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

Autor: Cecilia Balbi, Enrico Millo, Barbara Carnemolla, Lorenzo Tortolina, B. Biasotti, Melchiori A, C. Avignolo, Maria Pia Pescarolo, Paola Barboro, Gianfranco Fassina, Guido Cimoli, Silvio Parodi, Erika Nieddu, Nicoletta Castagnino, Davide Malacarne, Francesca Vasile, M. Mazzei, B. Licheri, Luca Bagnasco, Eligio Patrone, Paola Orecchia, D. Palomba, Cristina D'Arrigo, R. Ponassi, Gianluca Damonte, Stefania Pasa
Jazyk: angličtina
Rok vydání: 2005
Předmět:
Genetics and Molecular Biology (all)
Circular dichroism
Protein Denaturation
Protein Structure
Secondary
Hot Temperature
Magnetic Resonance Spectroscopy
Peptidomimetic
Stereochemistry
Molecular Sequence Data
Apoptosis
Breast Neoplasms
Fluorescence Polarization
Biochemistry
Protein Structure
Secondary
Cell Line
Proto-Oncogene Proteins c-myc
Structure-Activity Relationship
Protein structure
Drug Stability
Cell Line
Tumor
Genetics
Structure–activity relationship
Humans
Amino Acid Sequence
Molecular Biology
Peptide sequence
Fluorescent Dyes
Alanine
Tumor
Chemistry
Rhodamines
Circular Dichroism
Nuclear magnetic resonance spectroscopy
Cell Biology
Agricultural and Biological Sciences (miscellaneous)
Basic-Leucine Zipper Transcription Factors
Cell Division
Colonic Neoplasms
Dimerization
Fluorescein
Peptides
Biochemistry
Genetics and Molecular Biology (all)
Helix
Biotechnology
Zdroj: The FASEB journal 19 (2005). doi:10.1096/fj.04-2369fje
info:cnr-pdr/source/autori:Nieddu E.; Melchiori A.; Pescarolo M.P.; Avignolo C.; Bagnasco L.; Balbi C.; Barboro P.; Biasotti B.; Carnemolla B.; Castagnino N.; Cimoli G.; Damonte G.; D'Arrigo C.; Fassina G.; Licheri B.; Malacarne D.; Orecchia P.; Palomba D.; Pasa S.; Patrone E.; Ponassi R;Scapolla A.; Tortolina L.; Vasile F.; Mazzei M.; Parodi S./titolo:Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-myc/doi:10.1096%2Ffj.04-2369fje/rivista:The FASEB journal/anno:2005/pagina_da:/pagina_a:/intervallo_pagine:/volume:19
Popis: 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 Daa, 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 similar to 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 - sheet ( CD and NMR data).
Databáze: OpenAIRE
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