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

Erika Nieddu^*,1, A. Melchiori^#, M. P. Pescarolo^#, L. Bagnasco^#, B. Biasotti, B. Licheri, D. Malacarne^#, L. Tortolina, N. Castagnino, S. Pasa^,#, G. Cimoli, C. Avignolo, R. Ponassi, C. Balbi^#, E. Patrone, C. D’arrigo, P. Barboro^#, F. Vasile, P. Orecchia^¶,, B. Carnemolla^¶, G. Damonte, E. Millo, D. Palomba^||||, G. Fassina^||||, M. Mazzei^* and S. Parodi^,#

^* Department of Pharmaceutical Sciences, University of Genoa;
^# Laboratory of Experimental Oncology, National Cancer Research Institute of Genoa;
Department of Oncology, Biology and Genetics, University of Genoa;
Institute for the Study of Macromolecules, Genoa Section;
Department of Biophysical Sciences and Technology, University of Genoa;
Department of Experimental Medicine, Biochemistry Section c/o Center of Excellence for Biomedical Research, Genoa, Italy;
^|||| Xeptagen S.p.A., Napoli, Italy;
^¶ Laboratory of Cell Biology, National Cancer Research Institute of Genoa; and
Institute Giannina Gaslini, Genova, Italy

¹Correspondence: Department of Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, Genoa 16132, Italy. E-mail: erika.nieddu{at}unige.it

SPECIFIC AIMS

We have reported that a retro-inverso (RI) form of helix1 (H1) of Myc linked to a RI internalization sequence inspired from the third helix of Antennapedia (Int), RI-Int-H1-S6A,F8A, is endowed with antiproliferative and proapoptotic activity toward cancer cell lines MCF-7 and HCT-116. The activity apparently depended on the presence of the Myc motif.

The aims of the present work were to 1) synthesize variant molecules of our original lead [each side chain of the D-amino acids (D-aa) was exchanged with the methyl group of an alanine; we tested two D-proline substituted molecules; we tested a longer RI-Int-H1-S6A,F8A-loop-H2 peptido-mimetic molecule]; 2) investigate the Myc/Max dimerization domain at the level of circular dichroism spectroscopy (CD) and anisotropy to understand the relationships between physicochemical effects and biological activity of our molecules; 3) analyze the structure of our peptido-mimetic molecules at the level of circular dichroism spectroscopy and nuclear magnetic resonance (NMR) in relation to their different potencies in terms of antiproliferative activity; 4) check the molecules’ ability to cross the cytoplasmic membrane and concentrate inside living cells to determine whether differences in potency/activity were dependent on this factor or, rather, on their interactions with intracellular targets; and 5) investigate the pharmacokinetic properties in mice of this class of molecules.

PRINCIPAL FINDINGS

1. By ala-scan mapping with D-aa of the Myc-H1 analog part of our molecule, we demonstrated that its activity is sequence dependent
We found two D-aa to be necessary for antiproliferative activity (Lys in 4 and Arg in 5, numeration referred to L-form) and three D-aa (Glu in 2, Arg in 11 and Gln in 13, numeration referred to L-form) to reduce effectiveness. Substitution with a D-proline in position 5 or 7 caused a dramatic reduction in activity. A longer RI-Int-H1-S6A,F8A-loop-H2 peptido-mimetic molecule was much less active: formation of a ß sheet in this molecule (CD and NMR data) could probably explain this loss of activity.

2. Myc/Max heterodimer conformation was disturbed by our molecules at the four helix bundle level but not at the leucine zipper level
Side chains projecting toward the interior of the bundle were specifically required for this biochemical interference. Data on biologically activating or inactivating D-aa side chains suggested that biological activity was best explained in terms of a protein-protein interaction external to the Myc/Max heterodimer itself.

3. We tried to understand the folding necessary for our molecules
The H1 part of our peptido-mimetic molecules likely acts mostly as a random coil (CD data, NMR data, dynamic studies). We introduced a kink in our molecules (substitution with a D-proline in positions 5 or 7) or favored the formation of a ß-sheet structure; both were counterproductive.

4. We have confirmed the excellent capability of our molecules to cross the cytoplasmic membrane and concentrate inside living cells
No differences in biological activity were dependent on this parameter.

5. Our type of retro-inverso peptido-mimetic molecules composed of D-aa appeared very stable in cell cultures in vitro
Injected i.v. in mice, they appeared capable of reaching high concentrations in liver, lung, spleen, and kidney, but were almost undetectable in the brain; half-life 1 day.

The activity of our peptido-mimetic molecules on HCT-116 cancer cell line is shown in Fig. 1 .

View larger version (26K): [in this window] [in a new window]   Figure 1. Percent inhibition of growth induced in HCT-116 cells by our peptido-mimetic molecules at 10 µM defined as [(b0-b)/b0]·100, where [ln(N)= at2+bt+ln(N0)], according to equations 2 and 1, respectively, of on-line text.

