此应用程序的某些内容目前无法使用。
如果这种情况持续存在,请联系我们反馈与联系
1. (WO2018138106) METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF HEART FAILURE
注:相关文本通过自动光符识别流程生成。凡涉及法律问题,请以 PDF 版本为准

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT

OF HEART FAILURE

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of heart failure.

BACKGROUND OF THE INVENTION:

Heart failure is a common, costly, disabling, and potentially deadly condition. In developed countries, around 2% of adults suffer from heart failure, but in those over the age of 65, this increases to 6-10%. Heart failure is associated with significantly reduced physical and mental health, resulting in a markedly decreased quality of life. The failing heart becomes inefficient, resulting in fluid retention and shortness of breath, fatigue and exercise intolerance. Heart failure is defined by the symptom complex of dyspnea, fatigue and depressed left ventricular systolic function (ejection fraction < 35-40%), and is the ultimate endpoint of all forms of serious heart disease. Despite considerable advances in treatment, heart failure remains associated with high morbidity and mortality. So, there is a permanent need in the art for new molecules for the treatment of heart failure.

The evolutionarily conserved lipid kinase PIKfyve that synthesizes phosphatidylinositol 5-phosphate (PtdIns5P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), has been implicated in a variety of cellular processes, including cell proliferation, migration, tyrosine kinase receptor signalling and membrane trafficking (Shisheva, A. PIKfyve: Partners, significance, debates and paradoxes. Cell Biol Int 32, 591-604 (2008)). PIKfyve is ubiquitously expressed in mammals and the total knockout is embryonic lethal in mice (Ikonomov, O.C. et al. The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice. J Biol Chem 286, 13404-13413 (2011)). It contains a FYVE domain that binds to PtdIns3P on endosomes and is responsible for its intracellular localization (Sbrissa, D., Ikonomov, O.C. & Shisheva, A. Phosphatidylinositol 3 -phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomembrane localization. J Biol Chem 277, 6073-6079 (2002)). Expression of a PIKfyve dominant negative mutant (Ikonomov, O.C, Sbrissa, D. & Shisheva, A. Mammalian cell morphology

and endocytic membrane homeostasis require enzymatically active phosphoinositide 5 -kinase PIKfyve. J Biol Chem 276, 26141-26147 (2001)) (Ikonomov, O.C., Sbrissa, D., Foti, M., Carpentier, J.L. & Shisheva, A. PIKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis. Mol Biol Cell 14, 4581-4591 (2003)), epigenetic or pharmacological inhibition of PIKfyve (Rutherford, A.C. et al. The mammalian phosphatidylinositol 3 -phosphate 5 -kinase (PIKfyve) regulates endosome-to-TGN retrograde transport. J Cell Sci 119, 3944-3957 (2006)) (Jefferies, H.B. et al. A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding. EMBO Rep 9, 164-170 (2008)) induces the formation of enlarged endosomal vacuoles, indicating its critical role in the maintenance of the endo-lysosomal membrane homeostasis.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of heart failure. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Interestingly, the inventors showed that inhibition of PIKfyve improves cardiac function in different mouse models: high-fat-diet mice and mouse model of pressure overload-induced heart failure.

Concerning the mouse model of cardiomyopathy linked to obesity, the inventors provide in vitro and in vivo evidences that chronic inhibition of PIKfyve attenuates obesity-related cardiometabolic phenotype by reducing mitochondrial oxidative stress and apoptosis through the deacetylase SIRT3.

Concerning the mouse model of pressure overload-induced heart failure, in vivo studies showed that PIKfyve inhibitor reversed cardiac hypertrophy and cardiac dysfunction in aortic banding mice compared to vehicle-treated aortic banding mice.

Accordingly, a first aspect of the present invention relates to a method of treating heart failure in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a PIKfyve inhibitor.

In one embodiment, the heart failure is associated with metabolic diseases. In the context of the invention, a "metabolic disease" denotes a disease that disrupts normal metabolism such as obesity or diabetes for instance.

In one embodiment, the heart failure is associated with high blood pressure. As used herein, the term "high blood pressure" or "hypertension" refers to a medical condition in which the blood pressure is chronically elevated. In hypertension, systolic blood pressure is elevated. Diastolic blood pressure may also be elevated. An "elevated" blood pressure indicates a blood pressure which is above the accepted normal values for the age group of the subject, and/or which is in a range considered to be associated with adverse health outcomes.

In one embodiment, the heart failure is associated with coronary artery disease or heart attack. As used herein, the term "Coronary artery diseases", also known as "ischemic heart diseases" refers to the narrowing or blockage of the arteries and vessels that provide oxygen and nutrients to the heart, resulting in the restriction of blood flow to the heart. Coronary artery disease is the most common type of cardiovascular diseases and includes for example stable angina and unstable angina. As used herein, the term "myocardial infarction" or "heart attack", refers to the event that occurs when blood flow stops to a part of the heart causing damage to the heart muscle.

