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,
,1,
,
* Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy;
Laboratory of Experimental Oncology, National Cancer Institute (IST), Genoa, Italy;
I.S.O. Istituto Superiore di Oncologia, Genoa, Italy;
Department of Pharmaceutical Sciences, University of Genoa, Genoa, Italy; and
|| Structural and Computational Biology Unit, EMBL Heidelberg, Heidelberg, Germany
1Correspondence: Department of Oncology, Biology and Genetics, University of Genoa, L. go R. Benzi 10, Genoa 16132, Italy. E-mail: luca.bagnasco{at}unige.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 20
) we reported that retro-inverso peptidomimetic molecules inspired by the Helix-1 of c-Myc motif could be sequence-specific antiproliferative agents active in the low micromolar range. We also found that our peptides were not opening the four-alpha-helix Myc:Max bundle. Their antiproliferative activity in cancer cell lines needs the presence of side chains projecting outside of the bundle in the corresponding native H1 motif. This observation suggested interference with an external partner. In this study we investigated the INI1:Myc interaction. INI1 is a subunit of the SWI/SNF complex (component of the enhanceosome surrounding Myc:Max heterodimer). The INI1:Myc interaction was confirmed via pull down, ELISA, and fluorescence anisotropy assays. According to the length of INI1 fragments used, we calculated Kds ranging between 1.3x10–6 and 4.8x10–7 M. The three different techniques applied showed that the INI1:Myc interaction was also the target of our retro-inverso peptidomimetic molecules, which seem to bind specifically at INI1. A Myc binding, 21aa INI1 fragment (minimum interacting sequence), could inspire the synthesis of a new class of more selective c-Myc inhibitors.—Bagnasco, L., Tortolina, L., Biasotti, B., Castagnino, N., Ponassi, R., Tomati, V., Nieddu, E., Stier, G., Malacarne, D., and Parodi, S. Inhibition of a protein-protein interaction between INI1 and c-Myc by small peptidomimetic molecules inspired by Helix-1 of c-Myc: identification of a new target of potential antineoplastic interest.
Key Words: inhibitors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
.
The c-Myc N-terminal domain, called trans-activation domain (TAD), would bind bridge proteins with one of the three RNA polymerases, thereby playing a key role in regulating ribosome biogenesis and cell growth. Several genes downstream of c-Myc would be transcribed and finally translated into new proteins (3
4
5)
.
This action could be compared with other similar proteins (Mad family proteins) belonging, as Myc itself does, to the helix-loop-helix superfamily but missing the TAD that could interact and dimerize with Max, inhibiting Myc effects of cell growth and proliferation.
Papers describing genes downstream of c-Myc have appeared (6)
and continue to appear at high frequency in the literature (see on-line database on web site: http://www.myc-cancer-gene.org/site/mycTargetDB.asp). A reasonable possibility is that c-Myc is necessary but not sufficient for specific gene transcription. If other transcription factors concur to transcription specificity, then the thousands of genes potentially downstream of c-Myc could undergo a more finely regulated expression. Concerning the initial processes of gene transcription, a complex cell machinery, called enhanceosome, has been described by some investigators for different transcription factors; c-Myc could take part in similar complexes (7)
.
c-Myc is one of the most studied oncogenes. Its relevance in malignant transformation comes from the fact that its homeostasis has been directly or indirectly altered in the majority of all solid and hematopoietic tumors (8)
. During the last decade, many attempts using small drug-like molecules to inhibit c-Myc have been reported, and papers concerning the development of c-Myc inhibitors have been published (9
10
11
12
13
14)
.
A great number of other proteins besides Max have been found to interact directly with c-Myc, modulating its action both positively and negatively (15
, 16)
. For several years, our work has focused around c-Myc and the search of peptidomimetic c-Myc inhibitors (17
, 18)
.
