Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc

FASEB J. 2005 Apr;19(6):632-4. doi: 10.1096/fj.04-2369fje. Epub 2005 Jan 25.

Abstract

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apoptosis
  • Basic-Leucine Zipper Transcription Factors / chemistry
  • Breast Neoplasms
  • Cell Division / drug effects
  • Cell Line, Tumor
  • Circular Dichroism
  • Colonic Neoplasms
  • Dimerization
  • Drug Stability
  • Fluorescein
  • Fluorescence Polarization
  • Fluorescent Dyes
  • Hot Temperature
  • Humans
  • Magnetic Resonance Spectroscopy
  • Molecular Sequence Data
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / pharmacology*
  • Protein Denaturation
  • Protein Structure, Secondary*
  • Proto-Oncogene Proteins c-myc / analysis
  • Proto-Oncogene Proteins c-myc / chemistry*
  • Rhodamines / chemistry
  • Structure-Activity Relationship

Substances

  • Basic-Leucine Zipper Transcription Factors
  • Fluorescent Dyes
  • MYC protein, human
  • Myc associated factor X
  • Peptides
  • Proto-Oncogene Proteins c-myc
  • Rhodamines
  • rhodamine B
  • Fluorescein