As used herein, the term "heart failure" (HF) embraces congestive heart failure and/or chronic heart failure. The term "heart failure" denotes inability of the heart to supply sufficient blood flow to meet the body's needs. Heart failure occurs when the heart is damaged from diseases such as high blood pressure, coronary artery disease and heart attack, metabolic diseases, poor blood supply to the heart or a defective heart valve. Functional classification of heart failure is generally done by the New York Heart Association Functional Classification (Criteria Committee, New York Heart Association. Diseases of the heart and blood vessels. Nomenclature and criteria for diagnosis, 6th ed. Boston: Little, Brown and co, 1964; 114). This classification stages the severity of heart failure into 4 classes (I-IV). The classes (I-IV) are:

Class I: no limitation is experienced in any activities; there are no symptoms from ordinary activities.

Class II: slight, mild limitation of activity; the patient is comfortable at rest or with mild exertion.

Class III: marked limitation of any activity; the patient is comfortable only at rest. Class IV: any physical activity brings on discomfort and symptoms occur at rest.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.

As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. The term "treatment" encompasses the prophylactic treatment.

As used herein, the term "PIKfyve" has its general meaning in the art and refers to the lipid kinase phosphatidylinositol 3-phosphate 5-kinase type III that synthesizes PtdIns5P and PtdIns(3,5)P2 (Human Uniprot reference: Q9Y2I7 and mouse Uniprot reference : Q9Z1T6). The gene encoding PIKfyve is PIKFYVE gene (Human NCBI Gene ID: 200576 and mouse NCBI Gene ID: 18711). PIKfyve has been implicated in a variety of cellular processes, including cell proliferation, migration, tyrosine kinase receptor signalling and membrane trafficking (Shisheva, A. PIKfyve: Partners, significance, debates and paradoxes. Cell Biol Int 32, 591-604 (2008)). PIKfyve is ubiquitously expressed in mammals and the total knockout is embryonic lethal in mice (Ikonomov, O.C. et al. The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve -/-embryos but normality of PIKfyve+/- mice. J Biol Chem 286, 13404-13413 (2011)). It contains a FYVE domain that binds to PtdIns3P on endosomes and is responsible for its intracellular localization (Sbrissa, D., Ikonomov, O.C. & Shisheva, A. Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomembrane localization. J Biol Chem 277, 6073-6079 (2002).).

As used herein, the term "PlKfyve inhibitor" has its general meaning in the art and should be understood broadly, this expression refers to any natural or synthetic compound down-regulating the expression of PlKfyve, compound that blocks, suppresses, or reduces the biological activity of PlKfyve, or a protease that can degrade PlKfyve. The PlKfyve inhibitor is for instance a small organic molecule, an antibody, an aptamer, siR A, an antisense oligonucleotide or a ribozyme.

In some embodiments, the PlKfyve inhibitor is a small organic molecule. The term "small organic molecule" refers to a molecule 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 5000 Da, more in particular up to 2000 Da, and most in particular up to about 1000 Da.

In a particular embodiment, the PlKfyve inhibitor is STA-5326.

As used herein, the term "STA-5326" or "apilimod" refers to N-[(E)-(3-Methylphenyl)methylideneamino]-6-morpholin-4-yl-2-(2-pyridin-2-ylethoxy)pyrimidin-4-amine (CAS number: 541550-19-0). STA-5326 is known as inhibitor of PlKfyve (Cai X et al. PlKfyve, a class III PI kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll- like receptor signaling. Chem Biol. 2013 Jul 25;20(7):912-921).

In a particular embodiment, the PlKfyve inhibitor is YM201636.

As used herein, the term "YM201636" refers to 6-Amino-N-(3-(4-(4-morpholinyl)pyrido [3 '2 ' :4,5 ] furo [3 ,2-d]pyrimidin-2-yl)phenyl)-3 -pyridine carboxamide (CAS number: 371942-69-7). YM201636 is known as inhibitor of PlKfyve (Jefferies, H.B.J., et al. 2008. EMBO Reports 9, 164.).

In a particular embodiment, the PlKfyve inhibitor is APY0201.

As used herein, the term "APY0201" refers to (E)-4-(5-(2-(3-methylbenzylidene)hydrazinyl)-2-(pyridin-4-yl)pyrazolo [ 1 ,5 -a]pyrimidin-7-yl)morpholine (CAS number: 1232221-74-7). APY0201 is known as inhibitor of PlKfyve (Hayakawa et al,

Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL- 12/23 inhibitor, APY0201. Bioorg Med Chem. 2014 Jun l;22(l l):3021-9).

In a particular embodiment, the PIKfyve inhibitor is vacuolin-1 (Sano et al., Vacuolin-1 inhibits autophagy by impairing lysosomal maturation via PIKfyve inhibition. FEBS Lett. 2016 Jun;590(l l): 1576-85).

As used herein, the term "vacuolin-1" has its general meaning in the art and refers to 3-Iodobenzaldehyde[4-(diphenylamino)-6-(4-morpholinyl)-l,3,5-triazin-2-yl]hydrazine (CAS number: 351986-85-1).

In a particular embodiment, the PIKfyve inhibitor is AS2677131 (Terajima M et al., Inhibition of c-Rel DNA binding is critical for the anti-inflammatory effects of novel PIKfyve inhibitor. Eur J Pharmacol. 2016 Jun 5;780:93-105).

As used herein, the term "AS2677131" refers to rel-N-{6' -[(2R,6S)-2,6-dimethylmorpholin-4-yl]-3,3 -bipyridin-5-yl}-3-ethyl-2-methyl-lH-pyrrolo[3,2-b]pyridine-5-carboxamide.

