WO2021105384A1

WO2021105384A1 - Targeting the nls region of nupr1 protein to treat cancer

Info

Publication number: WO2021105384A1
Application number: PCT/EP2020/083670
Authority: WO
Inventors: Juan Iovanna; José Luis NEIRA; Mirna SWAYDEN; Patricia SANTOFIMIA; Wenjun LAN; Yi Xia
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université D'aix Marseille; Centre National De La Recherche Scientifique (Cnrs); Institut Jean Paoli & Irene Calmettes; Chongqing University; Miguel Hernández University Of Elche
Priority date: 2019-11-27
Filing date: 2020-11-27
Publication date: 2021-06-03

Abstract

The present invention relates to the treatment of cancer. NUPR1 is a 82-residue-long nuclear intrinsically disordered protein (IDP) that plays an important role in pancreatic ductal adenocarcinoma (PDAC) as well as other cancers since its genetic inactivation induces tumors growth arrest. The inventors have recently developed an efficient multidisciplinary strategy by combining biophysical, biochemical, bioinformatic and biological approaches for a molecular screening to select potential drug candidates against NUPR1. A family of TFP-derived compounds has been produced and the most active one, named ZZW-115, has more than 10 times efficient antitumor activity than TFP. More importantly, they demonstrated that targeting the NLS region (nuclear location signal region) of NUPR1 protein with small compounds, like TFP and TFP-derived compounds, could be an efficient method to treat patients with cancers. Thus, the invention relates to a compound which targets a region comprising the region consisting of amino acids 63-78 of SEQ ID NO:1 for use in the treatment of a cancer in a subject in need thereof.

Description

TARGETING THE NLS REGION OF NUPR1 PROTEIN TO TREAT CANCER

FIELD OF THE INVENTION:

The present invention relates to a compound which targets a region comprising the region consisting of amino acids 63-78 of SEQ ID NO: 1 for use in the treatment of a cancer in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Cancer is a major burden of disease worldwide. Each year, tens of millions of people are diagnosed with cancer around the world, and more than half of the patients eventually die from it. Globally, nearly 1 in 6 deaths is due to cancer.

In many countries, cancer ranks the second most common cause of death following cardiovascular diseases. With significant improvement in treatment and prevention of cardiovascular diseases, cancer has or will soon become the number one killer in many parts of the world. As elderly people are most susceptible to cancer and population aging continues in many countries, cancer will remain a major health problem around the globe.

This awareness led the World Health Assembly to pass, in 2017, the resolution Cancer Prevention and Control through an Integrated Approach to urge governments and WHO to accelerate action to achieve the targets specified in the Global Action Plan and 2030 UN Agenda for Sustainable Development to reduce mortality from cancer.

In spite of the recent advances, there remains a need in the art for novel anti-cancer drugs and novel targets to improve anti-cancer therapy.

SUMMARY OF THE INVENTION:

NUPR1 is a 82-residue-long nuclear intrinsically disordered protein (IDP) that plays an important role in pancreatic ductal adenocarcinoma (PD AC) as well as other cancers since its genetic inactivation induces tumors growth arrest. The inventors have recently developed an efficient multidisciplinary strategy by combining biophysical, biochemical, bioinformatic and biological approaches for a molecular screening to select potential drug candidates against NUPR1 . To this aim, they have employed a screening method based on measuring fluorescence thermal denaturation, and have identified the well-known antipsychotic agent Trifluoperazine (TFP), and its structurally related Fluphenazine hydrochloride, as ligands inducing significant differences in the temperature denaturation profile for NUPR1. Then, a strong antitumoral effect of the TFP has been demonstrated in vitro as well as in vivo studies.

On the basis of the biophysical and computational analysis, they have modeled this interaction and have determined that amino acids around Ala33 and Thr68 of NUPR1 are involved in the binding with TFP molecules. However due to: (i) the side-effects observed during treatments of mice with TFP; and (ii) the need to improve the anti-cancer activity of TFP they we started a multidisciplinary approach to optimize TFP, based on the synergy of in silico studies, chemical synthesis, and a plethora of biophysical, biochemical and biological analyses. A family of TFP-derived compounds has been produced and the most active one, named ZZW- 115, has more than 10 times efficient antitumor activity than TFP, on a large panel of primary PD AC-derived cells and several non-pancreatic cancer cells. These in vitro results are in good agreement with the findings of Isothermal Titration Calorimetry (ITC) measurements, in which ZZW-115 is the compound with the greatest affinity forNUPRl. Most importantly, ZZW-115 has shown a dose-dependent tumor regression in xenografted mice leading to almost a disappearance after 30 days of treatment with 5 mg/kg/day, in 5 independent PDAC models, including an immune-competent mice model, and, extraordinarily, with no apparent neurological effects. At the cellular level, they have demonstrated that ZZW-115 induces cell death by a combination of both necroptotic and apoptotic mechanisms. Importantly, these molecular mechanisms are similar to those observed in NUPR1 -deficient cells, and which can be respectively inhibited by Necrostatin-1 and Z-VAD-FMK.

