Strukturbasiertes Design und Entwicklung kovalenter Kinaseinhibitoren zur Umgehung der durch EGFR-T790M vermittelten Wirkstoffresistenz im nicht-kleinzelligen Lungenkarzinom

The epidermal growth factor receptor (EGFR), a member of the ErbB family, is a receptor tyrosine kinase that represents a key mediator in critical cellular signaling processes such as cell proliferation, cell cycle regulation as well as apoptosis. Therefore, dysregulation of EGFR plays a pivotal role in the onset and progression of cancer. Mutations in the gene encoding the EGF receptor have been discovered as oncogenic drivers in non-small cell lung cancer (NSCLC). These somatic kinase domain mutations accounting for increased tyrosine kinase activity are associated with increased cell proliferation and tumor growth. Specific inhibition of oncogenic EGFR mutant variants results in tumor remission. NSCLC patients harboring activating mutations such as the L858R point mutation or the exon 19 deletion showed a dramatic clinical response to reversible first-generation EGFR inhibitors gefitinib und erlotinib in the course of personalized medicine. However, acquired drug resistances limit the effective treatment with tyrosine kinase inhibitors. In more than 60 % of resistant cases, the relapse of disease is associated with an emerging secondary point mutation at the gatekeeper residue (T790M). However, covalent EGFR inhibitors demonstrated the potential to overcome T790M drug resistance by covalently targeting a unique Cysteine (Cys797) at the lip of the EGFR ATP-binding cleft. This thesis describes the structure-guided development of covalent T790M-specific EGFR inhibitors. Based on a reversible binding hit identified in a phenotypic screen, structure-based design led to the synthesis of pyrimidine-based inhibitors. They revealed a novel binding mode in EGFR and were demonstrated to covalently target the T790M drug-resistant mutant variant with low nanomolar inhibitory activities. Although the pyrimidine-based inhibitors targeted EGFR in a covalent and irreversible fashion, the strong inhibitory effects in biochemical evaluations could not be translated into cellular potency in cancer cell lines. Their moderate cellular efficacy is likely to result from insufficient cellular permeability and a dramatic efflux rates in conjunction with slow covalent bond formation as observed in kinetic studies. These insights triggered the structure-based de novo design of a new focused library of covalent T790M inhibitors of which a subset illustrated subnanomolar kinase inhibition as well as highly potent cellular efficacy in T790M drug-resistant H1975 cells. Moreover, these pyrazolopyrimidine-based inhibitors demonstrated a direct inhibitory effect on EGFR autophosphorylation and its downstream signaling targets in the H1975 cell line. Complex crystal structures with EGFR-T790M revealed a unique binding mode providing a structural explanation for the excellent potency of that series. Excellent selectivity profiles together with a suitable in vitro ADME/DMPK profile allowed an investigation of the pharmacokinetics of this compound class in vivo. In detail, high exposure by the intraperitoneal route could be achieved providing a promising plasma concentration that encourages further in vivo efficacy studies. This thesis also describes the establishment of the conceptual study CovClick (covalent in situ click chemistry) as basis for a novel structure-finding system to identify new covalent kinase inhibitors by target-guided synthesis (TGS). The CovClick approach is based on an irreversible modification of a unique cysteine in the binding site of the target protein by a covalent probe. This probe is equipped with an alkyne moiety and subsequently undergoes an in situ click reaction with azide fragments in close proximity. Utilizing structure-based design, a CovClick probe for MKK7 as model kinase was designed and synthesized. Protein-MS-analysis of the covalent probe demonstrated an irreversible binding mode in MKK7 that was confirmed by a complex crystal structure. An initial CovClick-Screen revealed a hit structure that was synthesized for further validation studies. Mass spectrometry experiments confirmed a covalent modification of MKK7 by the identified compound. This research provides the basis for further optimization studies and applications of the CovClick method to identify new covalent inhibitors