In our 2005 paper, we demonstrated sequence-specific activity (complete growth inhibition at 10 µM) for some peptidomimetic molecules, made of D-amino acids and inspired from the first helix of c-Myc, in c-Myc overexpressing sensitive cell lines (18)
.
These RI-peptides (19)
were fused to a retro-inverso internalization sequence discovered by our group, and could easily and efficiently cross through cell membranes and reach the nucleus. Moreover, being constituted by D-amino acids, they were insensitive to the activity of cell proteases, with long half-lives both in vitro and in vivo (18
, 20)
.
Based on circular dichroism (CD) and antiproliferative activity data (18)
, we suggested that our retro-inverso peptides could act externally of the "four alpha helix bundle" formed by the C-terminal domains of c-Myc and Max (21)
, acting as a wedge between helix 1 of c-Myc and an external protein belonging to the enhanceosome (or higher order structure surrounding c-Myc:Max).
Based on literature data and considerations of molecular modeling, we proposed the protein INI1 as a possible candidate (22)
.
In this work we demonstrate that our hypotheses were correct, with two independent molecular/biochemical methods showing that our retro-inverso peptides are specific in inhibiting the interaction between c-Myc and INI1. We had previously shown that some side chains of our retro-inverso peptidomimetic molecules were required for a partial interference at the level of the c-Myc:Max heterodimer, whereas different side chains were essential for a growth inhibitory activity. These last molecules are also capable of interference at the level of the c-Myc:INI1 protein-protein interaction.
By means of a third biophysical method based on fluorescence polarization, we have defined a minimum interacting sequence of INI1 with c-Myc; this sequence could be a starting point at the level of molecular modeling for future designs of c-Myc inhibitor peptides inspired by a INI1 motif and looking toward c-Myc.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, cDNA sequences encoding either the whole or part of the proteins were subcloned in pET-modified vectors and transformed in BL21 strain of E. coli. Bacterial pellets were harvested and lysed, and the proteins were purified on a Ni-NTA-column (Qiagen, Valencia, CA, USA). The C-terminal domain of c-Myc (b-HLH-zip), the RPT1–2 (aa 179–326) region of INI1, and the entire Max protein were produced in pETM11 vector. This vector contains a TEV (Tobacco Etch Virus) protease recognition site between the His tag and the recombinant protein start codon. After purification, the tag could be cut out using a recombinant His-tagged TEV protease; when needed, a second Ni-NTA column was used to further purify the protein from both cleaved His tag and the protease itself.
The whole INI1 protein was also produced as a nontagged protein, both in pETM13 and in pETM11.
Peptide synthesis
Peptides indicated in Table 1
were synthesized either with an Applied Biosystem 433A Peptide Synthesizer using a solid-phase technique or manually by using the standard method of solid-phase peptide synthesis, which follows the 9-fluorenylmethoxycarbonyl (Fmoc) strategy with minor modifications. Additional details are given in ref. 18
.
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The HLH-zip portion of c-Myc (QRR-H1-loop-H2-zip, aa 365–439) was obtained by chemical solid-phase synthesis. This L-peptide was fluoresceinated at the amino-terminal for fluorescence anisotropy studies (Myc-F).
The sequence of Myc-F is:
Fl•QRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA.
The peptide was then purified and characterized as described previously (18)
.
Pull down assay
The histidine-tagged b-HLH-zip domain of c-Myc was produced as described above.
To proceed for the pull down assay, this target was loaded onto a Ni-NTA-agarose column and the resin was washed extensively with urea 8 M at a gradient of pH ranging between 8 and 5.9, and to renaturate the recombinant protein, with 50 mM Tris/HCl pH 7. A small amount of resin was then used to assay the purity (by SDS-PAGE) and the concentration (by Bradford assay; Bio-Rad, Hercules, CA, USA) of the attached protein.
Resin-linked His-tagged b-HLH-zip domain of c-Myc at a final concentration of 1 µM was incubated with a bacterial lysate containing the whole untagged INI1 in blocking buffer (200 mM KCl, 20 mM HEPES pH 7, 5 mM DTT, 20 mM imidazole, 5 mg/ml BSA, 0.1% IGEPAL, Pefablock) for 60 min at 4°C with gentle rocking.