Since targeting NUPR1 by ZZW-115 is highly efficient for treating cancers, it is essential to determine the molecular mechanisms by which ZZW-115 exerts its anti-tumoral activity and eventually to determine other anticancer-associated functions. To address this question, the inventors used NUPR1 immunoprecipitation followed by mass spectrometry analysis to generate the NUPR1 interactome. To identify the partners of NUPR1, they stably transfected MiaPaCa-2 cells with a plasmid expressing the Flag-tagged NUPR1 fusion protein followed by its co-immunoprecipitation with antibodies against the Flag-tag and a LC-MS/MS proteomic analysis of the precipitate. They identified 656 proteins in the complex. As expected the majority of the partners are nuclear proteins. A bioinformatic analysis using the String protein-protein interaction database (https://string-db.org/) showed a significant enrichment of nucleocytoplasmic transporters (69 proteins; p=7.34e-34).

More importantly, they demonstrated that targeting the NLS region (nuclear location signal region) of NUPR1 protein with small compounds, like TFP and TFP-derived compounds, could be an efficient method to treat patients with cancers. Thus, the present invention relates to a compound which targets a region comprising the region consisting of amino acids 63-78 of SEQ ID NO: 1 for use in the treatment of a cancer in a subject in need thereof. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

Target of the invention and use thereof

A first object of the present invention relates to a compound which targets a region comprising the region consisting of amino acids 63-78 of SEQ ID NO: 1 for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment the invention also relates to a compound which targets at least one amino acids comprised into the region of amino acids 63-78 of SEQ ID NO: 1 for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment the invention also relates to a compound which targets a region comprising at least 2, 3 4 or 5 consecutive amino acids of the region consisting of amino acids 63-78 of SEQ ID NO: 1 for use in the treatment of a cancer in a subject in need thereof.

In another embodiment, the invention relates to a compound witch target the NLS region of NUPR1 (gene, mRNA or protein) for use in the treatment of a cancer in a subject in need thereof.

As used herein, the term NLS (for “Nuclear Location Signal”) region of NUPR1 denotes the amino acid sequence of NUPR1 that 'tags' this protein for import into the cell nucleus by nuclear transport. The NLS sequence of NUPR1 (protein) corresponds to the region consisting of amino acids 63-78 of SEQ ID NO:l. The NLS sequence ofNUPRl (cDNA) corresponds to the region consisting of nucleic acids 292 to 337of SEQ ID NO:2.

Amino acids sequences ofNUPRl (SEQ ID NO: 1): MATFPPATSA PQQPPGPEDE DSSLDESDLY SLAHSYLGGG GRKGRTKREA AANTNRP SPG GHERKLVTKL QN SERKKRGA RR

In a particular embodiment, the invention relates to a compound which targets a region comprising the region consisting of nucleic acids 292 to 337 of SEQ ID NO:2 for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment the invention also relates to a compound which targets at least one nucleic acids comprised into the region of nucleic acids 292 to 337of SEQ ID NO:2 for use in the treatment of a cancer in a subject in need thereof. In a particular embodiment the invention also relates to a compound which targets a region comprising at least 2, 3 4, 5, 6, 7, 8, 9, or 10 consecutive nucleic acids of the region consisting of nucleotides 292 to 337of of SEQ ID NO:2 for use in the treatment of a cancer in a subject in need thereof.

Nucleic acids of the cDNA of NUPR1 (SEQ ID NO: 2):

As used herein the term “compound witch target the NLS region of NUPR1” denotes all molecules which targets the NLS region or a part of the NLS region (on the gene, the mRNA or the protein) and inhibit the function of NUPR1 that is to say hampering its interaction with importins and consequently hindering its cytoplasmic to nuclear transport and hampering its nuclear functions. For example, the compound can be a small molecule or an intra-body.

According to the invention, the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer. In a particular embodiment, the cancer is a pancreatic cancer, liver cancer, melanoma, colon cancer, glioblastoma, osteosarcoma, prostate cancer and breast cancer.

In another particular embodiment, the cancer is a pancreatic cancer and particularly a Pancreatic Ductal AdenoCarcinoma (PDAC).

