Patent Application
20130217634
The present application claims priority to Italian Patent Application No. RM2012000060, file on Feb. 21, 2012, the content of which is hereby incorporated by reference in its entirety.
The present disclosure relates to p53 protein and in particular to compounds able to interfere with the activity of the MDM2/MDM4 heterodimer
The MDM2 and MDM4 are oncoproteins which are key negative regulators of the p53 tumor suppressor. Despite, several studies on the MDM2 and MDM4 contributions to the regulation of p53 stability and activity, development of approaches able to interfere with the activity of the MDM2 and MDM4 oncoproteins is still challenging.
Provided herein are peptides able to interfere with the MDM2/MDM4 heterodimer activity on p53.
In particular, a peptide having sequence of MDM2 or MDM4 interaction domain is described, as well as related nucleotide sequences, expression vectors, pharmaceutical compositions, medicaments, methods and systems.
The present disclosure now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with detailed reference to enclosed drawings, wherein:
The present disclosure concerns peptides able to interfere and in particular impair the inhibiting activity of MDM2/MDM4 heterodimer towards p53 and their use for cancer treatment. In particular, the disclosure concerns peptides able to inhibit MDM2/MDM4 heterodimer and maintain the association between MDM4 and p53 so to restore the p53 oncosuppressive function in cancer cells harboring wild type p53 protein, directing its function specifically towards an apoptotic outcome.
Inactivation of the oncosuppressor p53 by means of direct mutation of its gene is one of first genetic lesions recognized in human tumors (about 50% of human tumors). In tumors carrying wilde type p53, studies have evidenced alternative methods for p53 inactivation by deregulation of its partners, MDM4 and MDM2. The latter ones are two crucial regulators of the oncosuppressor p53. These two proteins carry out mainly inhibiting activity towards p53 by controlling both protein levels and transcriptional function.
The importance of MDM proteins on the inhibition of p53 activity resulted in development of different strategies aiming to disrupt the p53/MDM association.
For example WO 2008106507, WO201105219 patent applications are known and concern peptides inhibiting the interaction of p53 with MDM2 and MDM4, i.e. peptides aiming to break p53 binding with MDM4 and MDM2. According to WO201105219 patent application, the dissociation of p53 from MDM2 and MDM4 results in a biological response consisting both of proliferative arrest and cell death, although it is not specified whether the observed cell death is due to apoptosis.
WO2008106507 describes peptides able to bind with high affinity MDM2 and therefore, probably, inhibit the binding of MDM2 with all the members of the p53 family; however, such statement has not been experimentally proved. It is predicted, by computational analysis, that such peptides may bind MDM4 and therefore dissociate the same from p53. In vitro or in vivo experimental data have not been provided for a possible biological activity of these peptides. Therefore it is not predictable their effectiveness.
In addition, the strategy aiming to disrupt the binding of p53 to MDM4 and MDM2 does not consider recent studies suggesting that MDM4 binding to p53 potentiates its apoptotic activity. According to WO201105219 patent application, peptides would be able to inhibit the binding of p53 to MDM2 10 fold higher than to MDM4 (IC50=0.01 μM and 0.1 μM for MDM2 and MDM4, respectively). Therefore, it cannot be excluded that a part of the observed cell death, according to WO201105219, is due to the partial retention of MDM4-p53 complexes and not completely to the dissociation of p53 from MDM2 and MDM4.
WO200061193 patent application describes antisense compounds inhibiting MDM4 expression. Also in this case the importance of MDM4-p53 complex has not been taken into consideration for apoptosis induction.
Most promising results for reactivation of p53 are obtained using small molecules identified by high-throughput screening of compound libraries and drug design by computer analysis based on protein structure (Shangary S, Wang S. Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res. 2008 Sep. 1; 14(17): 5318-24).