In a particular embodiment, the PIKfyve inhibitor is AS2795440 (Terajima M et al., Inhibition of c-Rel DNA binding is critical for the anti-inflammatory effects of novel PIKfyve inhibitor. Eur J Pharmacol. 2016 Jun 5;780:93-105).

As used herein, the term "AS2795440" refers to N-(l -isopropyl-6-methyl-\ " ,2" ,6" -tetrahydro-3,3' :6' £" -terpyridin-5-yl)-2,3-dimethyl-lh-pyrrolo[3,2-b]pyridine-5-carboxamide.

In one embodiment, the PIKfyve inhibitor 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. 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. 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.

In a particular embodiment the PIKfyve inhibitor is an inhibitor of PIKfyve expression.

An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of a gene. Therefore, an "inhibitor of PIKfyve expression" denotes a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for PIKfyve. Typically, the inhibitor of PIKfyve expression has a biological effect on one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme

Inhibitors of gene expression for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of PIKfyve mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of PIKfyve, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding PIKfyve can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of gene expression for use in the present invention. Gene expression can be reduced by contacting the subject

with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that 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 Tuschi, 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 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 PIKfyve 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 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'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siR As 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 siR A or ribozyme nucleic acid to the cells. 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 nonessential 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 (A Laboratory Manual," W.H. Freeman CO., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 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 hematopoietic 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., SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 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. 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 microencapsulation.

In some embodiments, the PIKfyve inhibitor of the invention is administered to the subject with a therapeutically effective amount.

The terms "administer" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., PIKfyve inhibitor of the present invention) into the subject, such as by mucosal, intradermal, intraperitoneal, intravenous, subcutaneous, intramuscular, intra-articular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

By a "therapeutically effective amount" is meant a sufficient amount of PIKfyve inhibitor for use in a method for the treatment of heart failure at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the severity of the heart failure, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The compositions according to the invention are formulated for parenteral, transdermal, oral, rectal, subcutaneous, sublingual, topical or intranasal administration.

Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

In one embodiment, the compositions according to the invention are formulated for parenteral administration. 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.

In a preferred embodiment, the compositions according to the invention are formulated for intravenous administration. In another embodiment, the compositions according to the invention are formulated for oral administration.

In a preferred embodiment, the compositions according to the invention are formulated for intraperitoneal administration.

Typically the active ingredient of the present invention (i.e. the PIKfyve inhibitor) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

The term "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, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for

example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

In a particular embodiment, the PIKfyve inhibitor, for instance STA-5326, is formulated with mesylate salt.

In some embodiments, the PIKfyve inhibitor of the present invention is administered to the subject in combination with an active ingredient.

In some embodiments, the PIKfyve inhibitor of the present invention is administered to the subject in combination with a standard treatment. For instance, standard treatment of heart failure is angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, beta blockers, aldosterone antagonists or diuretics.

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: STA treatment reduces left ventricular hypertrophy and preserves cardiac function in a mouse model of pressure overload-induced heart failure.

Mice underwent aortic banding (AB) or sham operation and treated with STA (black bars) or with vehicle (white bars) for 4 weeks. Echocardiography was performed after 4 weeks of AB. Results are presented as mean+/-SEM from 6-9 mice per group.

(A) EF, ejection fraction; SF, shortening fraction; (B) LVPWd, left ventricle posterior wall thickness at end diastole; IVSTd, interventricular septum thickness at end diastole; (C) LVM/BW, left ventricle mass index on body weight ratio.

*P<0.05, **P<0.01 and ***P<0.001 between indicated conditions.

Figure 2: Inhibition of PIKfyve by STA reduces cardiomyoblast hypertrophic response and mitochondrial ROS production. (A) Rat H9C2 cardiomyoblasts were subjected to hypoxia (H) to induce cell hypertrophy or kept in normoxia (N) in the presence of STA or vehicle only (DMSO). Cell surface was quantified and presented as mean+/-SEM from 239-279 cells across 3 independent experiments. (B) qRT-PCR quantification of the expression level of the hypertrophic marker MHC from 3-6 independent experiments. (C-D) H9C2 cells were exposed to oxidative (C) or metabolic (D) stress as indicated and mitochondrial O2" production or mitochondrial H2O2 were assessed using the MitoSOX Red fluorescent probe and MitoPYl probe, respectively. Quantifications are shown. (E) Primary cardiomyocytes isolated from adult mice were treated as indicated and mitochondrial ROS production was measured by MitoSOX. Bonferonni's post-hoc test: ***P<0.001 between indicated conditions.

Figure 3. STA treatment prevents cardiomyoblasts apoptotic cell death. (A)

TUNEL staining of apoptotic H9C2 cells treated as indicated. Quantification of apoptotic cells from 3-4 independent experiments is shown. (B) Cell lysates from H9C2 cells treated as in A were probed with the indicated antibodies. GAPDH was used as a loading control. Quantification of Caspase 3 cleavage is shown from 3-4 independent experiments. (C) TUNEL staining of apoptotic cells treated with 2-deoxyglucose (2DG) in the presence or not of STA. Quantification is shown from 3-4 independent experiments. (D) Western-blot of H9C2 cells treated as in C. Hsp90 was used as a loading control. Quantification is shown. Results are from 4 independent experiments. Bonferonni's post-hoc test: ***P<0.001, **P<0.01 and *P<0.05 between indicated conditions.