As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. Particularly, the subject can suffer from a cancer and particularly a pancreatic cancer.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). In one embodiment, the compound of the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

Particularly, the compound of the invention is a compound of formula (I):

wherein:

R1 represents a linear or branched (C2-C5)alkyl group, optionally substituted with a

-NR3R4 group;

R2 represents a non-bonding pair or a -(CH2)1-5NR3R4 group, wherein when R2 does not represent a non-bonding pair, then the nitrogen atom to which R2 is attached is positively charged; and

R3 and R4 independently represent:

- a hydrogen atom;

- a -OH group;

- a linear or branched (C1-C6)alkyl group, said alkyl group being optionally substituted with one or several substituents chosen from a halogen atom, a -OH group and a -SH group;

- a linear or branched (C1-C6)alkoxy group, said alkoxy group being optionally substituted with one or several substituents chosen from a halogen atom, a -OH group and a - SH group; or

- a -COOH group; or R3 and R4 form, together with the nitrogen atom to which they are attached, a saturated or unsaturated (3- to 6-membered)heterocycle comprising, in addition to the nitrogen atom to which R3 and R4 are attached, 0, 1 or 2 additional heteroatom(s) chosen from a nitrogen atom and an oxygen atom; said heterocycle being optionally substituted with one or several substituents chosen from a halogen atom, a -OH group, a (=O) group and a linear or branched (C1-C6)alkyl group; and said compound of formula (I) being in all the possible tautomeric and isomeric forms: racemic, enantiomeric and diastereoisomeric, and also as addition salts with inorganic and organic acids or with inorganic and organic bases of the compound of formula (I).

In an embodiment, R1 represents a linear or branched (C2-C5)alkyl group, preferably a linear (C2-C5)alkyl group, in particular a linear (C2-C4)alkyl group and more particularly a - C2H5 group.

In another embodiment, R1 represents a linear or branched (C2-C5)alkyl group substituted with one -NR3R4 group, preferably a linear (C2-C5)alkyl group substituted with one

-NR3R4 group, in particular a linear (C2-C4)alkyl group substituted with one -NR3R4 group and preferably represents a -(C2H4)NR3R4 group.

In a particular embodiment, R1 represents:

- a -C2H5 group; or

- a -(C2H4)NR3R4 group, with R3 and R4 being as defined previously or as further defined here-after.

In another particular embodiment, the compound of the invention is a compound of formula (II):

wherein: n is an integer selected from the group ranging from 1 to 4; and R2, R3 and R4 are as defined previously or as defined here-after. In an embodiment of the invention, n is 1 or 2.

In an embodiment of the invention, n is 1.

In an embodiment, R2 according to formulae (I) or (II) represents a non-bonding pair.

In another embodiment, R2 according to formulae (I) or (II) represents a

-(CH2)1-5NR3R4 group, in particular a -(CH2)2NR3R4 group, R3 and R4 being as defined above.

In a particular embodiment, R2 according to formulae (I) or (II) represents a

-(CH2)2NR3R4 group wherein R3 and R4 independently represent a linear or branched

(C1-C6)alkyl group, preferably a linear (C1-C6)alkyl group and in particular a linear

(C1-C3)alkyl group.

In a particular embodiment, R2 according to formulae (I) or (II) represents a

-(CH2)2NR3R4 group wherein R3 and R4 both represent a -CH3 group.

In an embodiment of the invention, R2 represents a non-bonding pair or a

-(CH2)2N(CH3)2 group.

In every embodiment of the invention where R2 is different from a non-bonding pair, the nitrogen atom to which R2 is attached is positively charged.

In a particular embodiment, R3 and R4 are identical.

In an embodiment of the invention, R3 and R4 of formulae (I) or (II) independently represent a linear or branched (C1-C6)alkyl group or R3 and R4 form, together with the nitrogen atom to which they are attached, a saturated or unsaturated (3- to 6-membered)heterocycle comprising, in addition to the nitrogen atom to which R3 and R4 are attached, 0, 1 or 2 additional heteroatom(s) chosen from a nitrogen atom and an oxygen atom; said heterocycle being optionally substituted with one or several substituents chosen from a halogen atom, a - OH group, a (=O) group and a linear and branched (C1-C6)alkyl group.

In an embodiment of the invention, R3 and R4 independently represent a linear or branched (C1-C6)alkyl group, preferably a linear (C1-C6)alkyl group and in particular a linear (C1-C3)alkyl group. More particularly, R3 and R4 can both represent a -CH3 group or a -C2H5 group, and they preferably both represent a -CH3 group.

In another embodiment, R3 and R4 form, together with the nitrogen atom to which they are attached, a saturated or unsaturated (3- to 6-membered)heterocycle comprising, in addition to the nitrogen atom to which R3 and R4 are attached, 0, 1 or 2 additional heteroatom(s) chosen from a nitrogen atom and an oxygen atom; said heterocycle being optionally substituted with one or several substituents chosen from a halogen atom, a -OH group, a (=O) group and a linear and branched (C1-C6)alkyl group. In a particular embodiment, said (3- to 6-membered)heterocycle is saturated.

