Target-Identifizierung von 1,4-Dihydropyridinen als neuartige Inhibitoren des TGFβ-Signalwegs

The newly discovered b-annulated 1,4-dihydropyridines (1,4-DHPs) represent first-in-class small molecules for selective TGFβ inhibition. 1,4-DHPs exhibit a novel mode of action in a distinct but yet unknown mechanism to induce proteasomal degradation of TGFβ receptor type II. Nevertheless, the cellular target and the mode of action remain unknown. Building on SAR studies 1,4-DHP-based chemical probes have been designed. In the presented work, these probes were used for target identification and mechanistic studies through photoaffinity-labeling pulldown followed by a chemoproteomic analysis of enriched proteins.

Four photoprobes were characterized and validated as potent TβRII-degraders. Additionally, a PAL-pulldown protocol has been established. For this purpose, an optimized click reaction protocol was developed and further studies were carried out to enable an efficient Biotin-Streptavidin-based protein enrichment. Putative targets of the TβRII-degraders were identified using chemoproteomical analysis of photoprobe 1-dependant enriched proteins. Based on the enriched proteins it was shown that the TβRII-degraders most likely modulate specific protein-protein interactions (PPI). A siRNA-knockdown screen allowed the validation of putative targets.

In addition to target identification efforts for the 1,4-DHPs, two additional groups of possible TβRII-degraders were characterized. The 3,4-disubstituted indoles and the tetrahydrooxindoles were discovered via Scaffold Hopping based on the 1,4-DHP pharmacophore model. Both new indole scaffolds could be fully validated as TβRII-degraders.