Drug Design and Analytical Chemistry


New strategies in the fight against the widespread disease periodontitis

Periodontitis is an inflammatory disease of the periodontium that can lead to destruction of tissue and bone around the teeth. The disease is caused by bacteria that settle in plaque and gum pockets and trigger an inflammatory response. If left untreated, this leads to damage to the entire periodontium and even tooth loss. Various studies also show a direct link between periodontitis and other diseases such as cardiovascular disease and diabetes, as well as an increased risk of stroke.

The basis of the novel treatment concept is the inhibition of an enzyme that occurs almost exclusively in the bacteria that cause periodontitis and regulates the production of various virulence factors there. By selectively inhibiting these factors, the pathogenic germs can be specifically suppressed and the natural microbiome preserved. The use of classical antibiotics, on the other hand, leads to growth inhibition of all oral germs, which carries the risk of rapid and stronger recolonization by the pathogens.

The aim of the project is to test appropriate drug candidates for their efficacy and safety, thus creating the prerequisite for a clinical trial for initial testing in humans. The collaborative partners will address various regulatory aspects, including resistance formation, material compatibility, toxicity and safety.

The Fraunhofer IZI Department of Drug Design and Target Validation is contributing its expertise in the development and validation of bioanalytical methods for the comprehensive characterization of small molecule drugs.

In addition, toxicity and safety are being investigated both in vitro and in animal models as part of a GLP study.

Profiling of inhibitors of bacterial glutaminyl cyclases as drug prototypes of novel periodontal therapeutics (PInParo)

Studies show that there are clear links between the development of periodontitis and numerous serious systemic diseases, e.g. diabetes mellitus, arteriosclerosis and even Alzheimer's disease. The aim of the project is the preclinical development and characterization of drug prototypes for the treatment of periodontitis that address only those bacterial strains involved in the development of periodontitis. This includes characterizing them in terms of pharmacokinetic parameters, such as solubility, cytotoxicity, metabolic stability and membrane permeability, as well as their potential toxicity. Within the scope of the project, methods for determining metabolic stability towards liver microsomes and also inhibition of metabolic enzymes (cytochrome P450 monooxidases - CYPs) will be established at Fraunhofer IZI.

The collection of these data is of particular importance for the developments. In the case of systemic drug application, a certain stability must be present in order to maintain a plasma concentration required for the effect. In contrast, for local application, it is more appropriate to prefer a lower stability of the drug so that an absorbed drug is rapidly cleared to avoid systemic side effects. When inhibiting CYP isoenzymes, it is desirable that there is no inhibition by the drug candidate in vivo, as side effects may also occur here due to unintended resorption of the drug. The analytical methods established in the project will be used to obtain data that will provide valuable information for drug optimization and help to select suitable development candidates.

The project "Profiling of inhibitors of bacterial glutaminyl cyclases as drug prototypes of novel periodontitis therapeutics (PInParo)" is supported by the state of Saxony-Anhalt with funding from the European Regional Development Fund (ERDF).

Screening to identify aureolysin inhibitors as novel pathoblockers for Staphylococcus aureus therapy (AureoScreen)

Increasing resistance of pathogenic microorganisms is a major challenge for the treatment of infectious diseases. There are predictions that in the next few years the effectiveness of existing antibiotics will decline sharply, resulting in a significant increase in the mortality of bacterial infections.

So-called pathoblockers or antivirulence agents represent a new strategy for the treatment of infectious diseases. The aim is to develop active substances that do not kill bacteria but target properties that determine their pathogenic effect, the so-called virulence factors. Communication, nutrition and other relevant metabolic processes of pathogens that enable infection are thus to be impaired. An important group of these virulence factors are proteases, which are secreted by the bacteria and perform a wide variety of tasks. One such protease as a potential antivirulent drug target is aureolysin from Staphylococcus aureus, a metalloprotease that is considered among other enzymes to be an important virulence factor of resistant S. aureus strains. So far, however, hardly any inhibitors of aureolysin are known. Within the scope of the project, therefore, the first inhibitors are to be identified by means of focused screening. In the longer term, these first inhibitors should serve as lead structures to develop therapeutically applicable active substances.

The project "Screening to identify aureolysin inhibitors as novel pathoblockers for Staphylococcus aureus therapy (AureoScreen)" is supported by the state of Saxony-Anhalt with funding from the European Regional Development Fund (ERDF).