The resin was washed several times with Wash buffer (20 mM HEPES pH 7, 0.1 mM EDTA, 0.5 mM DTT, 4 mM MgCl2, 300 mM KCl, 20 mM imidazole, 0.05% IGEPAL, 10% glycerol), boiled for 5 min, run on a SDS-PAGE, transferred on a nitrocellulose membrane, and blotted with an anti-INI1 antibody (sc-13055 Santa Cruz; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). A secondary HRP anti-rabbit antibody was used for detection.
ELISA
Two different enzyme-linked immunosorbent assays were performed to study binding between INI1 and c-Myc. The first assay involves interaction of INI1 with immobilized c-Myc; the second involves interaction of c-Myc with immobilized INI1.
In the first case, 96-microtiter plate wells were coated with purified b-HLH-zip domain of c-Myc in 50 µl of PBS (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 4.3 mM, KH2PO4 1.4 mM) overnight at 4°C.
Wells were then rinsed with wash buffer (PBS, 0.05% Tween) and blocked with 200 µl of blocking buffer (PBS, 1% BSA) for 3 h at 37°C.
After rinsing three times with wash buffer, 100 µl of binding buffer (PBS, 0.05% Tween, 1% BSA) containing 2 µM of purified INI1 whole protein was added to the wells and let to react for 1 h at 37°C. Wells were rinsed again three times and 100 µl of binding buffer containing 200 ng/ml of a primary antibody anti-INI1 (sc-13055 Santa Cruz) was added and incubated for 1 h at 37°C. After three additional rinsings, 100 µl of the same buffer containing a secondary HRP anti-rabbit antibody was added and incubated for another hour at 37°C. Finally, 100 µl of ABTS substrate (2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was applied and the plate was kept in the dark until the color intensity of positive controls was maximum and the negative controls did not develop nonspecific reactions (
6–10 min). The ELISA plate was scanned in a Biotech ELISA reader at 450 nm.
For the second assay, the same method was applied, but INI1 was immobilized in the wells and incubated with c-Myc. Primary antibody anti-c-Myc (sc-40 Santa Cruz) was used to reveal the binding reaction.
Fluorescence anisotropy
The formation of the hetero-dimers was measured in terms of increased anisotropy (Polarion; Tecan Trading AG, Switzerland). All experiments were performed in polar buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA); the experimental volume was always 200 µl per well, and the plates used were 96-well black nonbinding plates (Corning Glassworks, Corning, NY, USA). Proteins were mixed at room temperature and maintained at target temperature (30°C) in the dark for the whole duration of the experiments.
Anisotropy measurements were performed at different incubation times to assess the stability of the reactions. The reported results refer to measurements performed after 60 min of incubation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, lane 5). To exclude a nonspecific antibody reaction, we applied to the resin bound to the tagged "bait" protein (b-HLH-zip of c-Myc), a binding buffer solution not containing the "prey" protein (INI1) (Fig. 1
, lane 4). As shown in Fig. 1
, only in lane 2, corresponding to the pull down of the prey protein INI1, it was possible to detect a signal similar in intensity to those of lanes 7 and 8 (positive controls). Thus, we can confirm that there is a direct, specific interaction between c-Myc and INI1.
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In our previous work (18)
we speculated that our peptides, active in inhibiting c-Myc at a cellular level, could interact externally to the c-Myc:Max four alpha helix bundle, probably acting between the helix 1 of c-Myc and an external protein such as INI1. Biological activity of our retro-inverso peptidomimetic molecules, in fact, required Helix-1 amino acid side chains looking outside of the four alpha helix bundle in the corresponding H1 motif of c-Myc.
