Since the two DARPins are structurally and biophysically similar, this pair of molecules can act as useful tools to systematically explore the two mechanisms of Ras inhibition in different cancer cell backgrounds. To understand the potential for inhibition of nucleotide exchange as a therapeutic approach to Ras-dependent tumours, there are a number of further studies that would be valuable using intracellular expression of DARPin K27. that potently inhibits the nucleotide exchange of Ras, which might facilitate the development of Solanesol Ras-targeted therapies. Wild-type Ras GTPases cycle between an active, guanosine 5-triphosphate (GTP)-bound state and an inactive, guanosine 5-diphosphate (GDP)-bound state1,2,3,4. This is mediated by nucleotide exchange factors, such as Son of Sevenless (Sos), which catalyse the exchange of GDP for GTP and GTPase activating proteins, which potentiate the poor intrinsic Solanesol GTPase activity of the Ras protein5. Molecular activation triggered by extracellular stimuli such as epidermal growth factor (EGF) increases intracellular Ras-GTP levels and consequent conversation with downstream effector proteins. Cancer causing mutations impair the GTPase activity of Ras, leading it to accumulate in the activated state6,7,8. Cancer cells expressing mutant Ras are dependent on Ras for proliferation and survival. The RAS gene family, comprisingHRAS,KRASandNRAS, is the most frequently mutated oncogene family found in human tumours, especially pancreatic, lung and colon cancers3,4,9. Despite the prevalence of RAS Rabbit Polyclonal to LDOC1L mutations in cancer, no therapies that directly target the oncoprotein are currently available in the clinic. The Ras molecule contains a number of sites essential to its function. The P-loop (residues G10-S17) is responsible for phosphate binding, whereas the Switch I (D30-D38) and Switch II regions (G60-E76) are critical for interactions with guanosine nucleotide exchange factors and effector proteins3,10,11. Three approaches demonstrated to directly modulate Ras function within the cell are: increasing hydrolysis rates of mutant Ras12,13, blocking the conversation between active Ras and downstream effectors such as Raf, or reducing the amount of active Ras within the cell by inhibiting nucleotide exchange. The theory of inhibition of the Ras/Raf conversation has been supported by studies with the small molecule Ras-mimetic rigosertib14, currently in phase III clinical trials for myelodysplastic syndrome, and a single domain name antibody iDab6, which binds to the switch 1 site15,16,17. When expressed within the cell, iDab6 inhibits signalling downstream of Ras, can revert the transformed phenotype and, in mouse models, reduces growth of Ras-driven tumours. In contrast, the effects of blocking nucleotide exchange on Ras function in cells are thought to be restricted by the low GTPase activity of Ras mutants. Peptides and small molecules that bind directly non-covalently to Ras and inhibit interactions with Sos have been reported18,19,20together with initial effects on pERK signalling. Recently, however, a potent small molecule ARS-853 has been described that specifically inhibits nucleotide exchange by G12C mutants of K-Ras by covalent modification at the cysteine residue. ARS-853 reduces the amount of active Ras, increases cleavage of PARP and triggers apoptosis21,22. The potency of ARS-853 Solanesol in reducing active Ras levels by >90% and substantially inhibiting both the ERK and AKT effector pathways has led some in the Ras field to re-evaluate the potential of nucleotide exchange inhibition, particularly for Ras mutants with intrinsically Solanesol higher rates of GTP hydrolysis23. To increase understanding of the effects of non-covalent inhibition of nucleotide exchange on multiple isoforms and mutant forms of Ras, we have isolated a pan-reactive antibody mimetic, Designed Ankyrin Repeat Protein (DARPin) K27, which potently inhibits nucleotide exchange, and also a comparator, DARPin K55, which inhibits the Ras/Raf conversation. DARPins are small (< 20 Solanesol kDa), antibody mimetic proteins which can bind to antigens with high affinity and specificity but, unlike antibodies, are particularly suited for targeting intracellular proteins since they lack cysteines and are highly stable allowing functional binding against intracellular targets24,25. Intracellular expression of the DARPins reported.