Figure 4: Endogenous SIRT3 is required for STA anti-oxidant and anti-apoptotic properties. (A) H9C2 cells were transfected with a control siRNA (siControl) or with a siRNA targeting SIRT3 (siSIRT3), and cells were treated as indicated. Mitochondrial O2" production was assessed using the MitoSOX Red fluorescent probe. Quantification is shown. (B) TUNEL staining of cells treated as in A. Quantification is shown. Bonferonni's post-hoc test: ***P<0.001 as compared with control cells; $$$P<0.001 between indicated conditions.

Figure 5: PIKfyve inhibition reduces cardiac hypertrophy and improves cardiac function in vivo. Obese mice were treated intraperitoneally with STA or vehicle only (Vehicle). (A-C) Echocardiographic measures of ejection fraction (EF, A), fractional shortening (FS, B), left ventricular posterior wall thickness at end diastole (LVPWd, C), intraventricular septum thickness (IVSTd, D) (E) Quantification of myocyte cross sectional area. (F) Quantification of the ratio heart weight on body weight (HW/BW) in vehicle- or STA-treated mice. (G-H) Expression levels of MHC (G) and BNP (H) were measured by qRT-PCR from heart tissues. (I) Heart cryosections were stained with Masson's trichrome to assess cardiac fibrosis. Quantification is shown. One-tailed Student test, *P<0.05; **P<0.01 ***P<0.001 from 4-6 mice per group.

Figure 6: Chronic STA treatment decreases cardiac oxidative stress and apoptosis in obese mice. (A) Quantification of mitochondria size from electron micrographs showing preservation of myocardial mitochondria structure in STA-treated mice. (B) Expression of several OXPHOS complexes was measured by western blot on heart lysates from vehicle- or STA-treated mice. Hsp60 was used as a loading control. Quantification of COX complexes expression is shown. (C) Mitochondria-derived O2" production was

measured on heart cryosections using MitoSOX Red by confocal microscopy. Quantification

of MitoSOX fluorescence is shown. (D) LPO activity was quantified in cardiac tissue. (E)

TUNEL-staining of heart cryosections showing apoptotic cells was carried out.

Quantification is shown. (F) Bax expression level was measured by qRT-PCR from heart

tissues. One -tailed Student test, *P<0.05; **P<0.01 ***P<0.001 from 4-6 mice per group.

EXAMPLE 1:

In order to confirm the anti-hypertrophic effects of STA, we have performed in vivo experiments in a mouse model of heart failure induced by pressure overload using aortic banding (AB). Four weeks after AB, cardiac function was assessed through echocardiographic measurements. Echocardiographic analysis revealed a significant increase in interventricular septum thickness (IVSTd) and LV posterior wall thickness (LVPW) after 4 weeks of AB in mice compared with sham-operated mice (Figures IB). Importantly, cardiac function was severely impaired in AB mice as measured by the decrease in fractional shortening (FS) and ejection fraction (EF) compared with sham-operated mice (Figures 1 A). In contrast, daily treatment (i.p., 2mg/kg/day) of AB mice with STA for 4 weeks reversed cardiac hypertrophy (Figure IB) and cardiac dysfunction (Figures 1A) as compared with vehicle-treated AB mice. In addition, the heart weight-to-body weight ratio (HW/BW) was significantly decreased in STA-treated AB mice compared to vehicle-treated AB mice, suggesting that STA attenuates cardiac hypertrophy (Figure 1C).

EXAMPLE 2:

Material & Methods

Reagents and antibodies. Antibodies used in this study are: anti-GAPDH, (sc-32233)

anti-HSP90 (sc-13119), anti-Hsp60 (sc-13115), anti-Drpl (H-300) and anti-caspase 3 (sc-

7148) from SantaCruz Biotechnology; anti-phospho-Serine (4A4) from Millipore; anti-

OXPHOS/COX (MS604/G2594) from Mitosciences; anti-acetylated-lysine (944 IS), anti-

SIRT3 (D22A3), anti-SIRTl (1F3) and anti-cleaved caspase 3 (9661) from Cell Signaling

Technology. Fluorescent Alexa-coupled secondary antibodies and DAPI were from Life

Technologies and HRP-coupled secondary antibodies from Cell Signalling Technology.

STA-5326 was purchased from Axon MedChem and was referred to as STA throughout this study. All other chemicals were from Sigma- Aldrich unless otherwise stated.

Molecular Biology. siRNA against SIRT3 were from Eurogentec. siRNA Universal negative control was from Sigma. siRNA against PIKfyve were from Sigma. Primers for qRT-PCR were from Sigma-Aldrich.

Quantitative RT-PCR analysis. Total RNAs were isolated from cultured mouse cardiac fibroblasts using the RNeasy mini kit (Qiagen). Total RNAs (300ng) were reverse transcribed using Superscript II reverse transcriptase (Invitrogen) in the presence of a random hexamers. Real-time quantitative PCR was performed as previously described (Alfarano, C. et al. Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin. Int J Obes (Lond) 39, 312-320 (2014).). The expression of target mRNA was normalized to GAPDH mRNA expression.

Quantification of PIKfyve product PI5P. PI5P was quantified using an in vitro mass assay as described (Pendaries, C. et al. PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection. EMBO J 25, 1024-1034 (2006).).

