Design and Synthesis of Covalent Inhibitors Targeting Cysteines in the Hinge Region of the Protein Kinases S6K2 and MPS1

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Dokumentart: PhDThesis
Date: 2025-03-10
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Pharmazie
Advisor: Gehringer, Matthias (Jun.-Prof. Dr.)
Day of Oral Examination: 2023-03-10
DDC Classifikation: 500 - Natural sciences and mathematics
540 - Chemistry and allied sciences
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Die Dissertation ist gesperrt bis zum 10. März 2025 !


The family of p70 ribosomal protein S6 kinases (or S6K) belongs to the group of AGC (protein kinase A, G and C) serine/threonine kinases and includes the two highly homologous isoforms S6K1 and S6K2. The family is named after the phosphorylation of the ribosomal protein S6, which, as a component of the 40S ribosomal subunit, plays an important role in protein synthesis and cell cycle progression. The exploration of S6Ks revealed further functions related to cell proliferation, transcription and signal transduction. Initially, a redundant biological functional profile of the isoforms was assumed, with the S6K1 isoform considered the prototypical form. This assessment led to a significant neglect of S6K2 in S6K-related studies. The preference for S6K1 was particularly evident in the development of isoform-selective inhibitors, which so far have only yielded S6K1-selective inhibitors. However, subsequent sparse studies on S6K2 function revealed the distinct roles of the isoforms, highlighting the particular role of S6K2 as a potential target of therapeutic intervention in cancer. In this work, we aimed to develop isoform-selective S6K2 inhibitors using an S6K2 isoform-specific cysteine (Cys150; Tyr174 in S6K1) in the hinge region of the ATP-binding pocket as a selectivity vector. For this purpose, suitable reversibly binding core scaffolds were linked to electrophilic aromatic fragments (so-called SNAr-warheads), which can form a covalent bond to nucleophiles such as a thiol. In this work, a variety of inhibitors with attached SNAr-warheads were synthesised that inhibited S6K2 with high potency (IC50 < 1 nM). Excellent selectivity over the S6K1 isoform as well as kinases with equivalently positioned cysteine was observed. The covalent binding mode was demonstrated by the significantly lower inhibitory potency of the unreactive control compounds and the non-promiscuous reactivity of the warheads was confirmed. The results validated the design hypothesis towards isoform-selective S6K2 inhibitors using SNAr warheads and represent an important step towards a high-quality chemical probe enabling the exploration of the biological function of S6K2 by pharmacological means. The monopolar spindle 1 (MPS1) kinase activates the spindle assemble checkpoint and regulates essential functions in monitoring correct chromatid separation during cell division. MPS1 is overexpressed in several cancers and leads to increased survival of aneuploid cancer cells. In this work, we aimed to develop MPS1 inhibitors that covalently address a cysteine in the hinge region (Cys604) of the ATP-binding pocket using SNAr-warheads to achieve high residence times. A series of compounds were synthesised that differed in their substitution at the SNAr-warhead. Trends in the structure-activity relationship were derived from the inhibition data obtained, but only low inhibition values were observed, suggesting no efficient covalent binding mode.

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