However, known therapeutic strategies show various disadvantages. For example, compounds dissociating MDM2 from p53 are ineffective in displacing MDM4 from p53 because of the intrinsic differences in p53 binding domains of MDM4 and MDM2. This could enhance apoptosis induction by MDM4-p53 complex. However, since such compounds do not inhibit the binding of MDM4 with MDM2, it can be predicted that the heterodimer still inhibits p53 activity. Moreover, association of MDM2 to MDM4 antagonizes the pro-apoptotic activity of MDM4 preventing this response. In addition growth arrest rather than apoptotic response often occurs, while thepreferred result in any cancer therapy is apoptosis (Toledo F, Wahl G M. MDM2 and MDM4: p53 regulators as targets in anticancer therapy. Int J Biochem Cell Biol. 2007; 39 (7-8): 1476-82. Epub 2007 Apr. 8; Shangary S, Wang S. Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res. 2008 Sep. 1; 14(17): 5318-24). Known methods, aiming to inhibit the interaction of p53 with MDM2 and/or MDM4, cause therefore activation of p53 not only towards apoptosis, as it would be preferred, but also towards proliferative arrest. In addition, the release of MDM2 from association with p53 results in higher degradative activity of MDM2 towards MDM4 and other proapoptotic molecules (as demonstrated by several recent papers).
All current methods failed to pass phase 1 of clinical trials, pointing out to existing limitations (Cheok C F, Verma C S, Baselga J, Lane D P. Translating p53 into the clinic. Nat Rev Clin Oncol. 2011 January; 8(1):25-37. Epub 2010 Oct. 26).
Based on these results, it emerges the need to provide new therapeutic methods for cancer treatment that overcomes the disadvantages of known methods.
Recent studies suggest that the MDM4/MDM2 heterodimer is the functional inhibitor of p53. This data suggest that agents aiming to inhibit MDM4/MDM2 heterodimer could represent new potential therapeutic agents. On the other hand, recent data suggest different activities of MDM2 and MDM4 under stress conditions. Under these conditions a weakening of association and degradation activity of MDM2 towards p53 mainly occurs (Marchenko ET to, 2007). In addition, upon a sub-lethal type of DNA damage, usually associated with growth arrest, MDM2 level increase and this results in degradation of MDM4 and some pro-apoptotic factors as HIPK2 (Rinaldo C, Prodosmo A, Mancini F, Iacovelli S, Sacchi A, Moretti F, Soddu S. MDM2-regulated degradation of HIPK2 prevents p53Ser46 phosphorylation and DNA damage-induced apoptosis. Mol Cell. 2007 Mar. 9; 25(5):739-50). On the contrary, following a severe DNA damage, usually associated to apoptosis, levels of MDM2 do not increase or even decrease (Ashcroft M, Taya Y, Vousden K H Stress signals utilize multiple pathways to stabilize p53. Mol Cell Biol 200020: 3224-33; Latonen L, Taya Y, Laiho M UV-radiation induces dose-dependent regulation of p53 response and modulates p53-HDM2 interaction in human fibroblasts. Oncogene 2001 20: 6784-93; Meng L H, Kohn K W, Pommier Y Dose-response transition from cell cycle arrest to apoptosis with selective degradation of Mdm2 and p21WAF1/CIP1 in response to the novel anticancer agent, aminoflavone (NSC 686,288). Oncogene. 2007, 26, 4806-16).
Based on these data, a recent model considers the up-regulation of MDM2 occurring during cell growth arrest, an event favoring this response in comparison to cell death (Shmueli A, Oren M. Mdm2: p53's lifesaver? Mol Cell. 2007 Mar. 23; 25(6):794-6). On the other hand, MDM4 stabilizes p53, preventing the MDM2-mediated degradation, and promotes p53 mediated apoptosis under stress conditions (Jackson M W, Berberich S J. MdmX protects p53 from Mdm2-mediated degradation. Mol Cell Biol 2000; 20:1001-7; Stad R, Little N A, Xirodimas D P, Frenk R, van der Eb A J, Lane D P, et al. Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms. EMBO Rep 2001; 2:1029-34; Mancini F, Gentiletti F, D'Angelo M, Giglio S, Nanni S, D'Angelo C, et al. MDM4 (MDMX) over-expression enhances stabilization of stress-induced p53 and promotes apoptosis. J Biol Chem 2004, 279:8169-80; Barboza J A, Iwakuma T, Terzian T, El-Naggar A K, Lozano G. Mdm2 and Mdm4 loss regulates distinct p53 activities. Mol Cancer Res 2008; 6:947-54).
