Erika Nieddu

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

c‐Myc is a transcription modulator proto‐oncogene. When overexpressed, it becomes an important contributor to the multi‐hit process of malignant transformation. In two earlier papers in this journal (see refs. 19, 20) we reported that retro‐inverso pep‐tidomimetic 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 enhanceo‐some 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 1.3×10−6 and 4.8×10−7 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. FASEB J. 21, 1256–1263 (2007)

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

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 α‐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 α‐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 µM total cell number was ∼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 α‐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 α‐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 β‐sheet (CD and NMR data).

F508del-CFTR Rescue: A Matter of Cell Stress Response

Cystic fibrosis (CF) is a common inherited fatal disease affecting 70,000 people worldwide, with a median predicted age of survival of approximately 38 years. The deletion of Phenylalanine in position 508 of the Cystic Fibrosis Transmembrane conductance Regulator (F508del-CFTR) is the most common mutation in CF patients: the deleted protein, not properly folded, is degraded. To date no commercial drugs are available. Low temperature, some osmolytes and conditions able to induce heat shock protein 70 (Hsp70) expression and heat shock cognate 70 (Hsc70) inhibition result in F508del-CFTR rescue, hence restoring its physiological function: this review sheds light on the correlation between these several evidences. Interestingly, all these approaches have a role in the cell stress response (CSR), a set of cell reactions to stress. In addition, unpredictably, F508del-CFTR rescue has to be considered in the frame of CSR: entities that induce - or are induced during - the CSR are, in general, also able to correct trafficking defect of CFTR. Specifically, the low temperature induces, by definition, a CSR; osmolytes, such as glycerol and trimethylamine N-oxide (TMAO), are products of the CSR; pharmacological correctors, such as Matrine and 4-phenylbutirric acid (4PBA), down-regulate the constitutive Hsc70 in favor of an up-regulation of the inducible chaperone Hsp70, another component of the CSR. The identification of a common mechanism of action for different types of correctors could drive the discovery of new active molecules in CF, overcoming methods clinically inapplicable, such as the low temperature.

Matrine modulates HSC70 levels and rescues ΔF508-CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP‐dependent Cl− channel located in the plasma membrane, and its malfunction results in cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Most CF patients carry the deletion of Phe508 (ΔF508 mutation); this mutation prevents the delivery of the CFTR to its correct cellular location, the apical (lumen‐facing) membrane of epithelial cells. Molecular chaperones play a central role in determining the fate of ΔF508‐CFTR. In this report, we show that the Matrine, a quinolizidine alkaloid, downregulates the expression of the molecular chaperone HSC70 and increases the protein levels of ΔF508‐CFTR in human alveolar basal epithelial cells (A549 cell line), stably transfected with a ΔF508‐CFTR‐expressing construct. Moreover, Matrine induced ΔF508‐CFTR release from endoplasmic reticulum to cell cytosol and its localization on the cell membrane. Interestingly, downregulation of HSC70 resulted in increased levels of ΔF508‐CFTR complexes with the co‐chaperone BAG3 that in addition appeared to co‐localize with the mutated protein on the cell surface. These results shed new light on ΔF508‐CFTR interactions with proteins of the chaperones/co‐chaperones system and could be useful in strategies for future medical treatments for CF. J. Cell. Physiol. 227: 3317–3323, 2012.

2-(dialkylamino)-4H-1-benzopyran-4-one derivatives modify chloride conductance in CFTR expressing cells.

Some 2-(diethylamino)-7-hydroxy-4H-1-benzopyran-4-one derivatives, potentially useful as activators of the cystic fibrosis transmembrane conductance regulator (CFTR), were prepared. The synthesized compounds were tested, together with others 2-(dialkylamino)-7-hydroxybenzopyran-4-one derivatives, by measuring their capacity to modify the kinetics of iodide influx in Fisher rat thyroid cells expressing wild type CFTR and the halide-sensitive yellow fluorescent protein. Among the tested compounds the dinitrile derivatives 8 and 9 are endowed with an activity comparable to the reference compound apigenin.

Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Some of the genetic mutations that cause cystic fibrosis (CF) impair the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− ion channel. This defect can be corrected with pharmacological tools (potentiators) that belong to various chemical families, including the 1,4‐dihydropyridines (DHPs). A small set of asymmetric 4‐aryl‐DHPs was synthesized, and each racemic couple was tested in a functional assay carried out on cells expressing the G1349D, ΔF508, and G551D mutants. The most active racemates were subjected to chiral separation by HPLC, and the pure enantiomers were tested to evaluate any gains in activity. Although three enantiomers demonstrated high potency (Kd values less than 0.09, 0.1, and 0.5 μM in G1349D, ΔF508, and G551D, respectively), in general, the screening of pure enantiomers did not produce a great diversity in potency values. It is probable that the degree of DHP asymmetry considered in our analysis is still insufficient with respect to that allowed in a putative DHP binding site in CFTR, so that the site could equally accommodate both enantiomers.

Phenylhydrazones as Correctors of a Mutant Cystic Fibrosis Transmembrane Conductance Regulator.

The phenylhydrazone RDR‐1 is endowed with moderate activity as F508del‐CFTR corrector; nevertheless, its simple structure enables stimulating developments in this class of correctors. Therefore, we synthesized a number of phenylhydrazones 3 by reacting phenylhydrazine derivatives 1 with furfural derivatives 2. By the same reaction, also the pyridine derivatives 4, the thiophene derivatives 5, and the hydrazides 6 and 7 were prepared. All compounds were tested as F508del‐CFTR correctors in the cystic fibrosis (CF) bronchial epithelial cell line CFBE41o‐, using corr‐4a and VX‐809 as controls. Some of the tested compounds emerged as interesting F508del‐CFTR correctors at 20 μM (3c) and 2 μM (5d). 3c and 5d administered together with VX‐809 produced a satisfactory additivity of action. When the structure of 5d was overlapped with RDR‐1 and five other established correctors, a shared central design was clearly visible. This fact may be of interest in the search for new F508del‐CFTR correctors.

Matrine in association with FD‑2 stimulates F508del‑cystic fibrosis transmembrane conductance regulator activity in the presence of corrector VX809

Cystic fibrosis is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the predominant mutation is termed Phe508del (F508del). Therapy for F508del-CFTR patients is based on the use of Orkambi®, a combination of VX809 and VX770. However, though Orkambi leads to an improvement in the lung function of patients, a progressive reduction in its efficacy has been observed. In order to overcome this effect, the aim of the present study was to investigate the role of matrine and the in-house compound FD-2 in increasing the action of VX809 and VX770. Fischer rat thyroid cells overexpressing F508del-CFTR were treated with matrine, VX809 (corrector) and/or with a number of potentiators (VX770, FD-1 and FD-2). The results demonstrated that matrine was able to stimulate CFTR activity and, in association with FD-2, increased the functionality of the channel in the presence of VX809. Based on these results, it may be hypothesized that FD-2 may be a novel and more effective potentiator compared with VX770.