Animal studies, experimental protocol and metabolic measurements. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985) and was performed in accordance with the recommendations of the French Accreditation of the Laboratory Animal Care (approved by the local Centre National de la Recherche Scientifique ethics committee) Two-month old wild-type male C57BL6/J mice purchased from Janvier Labs were fed a high fat diet (HFD, 45% fat) for 12 months. Animals were randomly divided into two groups (n=8 each). Mice received for 17 consecutive days intraperitoneal injections of STA (2mg/kg/day) or vehicle (DMSO), corresponding to a final DMSO concentration of 50% diluted in PBS. The dose of STA was selected on the basis of our preliminary animal

studies. The efficiency of PIKfyve inhibition was monitored by the quantification of cardiac PI5P (data not shown). Plasma glucose (Accu-check, Roche Diagnostics) was measured in fasted state. LPO (lipid hydroperoxyde) quantification was done as described before (Foussal,

C. et al. Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy. FEBS Lett 584, 2363-2370 (2010).) using an ELISA-based kit (Cayman). Triglycerides were quantified using enzymatic assay (TG enzymatic PAP 150, Biomerieux). Plasma insulin was measured using and ELISA-based kit (Mercodia). Mice genetically invalidated for SIRT3 have been described previously (Bochaton, T. et al. Inhibition of myocardial reperfusion injury by ischemic postconditioning requires sirtuin 3-mediated deacetylation of cyclophilin

D. J Mol Cell Cardiol 84, 61-69 (2015).). For the ISO acute treatment, echocardiographic analysis were performed on WT or SIRT3.KO mice (n=9 per group), the day before the treatment. ISO was intraperitoneally administrated (15mg/kg/day) for 3 days, mice were randomly segregated into two groups and injected for 4 days with ISO+STA (2mg/kg/day) or ISO+DMSO. Echocardiography was performed at the end of the protocol to assess cardiac hypertrophy.

Echocardiographic studies. Blinded echocardiography was performed as described (Alfarano, C. et al. Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin. Int J Obes (Lond) 39, 312-320 (2014).) on isoflurane anesthetized mice using a Vivid7 imaging system (General Electric Healthcare) equipped with a 14-MHz sectorial probe. Two-dimensional images were recorded in parasternal long- and short-axis projections, with guided M-mode recordings at the midventricular level in both views. Left ventricular (LV) dimensions and wall thickness were measured in at least five beats from each projection and averaged. Left ventricular posterior wall thickness at end diastole (LVPWd), fractional shortening (FS), ejection fraction (EF) and interventricular septal

thickness at end diastole (IVSTd) were measured. Fractional shortening and ejection fraction were calculated from the two-dimensional images.

Morphology. Ultrastructural studies of cardiac tissue by electron microscopy were done as before (Alfarano, C. et al. Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin. Int J Obes (Lond) 39, 312-320 (2014)). Briefly, cardiac tissue were fixed in cold 2.5% glutaraldehyde/1% paraformaldehyde, post-fixed in 2% osmium tetroxide, embedded in resin, and sectioned. Hematoxylin-eosin and Masson's trichrome stainings of heart cryosections were done according to standard methods.

Cell culture, transfection and treatments. The rat embryonic cardiomyoblastic cell line H9C2 (ATCC) was cultured in DMEM medium (Life Technologies) supplemented with 10% FBS and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. siR A transfection was performed with Lipofectamine RNAiMAX (Life Technologies) according to manufacturer's instructions. For hypoxic treatment, cells were pretreated for 30 minutes with STA (lOOnM) or DMSO (vehicle only) and then subjected to normoxia (5% CO2; 21% O2, balance N2) or hypoxia in a hypoxic chamber (5% CO2, 1% O2, balance N2) for 2h (for ROS measurement) or 16h (for apoptosis). To assess cell hypertrophy, the medium was replaced and cells were further incubated for 24h in normoxic conditions (reoxygenation) in the continuous presence of STA or DMSO. To induce metabolic stress, the cells were treated with 2-deoxy-D-glucose (2DG, 50mM) in complete medium for 4h (for ROS production) or 24h for apoptosis and for ER stress evaluation. As a positive control for ER stress induction, cells were treated with 2 μg/ml tunicamycin for 24 hrs. Primary cardiomyocytes were isolated from adult mice as described before (Boal, F. et al. Apelin regulates Fox03 translocation to mediate cardioprotective responses to myocardial injury and obesity. Sci Rep 5, 16104 (2015).).

Evaluation of cell viability, apoptosis and ROS production.

Cell viability was measured by MTT assay. Briefly, cells treated with STA or H2O2 for 2, 4 or 24hr were further incubated with 0.5mg/ml MTT for 2hr. After medium removal, cells were lysed with DMSO and cell viability was evaluated from absorbance at 570 nM. Apoptosis level was assessed using the DeadEnd Fluorometric TUNEL system according to manufacturer's instructions (Promega). Mitochondrial O2" and H2O2 were measured using MitoSOX Red indicator (Life Technologies) and MitoPYl (Sigma- Aldrich) respectively at ΙμΜ (for H9C2 cells) or 5μΜ (for primary cardiomyocytes) for 30min following live-cell imaging on a confocal microscope equipped with an incubation chamber with temperature control and CO2 enrichment. Mitochondrial superoxide levels on heart cryosections were assessed as described elsewhere (Sun, K. et al. Paradoxical role of autophagy in the dysplastic and tumor- forming stages of hepatocarcinoma development in rats. Cell Death Dis 4, e501 (2013)). Briefly, after incubating freshly prepared cryosections with MitoSOX, the sections were imaged by confocal microscopy. The fluorescence intensity was measured using Image J from 3 random fields of view across 5-6 mice per group.

