Covalent chemical probes for the neglected Ribosomal Protein S6 Kinase Beta 2 (S6K2)

• Applicant: Professor Dr. Matthias Gehringer
• Subject Area: Pharmacy
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 511101075

The family of p70 ribosomal protein S6 kinases comprises the two isoforms S6K1 (p70S6K) and S6K2 (p70S6Kβ), which act downstream of the PI3K-AKT-mTOR pathway. S6K1 has been thoroughly investigated as an anticancer target. Since early studies concluded that S6K1 and S6K2 have redundant functions with S6K1 being the dominant isoform, S6K2 has remained heavily understudied. However, more recent data shows that these isoforms have different roles in cancer biology. S6K2 has been associated with poor prognosis and therapy resistance in breast and prostate cancers. It has also been shown that survival of NRAS-mutant melanoma cells resistant to mitogen-activated protein kinase inhibitors crucially depends on S6K2, suggesting S6K2 inhibition as a treatment strategy. Several lines of evidence indicate that concomitant S6K1 inhibition abrogates the benefits of S6K2 inhibition. Thus, detailed pharmacological studies of S6K2 biology will require highly isoform-selective chemical probes. Due to the almost identical ATP binding sites of S6K1 and S6K2, design of selective inhibitors is challenging. We have recently developed the first isoform-selective S6K2 inhibitor. Covalent targeting of a poorly conserved cysteine, which is absent in S6K1, via nucleophilic aromatic substitution (SNAr) chemistry was key to success. Despite nanomolar potency and a promising selectivity profile, our prototype inhibitor has limitations including insufficient non-covalent binding affinity, moderate solubility and limited druglikeness. Moreover, and despite being very versatile covalent-reactive groups, SNAr electrophiles are underinvestigated in the context of covalent inhibitors and lack guidelines for their optimization. Here, we aim to develop high-quality chemical probes enabling the investigation of S6K2's role in health and disease. We will thoroughly optimize the covalent reactive group to make our compounds druglike. Investigation of structure-activity relationships and covalent inactivation kinetics will provide valuable insights which will help to broaden the scope and applicability of SNAr electrophiles in covalent inhibitor design. By optimizing the piperazine-linked "upper side chain" and the "hinge-binding motif" of our prototype compound, we will increase binding affinity and solubility. Inhibitor synthesis will be facilitated by the strategies developed in our preliminary work. Compound characterization will build on a broad array of biochemical, biophysical, and cellular assays and advanced inhibitors will undergo comprehensive selectivity profiling. In combination, our optimizations are expected to provide inhibitors fulfilling the community's stringent criteria for high-quality chemical probes. Accompanied by suitable negative control compounds, we plan to make our probe set openly available. This will lay the basis for pharmacological validation of S6K2 as a drug target and may open new avenues in cancer treatment and for combatting therapy resistance.

• DFG Programme: Research Grants
• International Connection: United Kingdom
• Cooperation Partner: Dr. Olivier Pardo
• Co-Investigator: Professor Dr. Stefan Knapp