title: Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53-MDM2 interaction.
abstract: Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable d-isomers to recapitulate biological activities of their parent l-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent l-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical l-peptide on its target protein binding has been estimated to be 3.0-3.4kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX-the two negative regulators of p53. We also prepared the retro isomer and d-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of l-stingin weakened its MDM2 binding by 720 fold (3.9kcal/mol); while enantiomerization of l-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural level.(1.)
authors: College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
authors: Chong
authors: C
authors: Li
authors: Changyou
authors: C
authors: Zhan
authors: Le
authors: L
authors: Zhao
authors: Xishan
authors: X
authors: Chen
authors: Wei-Yue
authors: WY
authors: Lu
authors: Wuyuan
authors: W
authors: Lu
mesh_terms: D002942:Circular Dichroism; D006801:Humans; D007536:Isomerism; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D010455:Peptides; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
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