Universität zu Köln

Lindenblatt, Dirk ORCID: 0000-0002-5561-1082 (2022). Structural Exploration of Different Binding Pockets Suitable to Affect Protein Kinases CK2α and CK2α’ With Peptides and Small Molecules. PhD thesis, Universität zu Köln.

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Abstract

The eukaryotic protein kinase CK2, previously known as casein kinase 2, is a ubiquitously expressed, acidophilic serine/threonine kinase belonging to a branch of the group of CMGC kinases. The enzyme features several peculiarities, one of which is its extraordinary pleiotropic character. A plethora of biological substrates have been described for CK2 to date and these are, among others, involved in cell proliferation, angiogenesis, apoptotic processes, viral infections, and DNA-damage repair. Several of these substrates play key roles in the development and progression of a diverse spectrum of diseases. The ubiquitous presence of CK2, combined with its unusual constitutive activity, presents a highly interesting pharmacological profile as a promising drug target. In particular, neoplastic diseases are significantly driven by high levels of CK2 and the importance of the search for suitable molecules to alter the enzymatic properties of CK2 is therefore evident and a subject of current research. This thesis also contributes to this field, focusing primarily on the investigation of the structural aspects of various protein-ligand interactions at different binding sites of the enzyme. An important role in these structural studies is accounted by CK2α’, a paralog of the catalytic subunit CK2α. Although the two paralogs are highly similar in many respects, CK2α’ has been neglected in CK2 research over the past decades due to its poor biochemical handleability and its insufficient crystallization properties. Therefore, in this work, for the first time, a crystallization protocol was developed that reliably yields CK2α structures with an atomic resolution of 1.0 Å and thereby outperforming all previously existing CK2α structures to date. This protocol has proven to be an extremely valuable crystallographic tool to study the precise binding mode of a wide variety of CK2 inhibitors from different substance classes, including high and low-affinity compounds. As an example, the exact binding site and binding mode of different 2 aminothiazole derivatives could be elucidated. These compounds belong to a class of CK2 inhibitors that were erroneously assumed to bind outside the cosubstrate pocket. In addition to crystallographic studies, organic syntheses were also conducted as part of the research for this thesis. This includes the synthesis of halogenated cyclic peptides which address the α/β interface area of the catalytic subunits, interfering with CK2β binding and thus with the tetrameric holoenzyme assembly. Furthermore, by conjugating with the cell-penetrating peptide sC18, it was possible to investigate the impact of some of these compounds on different cell lines. Moreover, different 4,5,6,7 tetrabromobenzimidazole derivatives were synthesized and studied. In particular, the bivalent inhibitor KN2, which simultaneously occupies the cosubstrate binding pocket as well as the recently discovered αD binding pocket, proved to be exceptionally high in affinity and outstandingly selective. The aspect of selectivity has always been a particular challenge for kinase inhibitors due to the high degree of conservation of the cosubstrate binding region among eukaryotic protein kinases. The inclusion of the αD binding pocket is currently one of the most promising approaches to overcome this challenge since the high plasticity in this region has exclusively been described for CK2. In this thesis, it was shown for the first time that this is not only true for CK2α, but rather for both paralogs. Finally, the crystallization successes with CK2α’ and an eight-month desalting procedure led to the discovery of a novel binding site, located close to the N-terminus. However, the suitability of this cryptic site for the design of future generations of CK2 inhibitors requires further studies.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Lindenblatt, Dirk UNSPECIFIED orcid.org/0000-0002-5561-1082 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-652457
Date:	2022
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	Natural sciences and mathematics Chemistry and allied sciences Life sciences
Uncontrolled Keywords:	Keywords Language Kinase UNSPECIFIED CK2 UNSPECIFIED Casein Kinase II UNSPECIFIED CK2α′ UNSPECIFIED CSNK2A2 UNSPECIFIED CK2α UNSPECIFIED CSNK2A1 UNSPECIFIED ATP-competitive inhibitors UNSPECIFIED Bivalent inhibitors UNSPECIFIED
Date of oral exam:	13 February 2023
Referee:	Name Academic Title Niefind, Karsten Prof. Dr. Baumann, Ulrich Prof. Dr. Wünsch, Bernhard Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/65245