Selective active agents and application systems for the treatment of bacterial infectious diseases of the periodontium

Working on the mass spectrometer
© Fraunhofer IZI
The stability and release of drug candidates was investigated using techniques such as mass spectrometry.

Periodontitis, commonly referred to as gum disease, is an inflammatory disease of the periodontium that affects almost every second adult in Germany. It is caused by a local infection involving specific oral microorganisms and the subsequent local immune response. Left untreated, periodontitis leads to tooth loss and bone loss in the jaw. Studies also show that chronic periodontitis drastically increases the risk of developing diabetes, cardiovascular diseases and rheumatoid arthritis.

All of the preparations used to date come with the major drawback that their active agent is a broad-spectrum antibiotic and / or antiseptic. This means they kill all the bacteria found in dental plaque, including desired commensal bacteria in the oral microbiome, which often leads to the treated areas being recolonized by the germs that cause the disease.

Together with Periotrap Pharmaceuticals GmbH, the Fraunhofer Institute for Microstructure of Materials and Systems IMWS and Martin Luther University of Halle-­Wittenberg, researchers at Fraunhofer IZI developed a pathogen-specific form of therapy. This is based on an agent that acts selectively to severely diminish the pathogenicity of the bacteria that cause periodontitis by inhibiting an essential enzyme: bacterial glutaminyl cyclase. The Fraunhofer IZI researchers primarily examined pharmacokinetic properties such as potential absorption of the active agent into the organism. Besides this, investigations were conducted into the stability and release of drug candidates from controlled release drug delivery systems. Extremely precise and sensitive mass spectrometry is employed in particular here, which enables even the lowest concentrations of an active agent to be measured in an organism. Research has also been conducted into a suitable route of administration that is as straightforward and pain-free as possible, and initial model systems have been tested. Maintaining the concentration of the agent at the target location presents a particular challenge as active agents are usually washed out quickly in the mouth and especially around the gum pockets due to mechanical influences and the constant flow of liquid. Once the research project is complete, Periotrap Pharmaceuticals GmbH will continue to work on preparing the therapeutic approach for the market.

The project “Controlled release drug delivery systems of innovative and selective drug molecules for the treatment of bacterial infectious diseases of the periodontium (ParoStop)” was supported by the Land of Saxony-Anhalt using funds provided by the European Regional Development Fund (ERDF).

Periotrap Pharmaceuticals GmbH; Fraunhofer Institute for Microstructure of Materials and Systems IMWS; Martin-Luther-University Halle-Wittenberg

Alternative metal binding groups in drugs for the treatment of periodontitis

In the context of drug development, despite enormous progress in computer-aided prediction of various parameters of drug candidates, one still has to rely on investigating as wide a variety of chemically diverse molecules as possible. This usually involves adding different substituents to a constant basic structure in order to investigate their influence on the activity of the compounds, but also their toxicity or pharmacokinetics. This requires a relatively large number of different derivatives, since even small chemical differences can have a major impact on the properties mentioned. Such compound libraries typically contain several dozen compounds, but can also grow to several hundred derivatives on an industrial scale. In order to enable a broad chemical variability, the so-called flow-chemistry is to be established within the scope of the project. This will make it possible to produce a large number of compounds simply and quickly in flow-through microreactors. This state-of-the-art technology will be established in a joint project with PerioTrap Pharmaceuticals GmbH. Basic scaffolds developed there will be further derivatized at the Fraunhofer IZI Department of Drug Design and Target Validation in order to advance the development of drug candidates.

The project, which started on December 7, 2020, will run until July 31, 2022, and is funded by the German Federal State of Saxony-Anhalt with money from the European Regional Development Fund (ERDF).

Project coordination: Kathrin Tan.

TRIGGER: King of hearts, joints and lungs; periodontal pathogens as etiologic factor in RA, CVD and COPD and their impact on treatment strategy

Extensive clinical and epidemiological data clearly shows that chronic periodontal disease (PD), the most prevalent infectious inflammatory disease of mankind, is strongly linked to systemic inflammatory diseases such as cardiovascular diseases (CVD), rheumatoid arthritis (RA), and chronic obstructive pulmonary disease (COPD). Taking into account that up to 30% of the adult population worldwide suffers from severe periodontitis, the impact of this disease on human health is immense and has been recognized by World Health Organization. Nevertheless, in many EU countries PD is a neglected disease, both by the population in general and health-care personnel. Often this negligence comes to the point that, like a hair-loss, the tooth-loss due to periodontitis is still considered as a normal inevitable event associated with aging. To combat this misconception and conceive novel approaches to prevent and/or treat CVD, RA, and COPD we will explore highly innovative ideas that these non-communicable diseases are at least aggravated, if not initiated, by periodontal infection. Results emanating from our project will: i) elucidate a relationship between the presence of specific periodontal pathogens and severity of systemic diseases; ii) show that extensive periodontal treatment improves clinical parameters of investigated systemic diseases; iii) reveal the impact of eradication of specific periodontal pathogen on the level of inflammatory markers; iv) develop novel, periodontal-pathogen selective bactericidal compounds based on specific enzymes that are essential for these pathogens vitality. This will reduce mortality and ameliorate the quality of life of CVD, RA, and COPD patients.

