Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.

The plasma membrane barrier greatly restricts intracellular delivery of macromolecules. Currently available methods suffer from various limitations, including low delivery efficiency, high cytotoxicity, or incompatibility with a wider range of macromolecules or cell types. To overcome these issues, stimuli‐responsive polymers such as the bio‐inspired, pH‐responsive poly(l‐lysine isophthalamide) grafted with l‐phenylalanine at a stoichiometric ratio of 50% (PP50) can be used. In mildly acidic environments, the pseudopeptidic polymer can permeabilize the plasma membrane overcoming the problem of payload entrapment in the endosomes and allowing for efficient intracellular delivery. It is demonstrated that PP50 is capable of intracellular delivery by simple co‐incubation at pH 6.5 with various macromolecules, including different‐sized Dextrans, green fluorescent protein (GFP), and an apoptotic peptide. The delivery process is fast, nontoxic, and compatible with multiple cell types, including adherent and suspension cell lines, primary human mesenchymal stem cells, and cells grown as spheroids. In addition, apoptotic peptide delivery by co‐incubation with PP50 is over three times more effective than delivery using other common methods, including poly(ethyleneimine) (PEI), cell penetrating peptides (CPPs), and electroporation. The findings suggest that payload delivery by co‐incubation with PP50 is a flexible, controllable method allowing delivery of various payloads to many different cell types in vitro.

Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a novel CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.