This proposal is centred on the development and application of chemical tools to probe molecular recognition of multiprotein complexes. Although much effort has been devoted to targeting protein-protein interactions using small molecules, these have focused to date on individual gene products or truncated domains, which however do not reflect the physiological organization and activity of many functional proteins. Few successes have been achieved, yet many of the key physical determinants of druggability of surfaces within native protein complexes have remained elusive. The aim of this project is to shed light upon this problem by chemically interrogating biological systems that rely on several subunits working in concert rather than on single proteins working alone. As model system we will investigate the Cullin RING Ligases (CRLs), the largest superfamily of multisubunit E3 ligases in humans. These enzymatic machines are responsible for the recognition, poly-ubiquitination and targeting of substrate proteins to the proteasome for degradation. Many members of this family have crucial roles in cellular physiology and homeostatis, are implicated in a wide range of diseases and are attractive targets for drug discovery. Two interdependent lines of enquiry will be followed. First, we will screen for and elucidate the binding of small molecular fragments and short peptides to identify new druggable surfaces and interfaces on CRLs and their components. Second, we will exploit the nature of the interactions to develop novel chemical probes of CRLs. As the probes are selected and optimised for binding rather than for a particular functional outcome, diverse mechanisms of action are envisaged beyond conventional disruption of the interaction. The successes of this interdisciplinary research will provide a step change in how we interrogate protein-protein interactions of functional and pathological pathways with impact in many areas of chemical biology and drug discovery.