Immunofluorescence, determination of mitochondria fragmentation and cell size measurement. Immunofluorescence was performed as previously described (Boal, F. et al. TOM1 is a PI5P effector involved in the regulation of endosomal maturation. J Cell Sci 128, 815-827 (2015).). For determination of mitochondria fragmentation, H9C2 cells were live-stained with MitoTracker Red CMXRos (Life Technologies) at 200nM for 15' 37°C. Fixed cells were then imaged by widefield microscopy. Mitochondria fragmentation was measured using a dedicated ImageJ plugin as described (Dagda, R.K. et al. Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J Biol Chem 284, 13843-13855 (2009)). For cell size measurement, three fields of view were randomly selected per conditions and cell surface was quantified using ImageJ.

Protein purification, immunoprecipitation and western blotting. Proteins from

cardiac tissues and H9C2 cells were extracted using RIPA buffer (50mM Tris-HCL (pH 7.5),

150mM NaCl, 0.1% SDS, 0.5% DOC, 1% Triton X-100, ImM EDTA and

protease/phosphatase inhibitor cocktails from Biotools) and quantified using the Bio-Rad

Protein Assay (Bio-Rad). For immunoprecipitation, lysates (250μg proteins) were incubated

overnight at 4°C with 5μΙ, of the specific antibody bound to G-protein sepharose 4 fast flow

(GE Healthcare). After extensive washes, bound proteins were eluted in Laemmli sample

buffer (50mM Tris-HCL (pH 6.8), 2% SDS, 6% glycerol, 0.2 mM DTT and 0.02%

bromophenol blue) and denaturated at 70°C for 15 min. Proteins were resolved by SDS-

PAGE and western blotting. Immunoreactive bands were detected by chemiluminescence

with the Clarity Western ECL Substrate (Bio-Rad) on a ChemiDoc MP Acquisition system

(Bio-Rad).

Statistical analysis. Data are expressed as mean ± SEM. Comparison between two

groups was performed by Student's one-tailed t-test while comparison of multiple groups was

performed by one-way ANOVA followed by a Bonferroni's post hoc test using GraphPad

Prism version 5.00 (GraphPad Software, Inc).

Results

STA treatment attenuates hypertrophic response and mitochondrial ROS

production in cardiomyoblasts. Cardiac hypertrophy is a potent predictor of cardiovascular

risk in obesity (Battiprolu, P.K. et al. Diabetic cardiomyopathy and metabolic remodeling of

the heart. Life Sci 92, 609-615 (2012).). To investigate the potential role of the lipid kinase

PIKfyve in hypertrophic responses to stress, we evaluated the effects of its pharmacological

inhibition by STA on hypertrophy in the cardiomyoblastic cell line H9C2. STA treatment of

H9C2 cells induced the formation of enlarged vacuoles (data not shown), a hallmark of

PIKfyve inhibition and a consequence of the swelling of endosomes (Ikonomov, O.C.,

Sbrissa, D. & Shisheva, A. Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PlKfyve. J Biol Chem 276, 26141-26147 (2001)) (Ikonomov, O.C., Sbrissa, D., Foti, M., Carpentier, J.L. & Shisheva, A. PlKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis. Mol Biol Cell 14, 4581-4591 (2003)) (Rutherford, A.C. et al. The mammalian phosphatidylinositol 3 -phosphate 5-kinase (PlKfyve) regulates endosome-to-TGN retrograde transport. J Cell Sci 119, 3944-3957 (2006)) (Jefferies, H.B. et al. A selective PlKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding. EMBO Rep 9, 164-170 (2008).). The formation of these vacuoles following PlKfyve inhibition by STA did not affect cell viability, even at high dose (up to 500nM) and for long incubation time (up to 24h) (data not shown). Strikingly, STA-treatment abrogated hypoxia-induced hypertrophic responses as shown by measuring the cell surface (Fig. 2A) and quantification of the hypertrophic marker MHC (Fig. 2B). Cell hypertrophy is closely linked to ROS production by mitochondria, a major site for ROS production (Sawyer, D.B. et al. Role of oxidative stress in myocardial hypertrophy and failure. J Mol Cell Cardiol 34, 379-388 (2002)), therefore we next investigated whether PlKfyve inhibition affects hypoxia-induced mitochondrial ROS production. Remarkably, H9C2 cells treated with STA presented a reduced level of mitochondrial produced O2" (Fig. 2C, MitoSOX) and H2O2 (Fig 2C, MitoPYl) in response to hypoxic stress. Interestingly, STA-dependent ROS inhibition was also revealed in condition of metabolic stress, a hallmark of obesity-linked decline in cardiac function. Indeed, as shown in Fig2D, PlKfyve inhibition by STA attenuated mitochondrial ROS production in response to metabolic stress induced by 2-deoxy-D-glucose (2DG). Strikingly, STA also prevented hypoxia-induced ROS production in primary cardiomyocytes isolated from adult mice (Fig. 2E).

