Within the framework of the LOEWE TRABITA project, Prof. Dr. Franz-Josef Meyer-Almes from the Chemical and Biological Technology faculty at Darmstadt University (h_da), is busily researching into new and innovative pharmaceutical active agents. The hope is that one day these could be used to treat breast cancer, diabetes or Chorea Huntington’s. A total of eleven work-groups formed from three hessian universities are participating in the TRABITA project. Their expressed aim is to establish the Rhine-Main region as a centre for pharmaceutical development.

By Christina Janssen, 10.1.2022

Franz-Josef Meyer-Almes is a particular strain of cave explorer. The caves explored by the h_da professor don’t depict any stone-age wall paintings, but they just might contain zinc ions. The biochemist examines incredibly tiny indentations in protein molecules that are smaller than a millionth of a millimetre.

Almost peculiar, one might feel, yet research into such 'cavities', which can only be made visible using highly complicated procedures, is a step into 'caves' harbouring enormous potential. The reason being that certain protein molecules in our bodies appear to be part and parcel of the development of chronic conditions. These include cancerous illnesses, diabetes as well as Chorea Huntington’s. If we could possibly find a way to dispatch these unwholesome proteins via a new generation of active agents, we could well have an opportunity to heal millions of sufferers around the globe. This is what the LOEWE-Projekts TRABITA aims to do.

This is where cave exploration can make its mark. In order for an active substance to have a chance at shutting down a harmful protein, it must be able to dock onto it. This is where the cavities come in. They are effectively keyholes for which the researchers must now find appropriate keys. This is difficult enough, given their size, but the task becomes even more arduous due to a very crafty trick that the proteins molecules pull: the cavities change, whilst hidden side-pockets can open or shut utterly without warning. No, the scientists are still not quite sure why, when or how. “It’s as if someone would come to your house each day and change the locks”, says Professor Meyer-Almes.

This accounts for the project’s name, TRABITA, which faintly recalls a Verdi opera, whereby the abbreviation stands for ‘Transiente Bindungstaschen’, or ‘Transient Binding Pockets’. It is precisely these cavities that Meyer-Almes and his team so fervently seek. Transient, because they only appear sporadically on a molecule. Meyer-Almes studies such ‘TRABITAs’ in a specific type of protein, the so-called Histone deacetylases, or ‘HDACs’. They interact with the DNA in a cell’s core, yet also with other proteins found in the cell’s fluid. This enables HDACs to control important processes in the cell. Should the highly complex protein network in a cell be unbalanced, this may, in a worst case scenario, lead to it becoming cancerous and malignant.

How glorious it would be for us to be able to simply insert a key into a protein lock and copy as many keys as we wished to. The active agent would be available – yet vital things are never simple in life. Prof. Meyer-Almes’ team has already developed and patented several molecules capable of blocking the conserved cavities of HDACs. However, to date Meyer-Almes has not managed to crystallise an active agent in a transient cavity. The hunt for effective protein inhibtors is one broken down into many  small steps. As the scientist stresses “you can’t let the frustration get you down“.

The first step will be to produce HDAC molecules in different variants. “We’re trying for 100 types“ explains Meyer-Almes, “this will enable us to determine how the tiniest variations in a protein at molecular level affect the interaction with any given active agent.“ The variants will be extracted from genetically modified strains of E. coli bacteria and bred in laboratories, which is frequently used in microbiology. Large flasks filled with the brownish bacterial soup are automatically warmed and stirred in a type of incubator over night, until the bacterial concentration is high enough. Afterwards, the bacteria are smashed using ultra-sound shock waves and the individual components finely separated using centrifuges and several chromatographic processes.