To confirm this hypothesis, we used some of our active peptides (see Table 1
in Materials and Methods) to dismantle the pull down reaction at concentrations of 1 µM, 10 µM, and 100 µM. An unrelated peptide (BH3 of Bak) was used as a negative control.
In Fig. 2
we show that only our active retro-inverso peptide RI-Int-H1-S6A,F8A was able to inhibit the binding between c-Myc and INI1 in a pull down assay, whereas L-Int-H1-S6A,F8A seemed inactive.
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This last outcome did not surprise us since it could be explained considering that the experiment was run in the presence of a raw bacterial lysate, probably rich in peptidases. The results of the subsequent ELISA assay were also compatible with an intrinsic weaker activity of the L-peptide with respect to the corresponding retro-inverso peptide.
To assess the activity of our L- and RI-peptides with a different technique and in a "peptidase-free" environment, we implemented an ELISA assay.
As for the pull down, we used similar negative controls to avoid unspecific reactions. Also in this case, results fitted with our hypothesis. Figure 3
shows a binding interaction between b-HLH-zip of c-Myc and INI1. Addition of our active L-peptide to the reaction buffer (Fig. 4
, upper panel) at concentrations ranging between 1 and 100 µM leads to a clear inhibition of signal intensity in a dose-dependent manner, indicating a binding inhibition between our two protein targets. Moreover, in this case we tested the sequence specificity of our peptide: in the lower panel of Fig. 4
the effects (at high concentration) of our RI active peptide and an RI unrelated peptide are shown, whereas 100 µM RI-Int-H1-S6A-F8A induces a close to 90% binding inhibition and 100 µM RI-Bak (BH3 of Bak) is completely inactive. In agreement with the pull down assay, our RI-peptide is much more potent than its corresponding L-form. This is also true at the cellular level (20)
.
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Finally, we run another set of ELISA assays in which other RI-peptides, found to be active at the cellular level (18)
, were tested.
Figure 5A shows a representative ELISA assay where the RI-Int-H1-S6A,F8A,Q13A peptide was used; results observed at the cellular level were confirmed.
Another set of experiments aimed at finding the minimum interacting sequence of INI1 with c-Myc was performed. The final goal of these studies is to conceive and synthesize new c-Myc-inhibiting molecules inspired by a specific INI1 sequence.
Data in the literature describe the fragment of INI1 corresponding to the region around the RPT1 (imperfect repeat motif conserved region) aa 183–243 as interacting with c-Myc (22)
.
To confirm these data, a fluorescence polarization test of interaction between Myc-F (see Materials and Methods) and a recombinant fragment of INI1, referred as RPT1–2 (aa 179–326) (22)
, was performed; Fig. 6
shows a clear interaction between these two protein fragments.
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A Kd was calculated using the equation proposed by Lundblad for simple binding interaction assays (23)
. For our graph, the best-fitting Kd was 1.3x10–6 M (SE=1.7x10–7 M). All the best-fitting computations and statistical analysis were performed using the SAS/STAT software, Version 8, of the SAS System for [Unix], copyright, SAS Institute Inc. (Cary, NC, USA).
Encouraged by these results, a set of seven consecutive fluoresceinated peptides, each 21 aa long and overlapping each other by 10 amino acids, encompassing the putative interacting fragment of INI1 (Table 2
), were synthesized as described in Materials and Methods.
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Each peptide was tested for interaction with c-Myc b-HLH-zip domain using the fluorescence anisotropy technique; only the fluoresceinated peptide INI_213–233 demonstrates a binding activity with b-HLH-zip, as shown in Fig. 7
. We calculated the Kd as described above and computed a Kd of 4.8x10–7 M (SE=9.8x10–8 M).
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We also demonstrated that this interaction is reversible by inhibiting the binding with the same nonfluoresceinated peptide. Figure 8
shows an inhibition curve obtained incubating a fluoresceinated INI1_213–233 and b-HLH-zip of c-Myc at the fixed concentrations of 7.5x10–7 M and 5.0x10–7 M, respectively, in the presence of an increasing concentration of "cold" INI1_213–233.