Therefore, although it is known that MDM2/MDM4 heterodimer inhibits p53 function, heterodimer dissociation has not been assumed a current suitable strategy in order to favor p53 apoptotic activity. This due to the fact that MDM2 release, i.e. the main p53 inhibitor, could however carry out inhibiting function against p53. For this reason, current strategies are directed to the dissociation of p53 from MDM2 and MDM4 and not to hetorodimer dissociation.
Applicants have demonstrated that MDM2 opposes the pro-apoptotic activity of MDM4 and that, when MDM4/MDM2 dissociation or partial dissociation at specific binding sites occurs as result of stress conditions, the p53-mediated apoptotic pathway is promoted, independently of MDM2. These results are coherent with the model proposed by Shmueli indicating p53 regulators not only for control of p53 activity, but also as having an important role in p53-mediated choice between cell life and death (Shmueli A, Oren M. Mdm2: p53's lifesaver? Mol Cell. 2007 Mar. 23; 25(6):794-6; Mitochondrial MDM4 (MDMX): An unpredicted role in the p53-mediated intrinsic apoptotic pathway. Mancini F, Moretti F. Cell Cycle. 2009 December; 8(23):3854-9). In particular, the inventors of the present disclosure observed that the over-expression of MDM4 mutant (C437G), unable of binding MDM2, induces the activation of p53, and this activation of p53 is not inhibited by concomitant expression of MDM2. Moreover, the over-expression of MDM4 (exceeding MDM2 levels) in a model of transgenic mouse causes an increase of cellular death (apoptosis) as a result of gamma irradiation and delays the tumor appearance induced by mouse exposure to genotoxic agents.
On the basis of these data, MDM4/MDM2 interaction region has been characterized. Amino acids involved in MDM4/MDM2 binding have been identified and tested by molecular mutagenesis. L430, A434 in MDM2 and L433, I489, F488 residues in MDM4, respectively, have been identified as relevant for MDM2/MDM4 complex.
Therefore, peptides belonging to the region of MDM4/MDM2 interaction and able to impair the inhibiting activity of MDM2/MDM4 heterodimer towards p53, that is to restore the p53 oncosuppressive function in cancer cells still harboring p53 wild type protein (approximately 50% of human tumors), and to maintain the proapoptotic function of MDM4 directing therefore the p53 response towards the apoptosis, have been prepared. Currently the majority of human tumors is characterized in that 50% express wild type and 50% express mutated p53, respectively. Therefore, independently from tumor type, this approach is directed to tumors expressing wild type p53.
In particular, three peptides of 12 amino acids (12 mer) have been prepared and tested at molecular and biological level. Since the peptides display some limitations for therapeutic application because of intrinsic instability thereof, chemical modifications necessary to increase the bioavailability and allow the in vivo administration have been carried out.
Techniques in order to increase the stability and bioavailability of peptides and allow administration thereof in vivo are well known to those skilled in the art. Particularly, above said peptides could be modified by means of insertion of hydrocarbon bridge suitable to increase the alpha helicity of said peptides (carbon staples). These modifications are able to promote cellular uptake, confer resistance to protease and increase global stability.