As Meyer-Almes emphasises “here we’re working in a so-called S1-Area, which means that these organisms do not, per se, pose any danger.“ He himself had once – unintentionally – been subjected to a bacterial soup shower, as a tube attached to large bio-reactor became dislodged and tore away. His laboratory coat was nonetheless sterilised – just to be on the safe side, “and I was permitted to do a bit of cleaning up“, not a particularly savoury task, as, being an intestinal bacteria, E.coli creates an appropriate scent. As Meyer-Almes confirms, it was rather horrid, but presented no health hazards. “I can assure you that the bacterial strains we breed can only survive in their specific nutrient solution. If I was to pour them down the drain – which we would never do, I hasten to add – they would simply die off.“

Alongside the elaborate and olfactory challenging protein production in Meyer-Almes‘ laboratories, potential active agents are also being synthesised. Some of the substances have already been shown to be capable of interfering with HDAC activity. These include the so-called hydroxamic acids. They attach themselves to the zinc ions in the cavities of the HDAC proteins. However, unfortunately, the hydroxamic acids are far too indiscriminate as they bind to any kind of zinc-based complex, “which means they could introduce unpleasant side-effects for patients“, explains the professor, “moreover, we suspect that hydroxamic acids could well be carcinogenic“. This would be like asking Beelzebub to drive out the devil.

Meyer-Almes’ team is therefore attempting to define and search for active agents which have a selective effect on only a limited range of HDAC proteins. The bio-chemist has a databank of around 15,000 substances in his laboratory, which is constantly being expanded. Meyer-Almes is in regular contact with researchers across the globe, who constantly develop new substances and then share with each-other. Meyer-Almes is presently testing a range of active agents sent to him by an Indian colleague, who had created them in her laboratory. “I had a really good look at the structure of these substances at a congress in Singapore where I thought, hmm, they might do the trick“. The colleague sent him a few samples. “And what do you know, some of them even worked!“ Intuition, along with trial and error, is abundant in research. “I reckon that intuition is one of the truly essential requirements for researchers. To my mind, if we don’t actually try out really way-out ideas, we won’t see any genuine progress.“

Yet how on earth can ‘cave explorer’ Meyer-Almes ascertain that something truly is happening between an active agent and protein? In addition to highly complicated technology a sure, experienced set of instincts, it seems, is called for. “For instance, we use an ITC unit.“ ITC stands for Isothermal Titration Calorimetry. Inside the inconspicuous white apparatus the requisite substances are mixed together with one-another. Once a chemical reaction takes place, the unit measures the tiniest fluctuations in temperature. “We’re talking about ten thousandth of a degree in tiny amounts of fluid.“ It is genuinely astounding what Meyer-Almes and his team are able to derive from the results. “If the temperature drops or rises, we already know that within this mixture heat is being either generated or consumed.“

An exact analysis of the titration curve can also reveal how strongly the protein and active agents are reacting with each-other. The researchers can also determine other thermodynamic aspects, thus supplying them with a kind of ‘calling card’. “We then combine and correlate all of this information in a sort of ‘thermodynamic binding signature’ which provides us with a model of the binding mode of the two molecules being tested. This enables us to understand how specific molecular groups influence this molecular interaction. This is a decisive method in developing active agents, for it is essential to know precisely how strongly an active agent binds to a given protein.“ Because, a strong interaction points towards the agent having promise. The absolute target is to develop substances which consist of as few atoms as possible, because such low molecular weight agents are particularly efficient and furthermore relatively uncomplicated to produce.

Agents which work via docking onto the ‘Transient Binding Pockets’ are usually found by serendipity. “If discovered, this aspect had certainly not been planned during the development of the medication“, according to Meyer-Almes. The scientists working on the TRABITA project are certainly delivering pioneering achievements. The project’s consortium is being led by TU Darmstadt, furthermore, the Goethe-Universität Frankfurt is also providing several work groups. The state of Hesse is funding the project over four years to the tune of 4.5 million Euros.

“We can almost certainly be sure to uncover fundamentally fresh insights, if only because the framework of TRABITA allows us to focus on a scientific issue which has, to date, only been seen as a side-issue by others.“ So will we ever fully comprehend Transient Binding Pockets? Meyer-Almes grins, “no idea, it would even be a huge success if we manage to understand partial aspects of the whole.“ The heart of research, as such. And as far as the ‘TRABITAs’ are concerned, this ought to be firmly anchored in a special research area: “it is our ambition to establish the Rhine-Main region as a sprawling centre for pharmaceutical research“, says Meyer-Almes. In his own, inimitable, cave exploring way … he certainly does his bit.

Translation: Paul Comley

Website of Prof. Dr. Franz-Josef Meyer-Almes: https://fbc.h-da.de/meyer-almes-franz-josef

Project website: www.chemie.tu-darmstadt.de/trabita/trabita_d/index.de.jsp