CONCLUSIONS AND SIGNIFICANCE

Our study is innovative because we worked with peptido-mimetic molecules 10-fold larger and more extended in space than small drug-like molecules. This type of molecule has the potential of a more selective docking at the level of a protein-protein interaction. Cell internalization and pharmacokinetic properties were quite good and encourage further study. Solid-phase synthesis of these molecules is feasible. In this new class of "druggable" molecules, modeling considerations seem especially appropriate for guiding the synthesis of new compounds.

Lys in 4 and Arg in 5 turned out to be necessary because the activity of related peptides without these side chains was strongly reduced compared with reference RI-Int-H1-S6A,F8A activity. These basic amino acids in Myc helix1 structure project toward the outside of the heterodimer and presumably are involved (perhaps together with some loop residues and basic helix basic side chains) in interactions with a third protein.

Our D-peptide amino acidic residues corresponding to those of H1 of Myc involved in "four--helix bundle" interactions (inside) could not find suitable binding sites in a third protein of the enhanceosome (perhaps the adaptor protein INI-1) and could be a steric or electrostatic hindrance for an acceptor surface approach by peptido-mimetic molecules; Gln 13, Arg 11, and Glu 2 of Myc-H1 (927, 925, and 916 of chain A of 1NKP) indeed seem be involved in these internal interactions, projecting their side chains toward the inside of the heterodimer. They probably do not link a third protein of the enhanceosome higher order structure present at the outside.

Our Ala-scan results suggest that Gln 13 and Arg 11 side chains could represent an obstacle, so that a reduction of these side chains to the methyl group of an Ala could fit with the higher biological activity of <Q13A> and <R11A> peptido-mimetic molecules. Similar considerations can be made for Glu in position 2. Substitution with Ala of these residues could enhance the number of molecules at the acceptor binding zone because interactions with nonspecific secondary sites would be reduced.

In R5P (R in 919 of chain A of 1NKP), we changed a useful residue (Arg) and introduced a kink, drastically reducing antiproliferative activity; in F7P (F in 921 of chain A of 1NKP), we changed a nonessential aa but introduced a kink in the structure, decreasing the possibility of a correct molecular docking toward the target protein.

We have synthesized a longer peptido-mimetic molecule in an attempt to obtain a wider zone of interaction: RI-Int-H1-S6A,F8A-loop-H2. However, such a molecule- containing a D-aa sequence of helix1-loop-helix2 is no longer made of two helices separated by an intervening loop, as it tends to assume ß-sheet conformation (CD and NMR data). Such an unwanted structure, very different from the c-Myc portion of Myc/Max heterodimer, is probably responsible for the low activity of this larger peptido-mimetic molecule.

A possible candidate acceptor of our active molecules could be INI-1. It has been reported that the part of Myc known as basic helix-helix1-loop-helix2 binds INI-1 between aa 181 and 240 (of INI-1): our peptido-mimetic molecules, mimicking helix1 of Myc, could interact with the same domain of INI-1.

Looking at the sequence of INI-1 portion (aa: 181-240) interacting with Myc, it is possible to note a high density of acidic residues, such as Glu and Asp. It is not unreasonable to think there could be good binding between some of these acidic side chains and the basic side chains of Myc, including adjacent Lys 4 and Arg 5 of the H1 motif.

We concluded that 1) the activity of our peptido-mimetic molecules is sequence specific and 2) our active molecules seem to act at the external level of the heterodimer, probably binding some acidic residues of a third protein (Fig. 2 ).

View larger version (65K): [in this window] [in a new window]   Figure 2. Schematic diagram. Enhanceosome: complex of proteins that are part of c-Myc:Max higher order structure. Different higher order structures can activate or inhibit c-Myc target genes. ip: example of protein interacting with c-Myc:Max heterodimer, an enhanceosome component; BRG1: transcriptional coactivator, ATPase subunit of hSWI/SNF ATPase remodeling complex; INI1: subunit of hSWI/SNF ATPase remodeling complex involved in chromatin remodeling; SWI/SNF complex: ATPase remodeling complex; Pol II Preinitiation Complex: RNA polimerase II complex. Not drawn to scale; does not depict all participants.

We have begun to study some basic pharmacokinetic properties of our peptido-mimetic molecules. To our RI-Int-H1-S6A,F8A lead molecule we added a ¹⁴C-labeled Gly. We were able to administer (i.v.) our drug and study concentration and half-life in different organs: these 30-mer peptido-mimetic molecules are capable of efficient internalization. Their half-life is in the 24 h range. In liver, lung, spleen, and kidney we found high concentrations of the lead molecule. Negligible levels were found in the brain. This could represent a new class of "druggable" molecules.

We will try to introduce modifications into the most potent of our molecules (e.g., <R11A> or <E2A>) in an attempt to significantly increase potency on a molar base.

An even more important goal will be to better define the target of our molecules, possibly making an [INI-1||Myc||Max] trimer or other [external protein||Myc||Max] trimers. This will provide a better understanding of the mechanism of action and allow testing of new peptido-mimetic molecules based on our models, such as peptido-mimetic molecules inspired from an external protein motif and directed to the dimerization domain of c-Myc, rather than the other way around.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2369fje;

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