It is therefore a specific object of the present disclosure a peptide belonging to (i.e. comprised in, taking part of) MDM4 or MDM2 sequence, at the levels of the interaction domain between MDM4 and MDM2, or mimetic peptides of said peptide, said peptide having a number of amino acids from 5 to 16, preferably from 8 to 12, and comprising at least one amino acid chosen in the group consisting of leucine, isoleucine or phenylalanine of MDM4 position 433, 489 or 488, respectively, or comprising at least one amino acid chosen in the group consisting of leucine or alanine of MDM2 position 430 or 434, respectively,
wherein said MDM4 sequence, at the levels of MDM4 and MDM2 interaction domain, is chosen between the following sequences from amino acid 473 to amino acid 490 and from amino acid 418 to amino acid 448, respectively:
and said MDM2 sequence, at the levels of MDM4 and MDM2 interaction domain, is the following sequence from amino acid 415 to amino acid 449:
Preferably, when said peptide belongs to MDM4, the peptide according to the disclosure comprises the aminoacids isoleucine and phenylalanine of MDM4 position 489 and 488, respectively.
According to a preferred embodiment, the present disclosure concerns a peptide or mimetic peptides thereof according to the disclosure, wherein said peptide has the following general formula I:
R-KVFI-R1 (I)
wherein
R is chosen from the group consisting of I, VI, LVI, QLVI (SEQ ID NO:13), IQLVI (SEQ ID NO:14), EIQLVI (SEQ ID NO:15), KEIQLVI (SEQ ID NO:16), KKEIQLVI (SEQ ID NO:17), CKKEIQLVI (SEQ ID NO:18), ICKKEIQLVI (SEQ ID NO:19), PICKKEIQLVI (SEQ ID NO:20), CPICKKEIQLVI (SEQ ID NO:21) or hydrogen of amino functional group of K;
R1 is chosen from the group consisting of A or hydroxyl of carboxylic functional group of I;
wherein said R and R1 are chosen so that said peptide ranges from 5 to 16 amino acids, preferably, from 8 to 12. Preferably, the peptide belonging to the sequence of MDM4, at the levels of MDM4 and MDM2 interaction domain, can consist of the following sequence from amino acid 479 to amino acid 490 of MDM4 or mimetic peptides thereof:
Again according to a preferred embodiment, the present disclosure concerns a peptide or mimetic peptides thereof according to the disclosure, wherein said peptide has the following general formula II:
R2-LPLNA-R3 (II)
wherein
R2 is chosen from the group consisting of S, SS, ESS, VESS (SEQ ID NO:22), SVESS (SEQ ID NO:23), ESVESS (SEQ ID NO:24), EESVESS (SEQ ID NO:25), KEESVESS (SEQ ID NO:26), DKEESVESS (SEQ ID NO:27), QDKEESVESS (SEQ ID NO:28), TQDKEESVESS (SEQ ID NO:29) or hydrogen of amino functional group of L;
R3 is chosen from the group consisting of I, IE, IEP, IEPC (SEQ ID NO:30), IEPCV (SEQ ID NO:31), IEPCVI (SEQ ID NO:32), IEPCVIC (SEQ ID NO:33), IEPCVICQ (SEQ ID NO:34), IEPCVICQG (SEQ ID NO:35), IEPCVICQGR (SEQ ID NO:36), IEPCVICQGRP (SEQ ID NO:37) or hydroxyl of carboxylic functional group of A;
wherein said R2 and R3 are chosen so that said peptide ranges from 5 to 16 amino acids, preferably, from 8 to 12. Preferably, the peptide belonging to the sequence of MDM2, at the levels of MDM4 and MDM2 interaction domain, can consist of the following sequence from amino acid 427 to amino acid 438 of MDM2 or mimetic peptides thereof:
An isolated nucleotide sequence encoding for peptide as above defined is a further object of the present disclosure.
Moreover, the disclosure concerns an expression vector comprising nucleotide sequence as above defined.
The disclosure refers also to a pharmaceutical composition comprising or consisting of at least one peptide or mimetic peptides thereof, or at least one nucleotide sequence, or at least one vector as above defined, as an active ingredient, in association with one or more pharmaceutically acceptable adjuvant and/or excipients.