Development of inhibitors for alternative beta-secretases as a new therapeutic approach for Alzheimer’s disease

© Fraunhofer IZI

The number of patients suffering from Alzheimer’s disease will increase dramatically, especially in the industrial nations. The major reason is the increasing lifespan, since age is the main risk factor for this disease. Thus, about 33 % of the whole population above 80 years is diagnosed with Alzheimer’s dementia.

In recent years pharma research was faced with a lot of failures in the development of novel drugs against Alzheimer’s disease that were based on the inhibition of an enzyme – beta secretase BACE-1 – being responsible for the release of a particular neurotoxic peptide (amyloid Abeta) as the main factor for the onset of the disease. The compounds exhibited a good efficacy in vitro, a good bioavailability and were well tolerated by the organism.

However, they failed in clinical trials due to a lack of efficacy in patients with spontaneous Alzheimer´s disease. Only in the case of a small sub population of 5 % of all patients that suffered from a genetically encoded variant of Alzheimer’s disease, the compounds showed promising effects in vivo.

This leads to the assumption of the existence of additional enzymes that might be able to release the neurotoxic amyloid peptide. Within this project we are investigating one of these alternative beta secretases. The aim is the computer aided and structure-based design and development of inhibitors of the isoenzymes Meprin alpha and beta. It was already possible to develop very specific acting compounds, exhibiting better activity than the known non-specific Meprin inhibitors. The IP is filed and we are now able to investigate the pathophysiological roles of Meprin alpha and beta more detailed.

The goal in this project will be the development of new therapeutic options for Alzheimer’s disease in close collaboration with the other units of the department.

Broadening the chemical space of metal binding groups

© Fraunhofer IZI

A lot of potential target enzymes with high medical interest contain a metal ion in their active site which is essential for the catalysis of the corresponding reaction. These metal ions are usually interesting pharmacophoric points for the development of new inhibitors because a binding to them leads very often to potent compounds. Unfortunately only a small amount of different potential metal binding groups are known, limiting the development of new drugs, because of their unselective binding not only to the target but also to other enzymes. One example for the discontinuation of the development of promising new drugs are matrix-metallo-protease inhibitors, where it was not possible to develop selective compounds among the enzyme class.

By the usage of a combination of semi-empirical, quantum chemical, ligand- and structure based approaches we are now able to overcome this limitations and to broaden the available chemical space for metallo enzyme inhibitors. In one example it was possible to extend the amount of the known 4 metal binding groups for a metal dependent acyltransferase by a number of 6, fully new and undescribed. This allows the development of a whole new world of active molecules.

MALDI-TOF mass spectrometry-based assays for the quantification of biomarkers

© Fraunhofer IZI

MALDI-TOF mass spectrometry is routinely used for the investigation of biomolecules of higher molecular weights, e.g. proteins. Examples are the sequencing of proteins and the investigation of different species of one protein or peptide. However, all of these tools are of a qualitative kind, because for a quantification of the proteins other mass spectrometry based tools are used. But these methods need more time for analysis and so the throughput is limited.

Nevertheless, for certain biomarkers the use of MALDI-TOF for quantification is a useful solution, because they need for the full ionization, and thereby quantification, more energy, than other methods are able to provide. As an example a MALDI-TOF based assay for the quantification of a relatively large protein of 4,8 kDa, used as a bio-marker was established. For this a sample preparation routine had to be developed that is easy to handle. For the determination of such critical parameter like LOD and LOQ an internal standard was synthesized (deuterated) and it was successfully used for the determination of a concentration range of 20 ng/µL to 100 ng/µL in human plasma that exactly represents the range, described for this biomarker in healthy persons and patients.

Completed projects

  • Mikro and nanoparticle applikation systems of drugs for dental cosmetic and therapeutic Applications
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