The P2X7 receptor (P2X7R) stands out among the purinergic receptors due to its strong involvement in the regulation of tumor growth and metastasis formation as well as in innate immune responses... Show moreThe P2X7 receptor (P2X7R) stands out among the purinergic receptors due to its strong involvement in the regulation of tumor growth and metastasis formation as well as in innate immune responses and afferent signal transmission. Numerous studies have pointed out the beneficial effects of P2X7R antagonism for the treatment of a variety of cancer types, inflammatory diseases, and chronic pain. Herein we describe the development of novel P2X7R antagonists, incorporating piperazine squaric diamides as a central element. Besides improving the antagonists' potency from pIC50 values of 5.7-7.6, ADME properties (logD7.4 value, plasma protein binding, in vitro metabolic stability) of the generated compounds were investigated and optimized to provide novel P2X7R antagonists with drug-like properties. Furthermore, docking studies revealed the antagonists binding to the allosteric binding pocket in two distinct binding poses, depending on the substitution of the central piperazine moiety. Show less
The main theme of this thesis, allosteric modulation effectuated through the sodium ion site of GPCRs, is inspired by the important role that this site appears to play in GPCR signaling. As... Show moreThe main theme of this thesis, allosteric modulation effectuated through the sodium ion site of GPCRs, is inspired by the important role that this site appears to play in GPCR signaling. As sodium ions are abundant under physiological conditions they may affect GPCR signaling considerably. Receptor activation causes a substantial rearrangement of the sodium ion site, suggesting an important role in this process. Chapter 2 reviews the current knowledge on allosteric modulation of amiloride and its derivatives binding to the sodium ion site of Class A GPCRs. Chapters 3 to 5 follow-up on the recent crystal structure of the adenosine A2A receptor with a sodium ion bound. Chapters 3 and 4 complement the crystal structure with additional results from combined biochemistry, biophysical, molecular dynamics, and mutational studies. Chapter 5 describes the synthesis of novel amiloride derivatives that bind in the sodium ion site but also protrude into the orthosteric binding site. In Chapters 3 to 5, radio-labeled ligands were used to quantify ligand binding to the receptor, and Chapter 6 describes an alternative approach towards ligand binding assays. Instead of using a radio-label, mass spectrometry was used to quantify binding of an unlabeled ligand to adenosine A1 and A2A receptors. Show less