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Finally we demonstrated that our peptidomimetic molecules RI-Int-H1-S6A,F8A; RI-Int-H1-S6A,F8A,Q13A and L-Int-H1-S6A,F8A, inspired by H1 of c-Myc, are able to interact with the INI1_213–233 fragment (Fig. 9
).
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At high concentrations, L-Int-H1-S6A,F8A (black squares) reaches a higher plateau level of anisotropy with respect to both RI-Int-H1-S6A,F8A (black triangles) and RI-Int-H1-S6A,F8A,Q13A (black circles). However, the inflection point for the three molecules binding with fluoresceinated INI1_213–233 is always 3x10–7 M (3.4x10–7 M for L-Int-H1-S6A,F8A, 2.9x10–7 M for RI-Int-H1-S6A,F8A, and 3.2x10–7 M for RI-Int-H1-S6A,F8A,Q13A, respectively). The difference among the three values is not statistically significant.
An intrinsic higher fluorescence anisotropy of the completely bound complex [fluoresceinated INI1_213–233]:[L-Int-H1-S6A,F8A] suggests a stiffer hetero-dimer structure further away from a spherical/ellipsoidal conformation with respect to the other two hetero-dimers, a complex endowed with a slower tumbling time (23)
.
When using the ELISA technique (Figs. 4
and 5)
with our three peptidomimetic molecules mentioned above, we were inhibiting the formation of a relatively large hetero-dimer (probably endowed with a significant degree of quaternary structure) [b-HLH-zip (88 aa)]:[INI1 (385 aa)]. This situation is quite different from that shown in Fig. 9
, where a direct binding study between short 21- and 30-aminoacid-long peptides is reported.
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Perhaps a slightly different ranking of the potency of our three molecules with the two methods is justified by the decidedly different experimental conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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suggested that our retro-inverso peptides, inspired by Helix-1 (H1) of c-Myc, in order to display biological antiproliferative activity required the presence of amino acid side chains projecting outward from the c-Myc:Max four alpha-helix bundle (making reference to the corresponding H1 of c-Myc motif).
In addition, CD data had clearly shown that the ability of some of our molecules to interfere with the c-Myc:Max four alpha-helix bundle never induced a complete separation of the two monomers and was not a requirement for biological activity. Literature reports (22)
referred to INI1, a component of the DNA remodeling complex SWI/SNF, as a protein binding to c-Myc perhaps at the basic helix-H1 region, external to the c-Myc:Max four alpha helix bundle. We therefore explored whether our peptidomimetic molecules could be active at the level of this protein-protein interaction.
We first wanted to confirm whether recombinant c-Myc and INI1 (whole proteins or fragments) were able to establish a protein-protein interaction. As shown in Figs. 1
and 3
, we have confirmed such interaction. At this point we wanted to explore whether our peptidomimetic molecules would interfere with this c-Myc:INI1 interaction. Since we could inhibit the binding between b-HLH-zip of c-Myc and the entire INI1 protein by means of our lead retro-inverso molecule RI-Int-F6A,S8A both in a pull down assay (Fig. 2
, lower panel) and by ELISA (Fig. 4
, lower panel), we could say that our hypothesis was correct. L-Int-F6A,S8A was apparently unable to inhibit that interaction at the level of a pull down assay (Fig. 2
, upper panel). This result could be explained at least in part by considering that a raw bacterial lysate enriched in recombinant INI1 was run on the column containing a histidine-tagged b-HLH-zip of c-Myc linked to the resin. This microenvironment could contain bacterial peptidases that might have partially but sufficiently digested the 30 amino acid-long L-Int-F6A,S8A. In fact, transferring this test to the "cleaner" ELISA environment showed a partial inhibition of the c-Myc:INI1 interaction by the L-peptide, but less efficient than the inhibitory activity of the corresponding RI-peptide (Fig. 4
, upper panel and lower panel, respectively).