PIKfyve inhibition prevents stress-induced cell apoptosis and mitochondrial structural damage. Mitochondrial damage and excessive ROS production may result in activation of apoptotic cascades and cell death (Tsutsui, H., Kinugawa, S. & Matsushima, S. Oxidative stress and heart failure. Am J Physiol Heart Circ Physiol 301, H2181-2190 (2011)). As shown in Fig. 3, in response to hypoxia, STA treatment of H9C2 cells attenuated apoptosis as shown by TUNEL staining (Fig. 3 A) and immonob lotting of cleaved Caspase 3, a bona-fide marker of apoptotic cascade activation (Fig. 3B). Importantly, STA-dependent anti-apoptotic activity was confirmed in conditions of metabolic stress induced by 2DG (Fig. 3C-D). Treatment with 2DG is known to interfere with oligosaccharide synthesis which results in ER stress (Read, D.E., Gupta, A., Ladilov, Y., Samali, A. & Gupta, S. miRNA signature of unfolded protein response in H9c2 rat cardiomyoblasts. Cell Biosci 4, 56 (2014)) (Xi, H. et al. 2-Deoxy-D-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion. Cancer Chemother Pharmacol 67, 899-910 (2010)) (Yu, S.M. & Kim, S.J. Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes. Exp Mol Med 42, 777-786 (2010)). Considering this well characterized mechanism, we next investigated the activation of this pathway in our cell system. 2DG or STA did not induce ER stress as shown by the lack of activation of eIF2, the downstream target of PERK, a major sensor pathway for ER stress (data not shown) (Hetz, C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13, 89-102 (2012)). More generally, we found that STA did not change the stress response induced by tunicamycin in H9C2 cells (data not shown). Altogether, our data indicate that STA is not involved in the regulation of the ER stress response and this phenomenon could not account for its anti-hypertrophic properties.

One of the hallmarks of apoptosis is the fragmentation of the mitochondrial network (Youle, R.J. & Karbowski, M. Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol 6, 657-663 (2005)). Therefore, we next examined whether inhibition of PIKfyve could affect stress-induced mitochondrial fragmentation. While control cells harbored an elongated and interconnected mitochondrial network, hypoxia and 2DG resulted in mitochondrial fragmentation (data not shown). Strikingly, PIKfyve inhibition by STA prevented mitochondrial fragmentation induced by both oxidative and metabolic stress suggesting STA-dependent preservation of mitochondrial integrity (data not shown). In order to confirm STA specificity towards PIKfyve, we resorted to siRNA-mediated silencing of PIKfyve in H9C2 cells. Silencing efficiency was monitored by RT-qPCR (data not shown). Depletion of PIKfyve recapitulated STA effect on the preservation of mitochondrial structures upon hypoxic stress (data not shown).

Mitochondrial fragmentation is mediated by recruitment of the small cytosolic GTPase dynamin-related protein 1 (Drpl) at the active fission site on the surface of mitochondria, which can be followed by immunofluorescence (Frank, S. et al. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell 1, 515-525 (2001)) (Smirnova, E., Griparic, L., Shurland, D.L. & van der Bliek, A.M. Dynamin-related protein Drpl is required for mitochondrial division in mammalian cells. Mol Biol Cell 12, 2245-2256 (2001)). Both oxidative and metabolic stress induced the self-assembly of Drpl in H9C2 cells. In these conditions, STA treatment reduced Drpl assembly, to the same extent as the Drpl -specific inhibitor Mdivi-1 demonstrating the implication of PIKfyve in the control of mitochondrial dynamics (data not shown).

Next, we investigated whether cell responses to oxidative and metabolic stress were linked to PIKfyve enzymatic activity. Both hypoxia and 2DG treatment induced an increase in PIKfyve product PI5P (data not shown). Strikingly, this PI5P synthesis was totally

abrogated by STA treatment providing the first evidence that stress-induced cellular responses are linked to PIKfyve activity.

PIKfyve induces mitochondrial ROS production and apoptosis through a SIRT3-dependent pathway. In cardiomyocytes the high density of mitochondria reflects the high energy demand to maintain contractile functions. Therefore, in order to maintain the redox cellular status and optimize the bioenergetic efficiency of the heart, the function of mitochondria is in turn tightly regulated. The NAD+-dependent lysine deacetylase SIRT3 has recently emerged has a key regulator of mitochondrial functions, through the control of the oxidative and metabolic status, mitochondrial dynamics and apoptosis (Huang, J.Y., Hirschey, M.D., Shimazu, T., Ho, L. & Verdin, E. Mitochondrial sirtuins. Biochim Biophys Acta 1804, 1645-1651 (2010)) (McDonnell, E., Peterson, B.S., Bomze, H.M. & Hirschey, M.D. SIRT3 regulates progression and development of diseases of aging. Trends Endocrinol Metab (2015)). In order to test whether PIKfyve inhibition affects cardiac SIRT3, we first localized endogenous SIRT3 in H9C2 cells subjected to oxidative and metabolic stress in the presence of STA. In control cells, SIRT3 was found mainly cytosolic (data not shown). Interestingly, a strong translocation of SIRT3 to the mitochondria was induced by STA treatment independently of stress stimuli (data not shown). This was further confirmed biochemically by subcellular fractionation. STA induced a strong enrichment of SIRT3 in the mitochondrial fraction of H9C2 cells (data not shown). Of note, it appears that STA-induced SIRT3 translocation is ROS-independent, as incubation with the exogenous ROS scavenger N-acetyl Cysteine (NAC) did not prevent SIRT3 translocation to the mitochondria induced by STA (data not shown). This is consistent with the fact that STA induces SIRT3 translocation to the mitochondria in normoxic conditions. Importantly, the localization of the nuclear SIRT1 was not altered by STA treatment (data not shown), suggesting a specificity towards SIRT3. In order to confirm the results obtained with the pharmacological inhibition of PlKfyve, we examined SIRT3 localization in H9C2 cells silenced for PlKfyve expression using siRNA. Knock-down of PlKfyve in H9C2 cells resulted in a strong translocation of SIRT3 to the mitochondrion without changes in SIRT1 localization (data not shown).