In a particular embodiment, said heterocycle is a 5- or 6-membered heterocycle.

In an embodiment of the invention, said heterocycle comprises, in addition to the nitrogen atom to which R3 and R4 are attached, 0 or 1 additional heteroatom chosen from a nitrogen atom and an oxygen atom. If present, said additional heteroatom can in particular be an oxygen atom.

In particular, R3 and R4 can form, together with the nitrogen atom to which they are attached, a heterocycle selected from the group consisting of:

In a particular embodiment, said heterocycle is not substituted or is substituted with a (=O) group.

In an embodiment of the invention, R3 and R4 independently represent:

- a -(CH3) group; or

- a -(C2H5) group; or form, together with the nitrogen atom to which they are attached, a saturated (5- or 6-membered)heterocycle comprising, in addition to the nitrogen atom to which R3 and R4 are attached, 0 or 1 oxygen atom; said heterocycle being non- substituted or being substituted with a (=O) group.

In a particular embodiment, R3 and R4 independently represent:

- a -(CH3) group; or

- a -(C2H5) group; or form, together with the nitrogen atom to which they are attached, a heterocycle selected from the group consisting of:

In a particular embodiment, R3 and R4 independently represent:

- a -(CH3) group; or

In a particular embodiment, the compound of the invention is a compound of formula (I) is of formula (II) wherein: n is 1 or 2;

R2 represents a non-bonding pair; and R3 and R4 independently represent:

- a -(CH3) group; or

In a particular embodiment, the compound of the invention of formula (I) is of formula (II) wherein: n is 1 or 2;

R2 represents a non-bonding pair; and R3 and R4 independently represent:

- a -(CH3) group; or

The compounds of the invention can in particular be selected among the following compounds:

as well as their tautomeric and isomeric forms: racemic, enantiomeric and diastereoisomeric, and also their addition salts with inorganic and organic acids or with inorganic and organic bases.

In a particular embodiment the compound for use according to the invention is selected from the group consisting of compounds A, B, C, D, F, G, I, J, K, L, M and N, in particular from the group consisting of C, D, F, G, I, J, K, L, M and N, and more particularly from the group consisting of C, I, F, K, L, M, N and D, as well as their tautomeric and isomeric forms: racemic, enantiomeric and diastereoisomeric, and also their addition salts with inorganic and organic acids or with inorganic and organic bases. More particularly the compound for use according to the invention is selected from the group consisting of compounds C, K, L, M and D, as well as their tautomeric and isomeric forms: racemic, enantiomeric and diastereoisomeric, and also their addition salts with inorganic and organic acids or with inorganic and organic bases.

In a particular embodiment the compound for use according to the invention is the compound C (call ZZW-115):

In a particular embodiement, the compound of the invention can be the Trifluoperazine (TFP), and its structurally related Fluphenazine hydrochloride (TFP-derived compounds).

In one embodiment, the compound of the invention is an antibody against NUPR1. In a particular embodiment, the compound of the invention is an intrabody anti-NUPRl. An intrabody is an antibody that works within the cell to bind to an intracellular protein. Due to the lack of a reliable mechanism for bringing antibodies into a living cell from the extracellular environment, this typically requires the expression of the antibody within the target cell, which can be accomplished in transgenic animals or by gene therapy. As a result, intrabodies are defined as antibodies that have been modified for intracellular localization, and the term has rapidly come to be used even when antibodies are produced in prokaryotes or other non-target cells.

In another embodiment, the antibody according to the invention is a single domain antibody against NUPR1. The term “single domain antibody” (sdAb) or " VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Then, for this invention, neutralizing aptamers ofNUPRl are selected.

In one embodiment, the compound according to the invention is a polypeptide.

In a particular embodiment the polypeptide is an antagonist ofNUPRl and is capable to prevent the function ofNUPRl for example its ability to bind to the DNA. Particularly, the polypeptide can be a mutated version of NUPR1 or a similar protein without the function of NUPR1.

In one embodiment, the polypeptide of the invention may be linked to a cell-penetrating peptide” to allow the penetration of the polypeptide in the cell.

The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri-functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half- life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).

In addition, to the polymer backbone being important in maintaining circulatory half- life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

In another embodiment, the compound according to the invention is an inhibitor of NUPR1 gene expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of NUPR1 expression for use in the present invention. NUPR1 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that NUPR1 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of NUPR1 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of NUPR1 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of NUPR1 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing NUPR1. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild- type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencapsul ati on .

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In order to test the functionality of a putative compound which targets the NLS region of NUPR1 a test is necessary. For that purpose, to identify such compound the subcellular localization of NUPR1 could be identified by using an immunohistochemistry or immunofluorescence method with antibodies directed against NUPR1. Moreover, a subcellular fractionnement could be used followed by the measure of the NUPR1 into the nucleous by a western blot method with an specific antibody directed against NUPR1.

