title: Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.
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).
authors.affiliation: Department of Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, Genoa 16132, Italy. erika.nieddu@unige.it
authors.forename: Erika
authors.initials: E
authors.lastname: Nieddu
authors.forename: A
authors.initials: A
authors.lastname: Melchiori
authors.forename: M P
authors.initials: MP
authors.lastname: Pescarolo
authors.forename: L
authors.initials: L
authors.lastname: Bagnasco
authors.forename: B
authors.initials: B
authors.lastname: Biasotti
authors.forename: B
authors.initials: B
authors.lastname: Licheri
authors.forename: D
authors.initials: D
authors.lastname: Malacarne
authors.forename: L
authors.initials: L
authors.lastname: Tortolina
authors.forename: N
authors.initials: N
authors.lastname: Castagnino
authors.forename: S
authors.initials: S
authors.lastname: Pasa
authors.forename: G
authors.initials: G
authors.lastname: Cimoli
authors.forename: C
authors.initials: C
authors.lastname: Avignolo
authors.forename: R
authors.initials: R
authors.lastname: Ponassi
authors.forename: C
authors.initials: C
authors.lastname: Balbi
authors.forename: E
authors.initials: E
authors.lastname: Patrone
authors.forename: C
authors.initials: C
authors.lastname: D'arrigo
authors.forename: P
authors.initials: P
authors.lastname: Barboro
authors.forename: F
authors.initials: F
authors.lastname: Vasile
authors.forename: P
authors.initials: P
authors.lastname: Orecchia
authors.forename: B
authors.initials: B
authors.lastname: Carnemolla
authors.forename: G
authors.initials: G
authors.lastname: Damonte
authors.forename: E
authors.initials: E
authors.lastname: Millo
authors.forename: D
authors.initials: D
authors.lastname: Palomba
authors.forename: G
authors.initials: G
authors.lastname: Fassina
authors.forename: M
authors.initials: M
authors.lastname: Mazzei
authors.forename: S
authors.initials: S
authors.lastname: Parodi
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry; D001943:Breast Neoplasms; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D003110:Colonic Neoplasms; D019281:Dimerization; D004355:Drug Stability; D019793:Fluorescein; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006358:Hot Temperature; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D005456:Fluorescent Dyes; C489427:MYC protein, human; C067850:Myc associated factor X; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D012235:Rhodamines; C029773:rhodamine B; D019793:Fluorescein