A peptide or mimetic peptides thereof, nucleotide sequence, vector or pharmaceutical composition, as defined above, for use in the treatment of tumors preferably expressing wild type p53, or associated pathologies like hyperproliferative benign syndromes represent a further object of the present disclosure.
Said peptides according to the disclosure can, for example, be administered by intravenous way, or using liposomal vectors directly deliverable to the tumor, or, at last, by means of nano-particles. In addition, said peptides can be delivered also by gene therapy using DNA expression vectors.
As above reported, peptides of the disclosure can be modified according to known methods in order to increase stability and bioavailability of the peptides and allow in vivo administration thereof. Particularly, said peptides can be modified by insertion of hydrocarbon bridge suitable to increase alpha helicity thereof (carbon staples). As reported in experimental section, peptides have been modified as below:
wherein the Ahx term indicates a linker. In addition, the peptides have been modified, for example, also with acetyl in the place of FITC-Ahx:
In addition, the present disclosure concerns the use of interaction domain between MDM4 and MDM2 for the screening of compounds able to dissociate at least one amino acid chosen in the group consisting of leucine, isoleucine or phenylalanine of MDM4 position 433, 489 or 488, respectively, or at least one amino acid chosen in the group consisting of leucine or alanine of MDM2 position 430 or 434, respectively. The compounds selected by the above screneening method are antitumoral compounds.
MDM4 sequence of interaction domain between MDM4 and MDM2 can consists of the following MDM4 sequence from amino acid 413 to amino acid 490:
MDM2 sequence of interaction domain between MDM4 and MDM2 can consists of the following MDM2 sequence from amino acid 411 to amino acid 491:
or the following MDM2 sequence from amino acid 428 to amino acid 491:
The peptides, compositions and related methods and systems herein disclosed are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
1.1 Materials and Methods
Tranfections. Transient transfections have been carried out using lipophilic vectors (“Lipofectamine 2000” for DNA, Invitrogen). This method is based on use of lipid vesicles (liposomes) mediating the transfer of DNA into eucaryotic cells.
Cells in 100 mm plates have been transfected with following vectors: pShutlle-hMDM4 (WT, C437G), pCMV-hMDM2 (WT), pCAG-p53.
Punctiform mutants have been obtained by PCR amplification with specific primers using plasmids containing hMdm4 and hMDM2 cDNAs and resulting products used for generation of plasmids containing hMdm4 and hMDM2 cDNAs using Qiagen mutagenesis kit.
Immunoprecipitation.
Cells have been lysed using Saito lysis modified reagent (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.5% Triton-X100, 5 mM EDTA). For p53 immunoprecipitation and BCL2 and MDM4 analysis EBC modified solution has been used (50 mM Tris-HCl, pH 7.4, 0.12 M NaCl, 0.4% NP-40, 1 mM EDTA, 1 Mm β-mercaptoethanol).
400 μg of total protein extract has been incubated with protein G (Invitrogen) for 2 hours under stirring at 4° C. in order not specific binding of proteins with said substrate can occur. Afterwards the cellular extract has been centrifuged, not specific complexes removed with precipitate and supernatant has been incubated for 16 hours with specific antibody. Successively, protein G has been added and incubated for 2 hour at 4° C. Finally, immunoprecipitate has been washed twice with Saito buffer and resuspended in 10 μl of loading buffer solution (loading buffer 4×: 0.6 M β-mercaptoethanol; 8% SDS; 0.25 M Tris-HCl, pH 6.8; 40% glycerol; 0.2% Bromophenol blue). Samples have been denatured at 95° C. for 5 minutes before loading on gel for electrophoresis and successive western blot analysis.
Blot Western Analysis.