In a particular embodiment, the invention also relates to a method for treating a cancer comprising administering to a subject in need thereof a compound according to the invention.

Combination of the invention

A second object of the present invention relates to a combination of a compound according to the invention and a genotoxic treatment for use in the treatment of a cancer in a subject in need thereof.

The inventors showed that when using the combination of an inhibitor of NUPR1 and a genotoxic treatment, particularly Temozolomide, 5-Fluorouracile, Gemcitabine, Oxaliplatin, and gamma-irradiation, some proteins, including p53, are hypo-SUMOylated and thus that the repair of the ADN in cancerous cells is strongly decreased.

Thus, the invention also relates to a combination of a compound according to the invention and a genotoxic treatment to decrease the SUMOylation of proteins implicated in the repair of DNA.

In another particular embodiment, the invention relates to a compound according to the invention and ii) a genotoxic treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

In a particular embodiment, the invention also relates to a method for treating a cancer comprising administering to a subject in need thereof a compound according to the invention and a genotoxic treatment.

As used herein, the term “a genotoxic treatment” denotes all genotoxic agent or gamma irradiation which can kill cancerous cells and thus treat cancer. According to the invention, a genotoxic treatment or genotoxic agent or gamma irradiation can damage the genetic information that is to say the DNA. The damages can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The use of these properties can be used to induce DNA damage into cancer cells. Any damage done to a cancer is passed on to descendent cancer cells as proliferation continues. If this damage is severe enough, it will induce cells to undergo apoptosis.

Particularly, the genotoxic agent can be selected in the group consisting in but not limited to Temozolomide (TMZ), 5-Fluorouracile (5-FU), Gemcitabine, Oxaliplatin.

As used herein, the terms “gamma radiation” denotes a penetrating electromagnetic radiation arising from the radioactive decay of atomic nuclei. It consists of the shortest wavelength electromagnetic waves and so imparts the highest photon energy. Gamma radiation can be used to treat some types of cancer, since the rays also kill cancer cells. In the procedure called gamma-knife surgery, multiple concentrated beams of gamma rays are directed to the growth in order to kill the cancerous cells. The beams are aimed from different angles to concentrate the radiation on the growth while minimizing damage to surrounding tissues.

In some embodiment, the genotoxic treatment is gamma irradiation, temozolomide (TMZ), 5-fluorouracile (5-FU), gemcitabine, oxaliplatin.

Therapeutic composition

Another object of the invention relates to a therapeutic composition comprising a compound according to the invention and/or a genotoxic treatment for use in the treatment of cancer in a subject in need thereof.

In the pharmaceutical composition according to the invention, the compounds of formula (I) can be identical or can be a mixture of different compounds of formula (I).

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.

For example, anti-cancer agents may be added to the pharmaceutical composition as described below.

Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).

Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.

Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.

Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol s, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.

In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

In still another embodiment, the other therapeutic active agent can be an opioid or non opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofmac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.

Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.

In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: ZZW-115 inhibited NUPR1 nuclear translocation. MiaPaCa-2 cells were treated with ZZW-115 (5 mM) for 6 hours. Immunofluorescence with rabbit anti-NUPRl primary antibody and Alexa 488-labeled goat anti-rabbit secondary antibody were used to reveal the localization of the protein. DAPI staining was used to detect nuclei and it was combined with the Alexa 488 fluorescence in the merged panel. (Magnification: 63x).

Figure 2: NUPR1 interacted with importin a.3 in vitro. (A) Fluorescence spectrum of the complex formed by importin a.3 and NUPR1 (●) and that obtained by the addition of the spectra of both isolated biomolecules after excitation at 280 nm (■). (B) Far-UV CD spectrum of the complex formed by importin a.3 and NUPR1 (●) and that obtained by the addition of the spectra of both isolated biomolecules (■). (C, D) ITC raw data (top: thermal power as a function of time) and titration curve or binding isotherm (bottom: ligand-normalized injection heats as a function of the reactants molar ratio) for the interaction between importin a.3 and NUPR1 in the absence (C) or presence (D) of ZZW-115.

Figure 3: ZZW-115 has strong antitumoral effect in vivo. NMRI-Foxnlnu/Foxnlnu mice (nude mice) xenografted with MiaPaCa-2 cells were separated into 2 groups of 6 mice and treated daily for 30 days with 0.5% DMSO in physiologic serum (control group) or 5 mg/kg ZZW-115 compound. Tumor volume was measured every 5 days. Individual tumour volume of each mouse is represented (by the different lines of the graphics).

