The p53 protein is known as the “Guardian of the Genome” and is a natural tumor suppressor. It reacts to cell stress and DNA damage by initiating transcription of genes which regulate the cell cycle, autophagy, metabolism, DNA repair, and apoptosis. This mechanism results in functional p53 having a natural tumor suppressing ability. This natural ability is impeded in virtually all cancers, making it an enticing target for therapies. Roughly half of all cancers contain mutated p53, with most mutations occurring in the DNA-binding domain, with the other half retaining wild-type p53. These WT p53 are often suppressed in one way or another in cancer, often by increased levels of the primary regulator MDM2. Disrupting the interaction of MDM2 and p53 has therefore been a long-standing goal in cancer research. This is made difficult by the fact that MDM2 binds p53 at the disordered transactivation domain (TAD), where traditional structural biology-based drug development methods fall short. Attention was instead turned to binding the ordered interface of MDM2 instead, yielding plenty of binders, but with limited clinical success.
Here we report a novel De Novo designed protein which directly binds the disordered p53 TAD and the process of its creation. Using a modified version of the Logos pipeline from Wu et al. [1], hundreds of potential binder designs were generated and screened for binding in silico. The top ten scoring designs were expressed and purified for further testing. Nine of the designs expressed correctly and bound their intended target, as evidenced by native mass spectrometry and biolayer interferometry (BLI). The binders function via three different conformations being induced in the otherwise disordered TAD; helical, strand, or stabilized coil. Keeping the TAD in these conformations allows the binders to form a large number of hydrogen bonds with it, resulting in strong binding affinities, with 13 nM being measured for the strongest binder using BLI. These proteins are small enough (19-29 kDa) that many forms of cellular delivery are feasible, with in vivo experiments ongoing to study the effect that might have.