Different amounts (70-100 μg for samples to be analyzed) of total protein lysates have been separated by polyacrylamide gel electrophoresis (29% acrylamide and 1% bis-acrylamide) at 12% final concentration. Loading solution has been added to samples (loading buffer 4×: 0.6 M β-mercaptoethanol; 8% SDS; 0.25 M Tris-HCl, pH 6.8; 40% glycerol; 0.2% Bromophenol blue) suitable to denature definitively the same (as result of disruption of disulfide bridges by β-mercaptoethanol) and confer a definitive negative charge to all the proteins (by conjugation with SDS). Samples are boiled at 95° C. for 5 minutes and successively loaded on gel. Electrophoretic run has been carried out at 100 V for approximately 2 hours in ionic buffer containing 25 mM Tris-HCl, pH 8.8, 200 mM glycine and 0.1% SDS.
At the end of electrophoretic run proteins have been transferred on PVDF filter (Millipore) under electric field in buffer consisting of 25 mM Tris Base, 200 mM glycine and 20% methanol. Transfer of proteins from anode to cathode has been carried out for 3 hours at 70 V.
After proteins transfer, the filter has been incubated under stirring for 1 hour in TPBS buffer 1× (10 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20) containing 5% skimmed milk powder (Bio-Rad) in order to block not specific antibody binding sites. The filter successively has been incubated for 2 hours with opportunely diluted primary antibody. At the end of the incubation the filter has been washed twice for approximately 10 minutes each with TPBS 1× and then incubated for 45 minutes with corresponding peroxidase-conjugated secondary antibody. After two washings for approximately 10 minutes with TPBS 1×, immunoreactivity has been evaluated using chemiluminescence reaction based on luminol oxidation (Amersham). Successively, chemiluminescence has been detected by autoradiography on sensitive plates (Kodak).
1.2 Effects of dissociation of MDM4 from MDM2 MDM2/MDM4 heterodimer is the main control tool for p53 levels and therefore inhibition thereof inside of cell under normal growth conditions (Wade and Wahl, 2009). Therefore it has been assumed that the dissociation of MDM4 from MDM2 can favor the implementation of pro-apoptotic function thereof, in addition to preventing degradation activity with respect to p53. The binding leading to heterodimer formation occurs at the level of “RING finger” domains of both the proteins (Tanimura et al., 1999). In order to point out the role of dissociation in the accomplishment of MDM4 pro-apoptotic activity, MDM4 mutant mutated at 437 cysteine residue has been used. Such mutation results in the impossibility to form a correct RING finger domain and thus MDM2 binding impossibility. Therefore, DNX cells have been transfected with constructs for expression of p53, MDM4 or MDM4C437G, and MDM2 proteins. Then Trypan Blue staining positive death cells have been counted and compared to number of total counted cells (
1.3 Preparation of Peptides Suitable to Disrupt the Interaction Between MDM2 and MDM4
For design of peptide suitable to disrupt the interaction between MDM2 and MDM4, initially an analysis at crystallized structure interfaces of MDM2 and MDM4 RING domains (pdb code: 2vjf) has been carried out. This analysis, in particular, has been carried out using a software program (SiteMap, Schrodinger Software) suitable to characterize binding pockets in proteins. As result, two small binding pockets at level of MDM2 and MDM4 N-terminal regions, respectively, have been detected.
Human MDM4 and MDM2 protein sequences, MDM4 and MDM2 amino acid sequences of MDM4 and MDM2 interaction domains and MDM4 and MDM2 amino acid sequences occurring in crystallographic structure of MDM4/MDM2 dimer are below reported:
Starting from N-terminal regions of MDM2 and MDM4, the detection of key amino acid set involved in mediating interactions occurring at interface of MDM2/MDM4 complex has been carried out. Among these residues, Leu430MDM2, Ala434MDM2, Leu433MDM4 and Ile489MDM4 have been detected as particularly important for stabilization of heterodimeric complex of RING domain (
Since the alpha helix conformation stability (helicity) is important parameter in order to favor a strong interaction between peptides and MDM2 and MDM4 RING domains, helicity of peptides 1 and 2 using simulations of molecular dynamics has been evaluated. These simulations have been carried out using Desmond software program and OPLS 2005 force field, as implemented according to Schrodinger software package. Particularly, simulations have carried out for 20 ns time length in aqueous solvent, at a temperature of 300° K, and according to NPT Berendesen protocol. Trajectories obtained by two simulations then have been analyzed through the calculation of secondary structure of peptides during said 20 ns. Resulting peptide helicities, expressed as percentage, has been 23.7% and 12.5% for peptide 1 and peptide 2, respectively. It has not been possible a similar analysis to be carried out for peptide 3.