Other RI-peptides found to be active at the cellular level in our earlier work (18)
have been ELISA tested and found to agree reasonably with previously observed biological results. For instance, at the biochemical level of the ELISA, RI-Int-H1-S6A,F8A,Q13A (Fig. 5)
is slightly less potent than RI-Int-H1-S6A,F8A (Fig. 4
, lower panel) but is slightly more active at the cellular level (18)
. Obviously the intracellular multiprotein complexity can finely modulate the biological effect beyond the simplified situation of a biochemical test.
During the last decade, much effort has been exerted in an attempt to find c-Myc inhibitors. These investigators (9
10
11
12
13
14)
tried to find small drug-like molecules capable of interfering at the level of the c-Myc:Max interaction. In this paper we reported inhibitors of a c-Myc interaction with a different partner. It could be important to define a new protein:protein interaction target useful for testing different families of potential c-Myc inhibitors.
For this reason we planned a series of experiments aimed at finding a minimum interacting sequence of INI1 with the carboxyl-terminal region of c-Myc.
The assessment via fluorescence anisotropy of the binding between the RPT1–2 motif of INI1 and a fluoresceinated HLH-zip fragment of c-Myc (Fig. 6)
suggested that a possible smaller fragment of INI1, still binding with c-Myc, could be searched within the RPT1–2 INI1 region.
To pursue this hypothesis, an array of seven fluoresceinated peptides, 21 amino acid long and overlapping by 10 aa, encompassing the major interacting region of INI1 with c-Myc, were synthesized. By means of the fluorescence anisotropy technique, they were tested to assess their binding with b-HLH-zip of c-Myc. Only the fluoresceinated peptide INI1_213–233 was able to interact (Fig. 7)
reversibly (Fig. 8)
with the above c-Myc domain, with a calculated Kd of 4.8 x 10–7 M. The calculated Kd (1.3x10–6 M) for the entire RPT1–2 region was slightly higher (Fig. 6)
. The c-Myc fragments used to test RPT1–2 and INI1_213–233 fragments were not identical, however. Only the c-Myc fragment binding the fluoresceinated INI1_213–233 fragment possessed a complete basic helix region. This could be a possible explanation for the better affinity observed for INI1_213–233 with respect to the larger INI1 fragment tested with a fluoresceinated QRR-helix-loop-helix-zip of Myc.
The results reported in Fig. 9
suggest that our peptides (both L- and RI-) interact directly with the INI1_213–233 fragment.
The results reported in Fig. 9
have prompted us to think that the RI-peptidomimetic molecules reported earlier (18)
may not have been optimally conceived considering they look at a modular protein, INI1, used by the cell for many different chromatin remodeling and accessibility functions rather than directly at a region of the c-Myc onco-protein. At the same time, the 21 aa INI_213–233 fragment could offer an inspiration at the level of modeling for conceiving and synthesizing molecules looking toward c-Myc, rather than toward INI1. This INI1:c-Myc interaction region is rich in acid aa on the INI1 side, basic aa on the c-Myc side, and is relatively poor in hydrophobic aa. It could turn out to be a favorable hot spot for the search of drug-like or more extended in space peptidomimetic inhibitors.
We set up a new well-defined and apparently robust biochemical target for screening (potentially including HTS of combinatorial libraries); this new target could be the starting point for searching a new class of c-Myc inhibitors: inhibitors of the INI1:c-Myc protein-protein interaction.
Looking at the problem of finding c-Myc inhibitors from a more general point of view, several proteins interact, for instance, with the important TAD domain of c-Myc. We think that the number of c-Myc protein-protein interactions of potential interest as targets for inhibitors is much larger than the Myc:Max heterodimerization region (probably the most evolutionary conserved region). In this work we suggest a new possibility and invite a look at a higher order structure surrounding c-Myc.
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ACKNOWLEDGMENTS |
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Received for publication August 18, 2006. Accepted for publication November 9, 2006.
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