EXAMPLE:

Material & Methods

Flag-NUPRl co-immunoprecipitation

MiaPaCa-2 cells, expressing Flag-NUPRl or Flag-GFP, were plated in 10 cm2 dishes. When MiaPaCa-2 cells expressing Flag-NUPRl or Flag-GFP reached 60% confluence were lysed on ice by using HEPES based lysis buffer containing 10 mM NEM (N-Ethylmaleimide, Sigma 04259) and a proteases inhibitor cocktail (1:200) (Sigma P8340). Lysates were centrifuged for 10 min at 14000 rpm at 4°C. Protein concentration of the supernatant was determined by using Protein Assay (BioRad), and equal amounts of total protein were used to incubate with 30 μl of anti-Flag M2 coated beads under rotation for 2 hours at 4°C. Beads were then washed three times with cold lysis buffer and proteins were eluted using 250 μl ammonium hydrogen carbonate buffer containing 0.1 μg/μl of Flag peptide for 90 min while rotating at 4°C. After a short spin, the supernatant was recovered by using a Hamilton syringe. Protein samples were then concentrated by using Amicon Ultra-0.5 centrifugal filter devices (Millipore) according to manufacturer’s instructions. Eluted proteins were collected and analyzed by mass spectrometry.

Immunofluorescence staining

Immunofluorescence staining was performed on MiaPaCa-2 cells in the presence or not of ZZW-115. The rabbit anti-NUPRl primary antibody (homemade) was utilized (diluted 1:200) Signals were detected with a LSM 880 controlled by Zeiss Zen Black, 63x lens. Co localization analysis and measurement of both channels was performed by Image J (Fiji) software.

Protein expression NUPR1 was expressed and purified as described (Encinar et al., JBC 2001). A codon- optimized vector containing residues 1-520 of importin α3 was synthetically produced by Nzytech (Lisbon, Portugal). The modified protein was the same except for the removal of the first N-terminal seventy residues. Both proteins were expressed in BL21 E. coli strain at 37°C, overnight, in LB medium, after induction with 1 mM (final concentration), when the culture had reached and absorbance at 600 nm between 0.6-1.0. Purification was similar to that described for NUPR1, except that the final polish purification step for both constructs was carried out with a Superdex G200 16/60 in buffer Tris (50 mM ,pH 8.0) with 200 mM NaCl, running on an AKTA FPLC (GE, Barcelona, Spain) by following the absorbance at 280 nm.

Circular dichroism (CD) and fluorescence spectroscopies

The experimental set-up for CD and fluorescence spectroscopies was the same described previously. The temperature was 25 °C for both techniques, and protein concentrations were: 10 mM of NUPR1 and 5 mM of importin α3 for the fluorescence experiments; and 20 μM of NUPR1 and 5 μM of importin α3 for the CD experiments. For the thermal denaturation experiments the concentration of both proteins was the same, as well as that of ZZW-115. The response was 8 s, the scan speed was 60°C/h, and the band width was 1 nm. Data were collected every 0.2°C.

Isothermal titration calorimetry (TTC)

Importin a.3 (100-110 μM was loaded into the syringe and NUPR1 (5-10 μM) into the calorimetric cell in buffer Tris 50 mM, pH 8. Reverse titrations (NUPR1 in the syringe and importin a.3 in the calorimetric cells) were also carried out, but direct and reverse titrations provided similar thermodynamic binding parameters. The temperature for all the experiments was 25°C. The binary experiments (interaction of NUPR1 with importin α3 ) were analyzed applying a model considering a single binding site (1:1 stoichiometry for the NUPRl/importin α3 interaction). For experiments in the presence of ZZW-115, a concentration of the compound of 100 μM was kept constant during the titration. These ternary experiments were analyzed in two ways: (i) considering an apparent quasi-binary system with a single binding site displaying apparent thermodynamic binding parameters implicitly dependent on the concentration of ZZW-115; and (ii) considering an exact ternary system with a single binding site displaying intrinsic thermodynamic binding parameters explicitly dependent on the concentration of ZZW- 115 through cooperative interaction parameters. The concentration of ZZW-115 in the calorimetric cell was much higher than its dissociation constant for its interaction with NUPR1, and the data analysis with both models gave similar values for the reduction in affinity for the NUPRl/importin a.3 interaction caused by the presence of ZZW-115.

Results

The cytoplasm to nucleus transport of NUPR1

The nuclei separate physically the cytoplasmic translation machinery from transcription, and then gene expression and DNA function relies on a tight control of the exchange of proteins and mRNA between cytoplasm and the nucleus. Active transport between the nucleus and the cytoplasm occurs via a family of nuclear transport receptors known as importins (or karyopherins), together with other proteins including nucleoporins (NUPs). The importin pathway is initiated by recognition of proteins, than have a classical nuclear location signal (NLS) in their sequences, by importin a. This complex is transported through the pore in the nucleus by importin b, thus forming a larger complex, which interacts with the FG-rich (Phe and Gly (FG)-rich nucleoporin repeats) regions belonging to NUPs. NUPR1 contains a canonical bipartite domain of positively charged amino acids, typical of NLS, localized from residues 63 to 78.