1.4 Study on Apoptotic Effectiveness of Peptide 1 and Peptide 3
SEQ ID NO: 1-3 peptides have been modified by addition of FITC at N-terminal residue followed by a spacer. Amide group, in order to increase peptide stability, has been attached at C-terminal residue:
Ahx term indicates a linker.
Below properties thereof are shown.
Peptide 1 (SEQ ID NO: 1): the peptide has been tested by immunoprecipitation assays and detection of cell death.
It has been investigated whether peptides were able to enter the cell simply by addition thereof at given concentration (5 μM) to cell culture medium and analyzing for the presence thereof by confocal and fluorescence microscopy, each peptide being labeled with green fluorophore (FAM). It has been observed that peptides were detected in cells only after transfection by liposomes. Successively, the ability of peptides to disrupt the binding by MDM4 or MDM2 immunoprecipitation after peptide transfection in HCT116 cancer cell line, and detection of presence of corresponding partner in immunocomplex by western blot assay have been evaluated. Two HCT116 wild type and HCT116 p53−/− syngeneic cell lines have been used in order to understand also observed effect dependence on the presence of p53. SEQ ID NO:1 peptide proved to be suitable to dissociate said two proteins and induce a significant apoptotic response (
Peptide 3 (SEQ ID NO: 3): analogously to peptide 1, also for this peptide the ability to penetrate the cell has been evaluated by counting the number of cells positive for the presence of FITC fluorophore. This peptide has been proved to be internalized in cells both by liposomal vectors and independently although with lower efficiency. Successively, ability thereof to dissociate MDM4/MDM2 complex has been analyzed by immunoprecipitation experiments. Applicants have observed that the peptide is highly effective in dissociating said two molecules while does not dissociate p53 from MDM4 (
Moreover, this peptide has been proved to be highly effective in inducing cellular apoptosis in specific way since another peptide unable to dissociate MDM4/MDM2 does not induce cell death (not shown data). Apoptotic effect in addition is dependent on p53 since induced death is meaningfully lower in p53 lacking than harboring cells (
Further experiments have been performed to define the role of peptide 3 composition in its molecular and biological activity. These experiments have demonstrated that aminoacids Isoleucine at position 489 and Phenylalanine at position 488 of MDM4 protein are essential for the ability of Peptide 3 to inhibit MDM4/MDM2 activity and apoptosis-promoting function. Indeed, a peptide with two substitutions at position 10 and 11 of peptide 3 (Peptide 3D, KEIQLVIKVAEA (SEQ ID NO:38) was completely ineffective in inhibiting ubiquitination of p53 and inducing apoptosis (
In addition, since SC3A and SC3B have different solubility and helical content (as estimated by AGADIR software), these data reinforce the importance of targeting aa A488 and 1489, independently of other peptide properties.
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the peptides, compositions, arrangements, devices, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, Examples and Annex A is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence.
Further the sequence listing filed concurrently with present description as txt file P1202-US-2013-02-21-Sequence Listing_ST25, forms integral part of the present disclosure and is incorporated herein by reference in its entirety.
The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein can be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.
In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims
| Number | Date | Country | Kind |
|---|---|---|---|
| RM2012A000060 | Feb 2012 | IT | national |
