Photo-Control of RAS Nucleotide Exchange Reaction using Peptide Inhibitor Modified with Spiropyran Derivative

Abstract

Ras is a kind of GTPase protein, and it regulates cellular signal transduction. For example, some information such as cell growth and cell differentiation from in vitro to in vivo of downstream by cycling between GTP active form and GDP inactive form. GDP-GTP exchange of Ras is induced by binding of GEF(Guanine Exchange Factor) to Ras. Recently some kinds of peptide which bind to GEF binding region or allosteric binding region. Then, they cause inhibition of nucleotide exchange, were reported. It is known that mutated Ras (G12D etc.) continues transducing permanently, so it is the factor of making cancer cells. Then, it is called cancer gene. From this, the study that develops molecule target medicine only abnormality protein is recognized and controlled is attempted so as not to affect normal cells. Photochromic molecule, spiropyran isomerize between ring-closed spiro (SP) and ring-opened merocyanine(MC) forms photo-reversibly upon visible and ultraviolet lights. It is known that MC form of spiropyran has a polarity, so it exhibits electric interaction each other at peptide. Therefore, peptide also change the secondary structure between alpha-helix and random coil. In this study, we designed and synthesized peptide inhibitor of Ras based on interface structure between Ras and GEF. The synthesized petide RRFCGIYCTNACKTCEGNRR composed of four cysteine residues. These cysteine residues were modified with SP-maleimide(SP-MA) stoichiometrically. The secondary structural change of the SOS mimic peptide induce by photo-isomerization of SP-MA were examined by CD spectrum. The photo-control of nucleotide exchange of Ras using the peptide inhibitor was examined. We also synthesized several peptide inhibitors which have different number of cysteine residues and examined the inhibitory effect of the peptides.