Therapeutical Principles

G protein-coupled receptors, receptor kinases and arrestins

Adrenergic receptors play a pivotal role in the regulation of many physiological functions of the cardiovascular system. Catecholamines such as adrenaline and norepinephrine bind to these receptors which are common in many organs of the body. These receptors are activated by specific ligands, and can then be specifically inactivated by proteins which belong to the families of receptor kinases and of arrestins. The team has developed a number of compounds which bind to these proteins, and regulate the propagation of hormone stimuli to intracellular signalling pathways.

Additionally, the group of W. Koch and the 2012 Nobel prize awardee Robert J. Lefkowitz, in whose group Martin Lohse had worked for several years, investigated gene transfer of the C-terminal fragment „ßARKct“ of the ß-adrenergic receptor kinase-1 (ßARK-1, GRK-2) for its effect on heart muscle cells. They observed an improvement of myocardial function in heart failure in several animal models. Our team has extended these observations to an in vivo gene transfer model and to a related signalling protein, phosducin. Moreover, novel transmitters and pathomechanisms were described in heart muscle cells. A specific phosphorylation mode of the extracellular-regulated kinases ERK1/2 causes a deleterious impact on heart muscle cells to induce hypertrophy. New insight into the intracellular compartmentation of second messengers, especially cAMP, was obtained in cooperations. Methods to detect this signal activation and signal spreading optically on a millisecond scale were developed.

Specifically, novel assays were established which allow to determine molecular arrestin activation. Thereby, signal pathway-specific  receptor agonists (“biased ligands”) can be identified.

References on receptor pharmacology and signalling

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Identification of novel therapeutic targets

Gene transfer to the heart and cardiomyocytes

A broad-range screening program in relevant models of human cardiovascular disease has allowed to identify a number of novel targets in cardiovascular diseases – heart failure (by investigating heart muscle cells – cardiomyocytes), atherosclerosis (endothelial and vessel wall cells) and thrombosis (platelets).

The focus was on the direct, unbiased comparison of molecular interventions in various signalling pathways. This comparison tested for effects on disease as end points, but was not primarily driven by disease hypotheses.

 

Adenoviral or AAV-mediated gene transfer was used to directly test potentially relevant proteins in heart failure in rabbits which suffered from dilated cardiomyopathy. The method does not require any time consuming generation of transgenic mouse lines and no cross-breeding.

The effect on disease-relevant parameters was investigated by echocardiography and catheter-based intracardiac measurements of hemodynamics (method description in: Weig HJ, Laugwitz KL, Moretti A, Kronsbein K, Städele C, Brüning S, Seyfarth M, Brill T, Schömig A, Ungerer M. Enhanced cardiac contractility after gene transfer of V2 vasopressin receptors in vivo by ultrasound-guided injection or transcoronary delivery. Circulation. 2000; 101(13):1578-1585).

Among a larger number of potential target proteins, some especially relevant proteins were identified. The figure shows that activation of some proteins improved contractility and outcome of heart failure (marked in green), while inhibition of some others worsened both parameters (marked in red). These proteins belong to the families of activating receptors, of calcium-regulating proteins, of apoptosis-(cell death)-regulating proteins, or of contractile proteins. Further work demonstrated that agents which were developed to target these proteins hold promise for the treatment of patients with heart failure.

Gene transfer to megacaryocytes/platelets

Retroviral gene transfer to megakaryocytes resulted in recombinant platelets (thrombocytes) which could be harvested and used for experimentation ex vivo and vivo, e. g. measuring their activation, aggregation and secretion, as well as thrombus formation (method described in: Ungerer M, Peluso M, Gillitzer A, Massberg S, Schulz C, Heinzmann U, Münch G, Gawaz M. Generation of functional culture-derived platelets from CD34+ progenitor cells to study transgenes in the platelet environment. Circulation Research 2004; 95:e36-e44)

Among a larger number of potential target proteins, some especially relevant proteins were identified. The figure shows that activation of some proteins diminished platelet activation and thrombus formation (marked in green), while inhibition of some others worsened both parameters (marked in red). These proteins belong to the families of adhesion receptors, of secretion-regulating proteins, or of serin-/threonin kinases.

Gene transfer to the endothelium and wall of blood vessels

After adenoviral gene transfer to cultured human vascular endothelial cells and to atherosclerotic arteries in vivo activation of endothelial cells and vasoreactivity was tested after a volume or parasympathic challenge by ultrasound measurements in vivo (method described in in: Bültmann A, Herdeg C, Li Z, Münch G, Baumgartner C, Langer H, Kremmer E, Geisler T, May A, Ungerer M, Gawaz M. Local delivery of soluble platelet collagen receptor glycoprotein VI inhibits thrombus formation in vivo. Thromb Haemost 2006: 95:763-766).

Also these investigations showed that activation of some specific proteins (as outlined in the Figure) diminished endothelial activation and inflammation, and improved vasoreactivity (marked in green), while inhibition of some others worsened these parameters (marked in red). These proteins belong to the families of cholesterol transport proteins, of reactive oxygen-regulating proteins, or of calcium channels.

References

Therapy of receptor-directed auto-immune disease by „inverse vaccination“/hyposensitization

Autoimmune diseases are frequently caused by specific auto-antibodies, which are probably being generated in response to a false pattern recognition of “self“ surface structures as „alien“ and dangerous, e.g. because co-stimulatory signals are being recognized simultaneously by the immune system, or because of pathogen cross-reactivity.

Since many years, allergies have been successfully treated by hyposensitization (=specific immune therapy). During this therapy, increasing allergen doses are being injected subcutaneously, or (in a few cases) administered as sublingual medication. This therapy leads to consecutive symptom improvement. Sometimes, allergen preparations are not homogenous because they were extracted from crude extracts.

Accordingly, our team seeks for even more specific agents for such an “inverse” vaccination, which mimic relevant epitopes (i.e. the targets of antibodies) in their 3D structure, and are composed of natural amino acids. These agents are especially pure, and can therefore be used for the intravenous therapy of systemic auto-immune diseases.

Therapy of thrombo-embolic diseases by activation-specific platelet inhibition

Platelets play a pivotal role in many acute or chronic cardiovascular diseases. They are pivotally involved in the formation of arterial thrombi, which can then further embolise to peripheral tissues, and most damagingly, to the brain.

Such arterial thrombi originate from endothelial erosions and rupturing plaques, where subendothelial matrix is being exposed to the arterial blood. At such sites, collagen activates platelets. An important process during local platelet aggregation is direct activation of glycoprotein (GP) VI, and via collagen bound von Willebrand factor.

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