Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange

Sandrine Guillard,Nicolas Bery,Judit Debreczeni,Jason Breed,Ronald Jackson,Paulina Kolasinska-Zwierz,Jing Zhang,Jon Tart,Bina Mistry,Christopher Phillips,Rose Marwood,Pawel Stocki,Ross Overman,Terence Rabbitts,Ralph Minter

doi:10.1038/ncomms16111

Abstract

Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies. Inhibition of Ras nucleotide exchange is a promising new approach but better understanding of this mechanism of action is needed. Here we describe an antibody mimetic, DARPin K27, which inhibits nucleotide exchange of Ras. K27 binds preferentially to the inactive Ras GDP form with a Kd of 4 nM and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 significantly reduces the amount of active Ras, inhibits downstream signalling, in particular the levels of phosphorylated ERK, and slows the growth in soft agar of HCT116 cells. K27 is a potent, non-covalent inhibitor of nucleotide exchange, showing consistent effects across different isoforms of Ras, including wild-type and oncogenic mutant forms.

Highlights

Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies
Wild-type Ras GTPases cycle between an active, guanosine 50-triphosphate (GTP)-bound state and an inactive, guanosine 50-diphosphate (GDP)-bound state[1,2,3,4]. 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 weak intrinsic GTPase activity of the Ras protein[5]
Designed Ankyrin Repeat Protein (DARPin) K27 was selected as the nucleotide exchange inhibitor and DARPin K55 as the Ras/Raf inhibitor

Summary

Introduction

Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies. Three approaches demonstrated to directly modulate Ras function within the cell are: increasing hydrolysis rates of mutant Ras[12,13], blocking the interaction between active Ras and downstream effectors such as Raf, or reducing the amount of active Ras within the cell by inhibiting nucleotide exchange. 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 a comparator, DARPin K55, which inhibits the Ras/Raf interaction. Intracellular expression of the DARPins reported in this paper has allowed investigation of the effects of a potent inhibitor of nucleotide exchange at Ras on levels of active Ras and Ras function

Methods

Results

Conclusion