The interactome of NUPR1 revealed that it was bound to some components of nuclear pore including several importins (KPNA1, KPNA2, KPNA3, KPNA4 and KPNA6) and 17 NUPs. As Thr68 belongs to the NLS region of NUPRl and it is involved in binding to ZZW- 115, we hypothesized that ZZW-115 can hamper the interaction between NUPRl (through its NLS) and importins, and then, it can block the NUPRl nuclear translocation. Therefore, by using NUPRl immunofluorescence staining, we have studied the potential impact of ZZW-115 on the intracellular location of NUPRl. Consistently, we demonstrated that treatment with ZZW-115 inhibited almost completely the translocation of NUPRl from the cytoplasm to the nucleus from 78% in control cells to 16% in ZZW-115 treated cells (Figure 1). This result led us to the conclusion that ZZW-115 can inactivated NUPRl by preventing its translocation into the nucleus, where it is presumed to play its essential roles regarding cell survival, especially in cancer cells.

NUPRl and importin α3 bound in vitro

As we had identified a physical interaction between importins and NUPRl in our interactome assay, we decided to test this interaction in vitro by using spectroscopic techniques. Among the different isoforms of importins, we used importin α3 (KPNA4). We tested the binding first by using fluorescence and circular dichroism (CD). As NUPRl has only two tyrosines (Tyr30 and Tyr36) the changes observed in the fluorescence spectrum must be due to changes in the environment around at least one of the 6 tryptophans in importin α3 (Figure 2). Conversely, the far-UV CD spectra did not show any change, suggesting that the secondary structure of importin α3 did not change upon binding (Figure 2), as it has been shown when other importins bind to otherwise fully- folded proteins; furthermore, the CD results suggest that NUPR1 remained disordered upon binding. Then, we showed that in vitro there was binding between NUPR1 and importin α3.

To determine qualitatively, and in an easy and fast way, that the presence of ZZW-115 shifted the binding equilibrium between importin α3 and NUPR1, we carried out thermal denaturations followed by CD of a variant of importin α3, which does not have at the N-terminal region, the polypeptide patch which acts as an auto-inhibitory domain, hampering the binding of the NLS of any protein. In the presence of this variant, the apparent thermal denaturation midpoint of isolated importin α3 is around 317 K; in the presence of equimolar amounts of NUPR1, the apparent thermal denaturation midpoint is 325 K, as it could be expected from the presence of a ligand (NUPR1) which binds to a target (importin α3) and thus stabilizes it. Conversely, in the presence of equimolar amounts of ZZW-115, the apparent thermal denaturation point moves to 322 K, that is closer to that of isolated importin (Figure 2).

We carried out isothermal titration calorimetry (ITC) experiments in the absence and in the presence of ZZW-115. The results indicate that: (i) the affinity ofNUPRl for importin a.3 (association constant of 6.9 x 105 M-l, and dissociation constant of 1.4 mM) was similar to that shown by NUPR1 towards other biomolecules and for ZZW-115 (association constant of 4.7 x 105 M-l and dissociation constant of 2.1 μM); and (ii) in the presence of ZZW-115, a 25-fold reduction in the affinity between NUPR1 and importin a.3 was observed.

ZZW-115 shifted importins of the cargo-importin complexes

NUPR1 is a small IDP protein with a typical NLS sequence at its C-terminal region. Due to its disordered nature (and thus a large hydrodynamic radius), it needs to be transported from the cytoplasm to the nucleus to play its critical role in cancer cells. By using a proteomic- based strategy we found that NUPR1 binds to proteins involved in the nuclear pore complex (NPC). The initial step of the nuclear transport process is the formation of importin-cargo complex in which importins bind cargo molecules after recognition of their NLS. Then, the N- terminus of importin-a (importin-a binding domain) binds to importin-a. After the formation of importin(s)-cargo complex, this can pass through the nuclear pore. Each importin isoform has its own specific cargo molecules. We explored in vitro the binding between the isoform importin α3 and NUPR1. The binding affected some of the aromatic residues of importin, but it did not cause any change in the secondary structure of both proteins; that is, NUPR1 remained disordered upon binding, forming a fuzzy complex (as it happens in the presence of other partner biomolecules. Our thermal denaturation data (even though the obtained thermal midpoints are apparent due to the irreversibility of thermal denaturations) indicate that ZZW- 115 arrests NUPR1 shifting the binding equilibrium between the latter and importin α3.

