Identification and characterisation of cyclic peptide inhibitors of Ras/PI3K and Ras/Raf protein complexes


Year:

Session type:

Theme:

Mohamed (Soly) Ismail1,Mohamed Ismail1,Julian Downward1
1The Francis Crick Institute

Abstract

Background

Ras oncogenic mutations are present in almost 25% of human cancers – indicative of their involvement in important signalling pathways that regulate many cellular functions such as: cell growth, proliferation, differentiation and survival. PI3K and Raf Kinases control two of the main effector signalling pathways that are activated by Ras proteins. There have been long efforts in discovering drugs for Ras mutants however these drugs either lacked strong binding affinity or specificity to Ras mutants. Here, in collaboration with Ali Tavassoli’s lab at Southampton University, we apply a new approach for discovering inhibitors that can block the Ras/Raf and Ras/PI3K interactions. The technique gives a great advantage of the possibility to discover inhibitors against Ras, Raf or PI3K proteins at the same time, rather than following the traditional route of screening drugs against Ras in isolation. In addition, because the effector-binding site of Ras is negatively charged and both of the binding sites of Raf and PI3K with Ras are positively charged – this opens up the opportunity for both negatively and positively charged inhibitors to compete with both interactions. The technique we are applying combines two methodologies: The “split-intein cyclisation of peptides and proteins” (SICLOPPS), for producing random cyclic peptides in vivo; and a bacterial reverse two-hybrid system (RTHS), can be used for high throughput screening (HTS). SICLOPPS has the capacity to produce millions of random cyclic peptides, which significantly increases the chances of finding suitable inhibitors, to interrupt protein complexes. Alongside this the RTHS provides a time efficient and effective technique to screen such a high number of peptides. We have developed a robotic HTS process that allows the screening of thousands of peptides per day. Initial characterisation of positive peptides that disrupt the interaction of the Ras/Raf complex is currently in progress.