Microfluidic channel 10 micrometers wide with several narrow areas, resulting in concentration gradients. © Rosenau/Kay Gottschalk/Uni Ulm

Cell-free bioproduction with immobilized enzymes involves the use of so-called nano-3D printers that produce three-dimensional topographies on surfaces to which the proteins can then bind. Such 3D-patterned polymer surfaces can be equipped with different properties. This method enables the scientists to attach enzymes to specific areas of the microfluidic channels. The microfluidic channels resemble a protein degradation mini-reactor, which needs to be spotlessly clean. In addition to mini-reactors, the scientists also require specific analytical tools, including electrospray ionisation, which is a technique used in mass spectrometry that enables the number, type and sequence of the peptides in a sample to be determined.

Physicist to help develop concept

The scientists from Ulm also have plans to use the prospective funds for developing such microfluidic reactors. These reactors will be able to carry out digestions continuously and in parallel and draw up and analyze samples. The goal is to turn the printable biotechnology principle into a reactor concept that enables the sequential digestion of proteins. The reactors will have integrated controls which will enable the scientists to define the conditions, e.g. the speed at which the substrates are pumped past the proteases and the order in which the enzymes break down the substrates and how long this process continues.

This combinatorial protein digestion, along with sampling and analysis, will then be automated. Rosenau will be working with Prof. Dr. Kay-E. Gottschalk who will be instrumental in implementing this novel reactor concept. Gottschalk is an experimental physicist with expert knowledge in microfluidics and surface modification who is already working with Rosenau on another BMBF-funded project.

A bet on probability

Rosenau will initially use albumin for his investigations as it is already known to be a rich source of bioactive peptides. The more peptide compounds the researchers are able to find, the greater the probability of finding a peptide lead structure. In addition to Rosenau’s novel approach, 20 or so medical research groups from Ulm are already focused on screening body fluid peptide libraries for the presence of naturally occurring bioactive peptides. With his approach, which covers the entire sequence space, Rosenau increases the chance of finding a peptide that is suitable as a drug. Rosenau is well aware that the approach is nothing short of a shotgun-like approach. Nevertheless he is using a far larger number of “shotgun pellets” than ever before and therefore has a higher probability of finding peptides with a pharmaceutical effect.

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