Altogether, these data show that ZZW-115 was bound to the NLS region of the NUPR1, and then competes with it when importin α3 is present. Until recently, the palette of nuclear transport inhibitors had been limited to XPOl by Leptomycin B (LMB), however LMB failed in Phase I clinical trials due to its toxicity. Since the discovery of LMB, an increasing number of new inhibitors of nuclear transport mediated by importins have been reported, but retaining its persistent toxicity due to its lack of specificity; whereas, on the contrary, a limited number of the inhibitors that target specific cargos have been reported so far. The first cargo-specific nuclear transport inhibitor described was mifepristone as a specific inhibitor of recognition by importin a/b of HIV-1 integrase with no other importin a/b-recognized cargos. There has been a limited progress in the last few years in identifying and characterizing nuclear transport inhibitors. In terms of clinical applications, their high toxicity limited their use. Since ZZW- 115 specifically binds NUPR1 at residues involved in its NLS region, it shifts importins in a cargo-specific manner. We assume that its toxicity will be low, if any, because there is not binding to the importin (in fact, thermal denaturations followed by CD, indicate that the binding of ZZW-115 to the intact importin α3 does not exist). Screening and identification of high affinity compounds, using the NLS specific cargos used as bait, could be a worthy strategy to identify new targets of nuclear proteins.

ZZW-115 inhibits the growth of pancreatic xenografted tumors in vivo The anticancer effect of the ZZW-115 compound was used to treat MiaPaCa-2 cell- xenografted mice. Ten million of cells were subcutaneously injected in mice. When tumors reached 200 mm3 we started a daily treatment for 30 days with 5 mg/kg ZZW-115 (Figure 3). The control group received an equivalent volume of vehicle solution. As expected, tumor volume increased in an exponential manner in control mice (from 217.7 ± 16.7 mm3 to 1790.7 ± 97.0 mm3 during the 30 days of observation). In contrast, when the mice were injected with 5 mg/kg ZZW-115, the tumors stopped growing a few days after treatment and their size decreased progressively, almost disappearing at the end of the treatment (from 205.9 ± 23.7 mm3 to 134.2 ± 106.8 mm3; 27.4 ± 4.6 mm3 when excluding the outlier mouse).

We concluded that ZZW-115 treatment could reduce tumor size for the xenografted tumors by inhibiting its nuclear translocation. Conclusions

We have demonstrated that targeting the NLS region of NUPR1 protein with small compounds, like TFP and TFP-derived compounds, is a very efficient strategy to: first, hampering its interaction with importins and consequently hindering its cytoplasmic to nuclear transport; and, second, hampering its nuclear functions which are essential for cancer cells survival. Therefore, we presume that all compounds capable of interacting with this NLS region of NUPR1 may have the same beneficial consequences for cancer treatment. The NLS region of NUPR1 can become an important target to treat patients with cancers. This is why the NLS region of the NUPR1 should be protected as a therapeutically target for treating cancers.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A compound which targets a region comprising the region consisting of amino acids 63-78 of SEQ ID NO: 1 for use in the treatment of a cancer in a subject in need thereof.

2. A compound according to claim 1 wherein said compound targets at least one amino acids comprised into the region of amino acids 63-78 of SEQ ID NO: 1.

3. A compound according to claim 2 wherein said compound targets a region comprising at least 2, 3 4 or 5 consecutive amino acids of the region consisting of nucleotides 63- 78 of SEQ ID NO: 1.

4. A compound according to claims 1 to 3 wherein the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

5. A compound according to claim 4 wherein the cancer the cancer is a pancreatic cancer or a Pancreatic Ductal AdenoCarcinoma (PD AC).

6. A compound according to claims 1 to 5 wherein said compound of NUPR1 is a compound of formula (I):

wherein:

R1 represents a linear or branched (C2-C5)alkyl group, optionally substituted with a - NR3R4 group;

R3 and R4 independently represent:

- a hydrogen atom;

- a -OH group;

7. The compound according to claim 6, wherein R1 represents: - a -C2H5 group; or

- a -(C2H4)NR3R4 group, with R3 and R4 being as defined in claim 1.

8. The compound according to claim 6, wherein said compound is of formula (II):

wherein: n is an integer selected from the group ranging from 1 to 4; and R2, R3 and R4 are as defined in claim 1.

9. The compound according to claim 6, wherein said compound is selected from the following compounds:

10. The compound according to claim 6, wherein said compound is selected from the following compounds:

11. The compound according to any one of claims 6 to 10, wherein said compound is the following compound:

12. A combination of a compound according to claims 1 to 11 and a genotoxic treatment for use in the treatment of a cancer in a subject in need thereof.

13. A method for treating a cancer comprising administering to a subject in need thereof a compound according to claims 1 to 11.

14. A therapeutic composition comprising a compound according to claims 1 to 11 and/or a genotoxic treatment for use in the treatment of cancer in a subject in need thereof.