Cytosolic localization of SIRT3 is a matter of debate in the literature, as SIRT3 has been largely described as a mitochondrial protein, thanks to a strong mitochondrial-localization signal (Pillai, V.B., Sundaresan, N.R., Jeevanandam, V. & Gupta, M.P. Mitochondrial SIRT3 and heart disease. Cardiovasc Res 88, 250-256 (2010)). However, SIRT3 is a nuclear-encoded protein and therefore needs to be translocated into the mitochondrial matrix to deacetylate its targets (Schwer, B., North, B.J., Frye, R.A., Ott, M. & Verdin, E. The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase. J Cell Biol 158, 647-657 (2002)). We have performed comparative immunofluorescence study in H9C2 cells vs. cancer HeLa cells. While SIRT3 harbors a strong mitochondrial pattern in the cancer HeLa cells, it appears more cytosolic in H9C2 cells (data not shown). This observation was confirmed by quantification of the Pearson's coefficient, suggesting that the localization of endogenous SIRT3 is cell-type dependent. Our results are consistent with previously described SIRT3 localization in neonatal rat cardiomyocytes, where the authors clearly showed that SIRT3 is partly localized in the cytoplasm, and not exclusively in the mitochondrial matrix in basal conditions (Sundaresan, N.R., Samant, S.A., Pillai, V.B., Rajamohan, S.B. & Gupta, M.P. SIRT3 is a stress-responsive deacetylase in cardiomyocytes that protects cells from stress-mediated cell death by deacetylation of Ku70. Mol Cell Biol 28, 6384-6401 (2008)).

We next investigated if PlKfyve inhibition could alter the metabolic status of H9C2 cells. STA-treatment did not affect the consumption of the key metabolites D-Glucose, D-Galactose and D-Glucose-l-P (data not shown).

Next, in order to determine whether SIRT3 is involved in PIKfyve regulation of mitochondrial ROS generation and apoptosis, we resorted to its silencing using specific siRNA. Knock-down efficiency was confirmed by RT-qPCR (data not shown). We hypothesized that SIRT3 depletion may result in the loss of STA properties. As shown in Fig. 4A, in conditions of metabolic stress, SIRT3 silencing totally prevented STA effect on mitochondrial ROS production. In addition, SIRT3 depletion abrogated the anti-apoptotic effect of PIKfyve inhibition (Fig. 4B).

So far, our results point to an unprecedented role of PIKfyve in the control of cardiomyoblast hypertrophy, mitochondrial ROS production and apoptosis through the control of SIRT3 pathway.

STA treatment reduces cardiac hypertrophy and improves cardiac function in obese mice. Considering the in vitro effects of STA on cellular responses to oxidative and metabolic stress, we next examined whether PIKfyve inhibition could improve cardiometabolic phenotype in a mouse model of chronic high fat diet (HFD) feeding. Indeed, cardiomyopathy induced by obesity is characterized by cardiac hypertrophy, excessive ROS production and apoptosis, culminating in reduced cardiac function (Barouch, L.A. et al. Cardiac myocyte apoptosis is associated with increased DNA damage and decreased survival in murine models of obesity. Circ Res 98, 119-124 (2006)) (Bournat, J.C. & Brown, C.W. Mitochondrial dysfunction in obesity. Curr Opin Endocrinol Diabetes Obes 17, 446-452 (2010)) (Fuentes-Antras, J. et al. Updating experimental models of diabetic cardiomyopathy. J Diabetes Res 2015, 656795 (2015)) (Aurigemma, G.P., de Simone, G. & Fitzgibbons, T.P. Cardiac remodeling in obesity. Circ Cardiovasc Imaging 6, 142-152 (2013)). Mice fed a HFD for 12 months developed glucose intolerance, insulin resistance and morphometric changes as compared with normal diet (ND) fed mice (Table 1). Echocardiographic analysis revealed ventricular hypertrophy as shown by elevated LVPWd and IVSTd (Table 2) and cardiac

dysfunction characterized by the decreased cardiac ejection fraction (EF) and left ventricular fractional shortening (FS) in HFD-fed mice as compared to ND-fed mice.

In HFD-fed mice, treatment with STA prevented structural lesions of cardiac tissue as compared with vehicle-treated mice (data not shown). Importantly, chronic PIKfyve inhibition resulted in improved cardiac function in obese mice, as shown by the increased EF (Fig. 5A) and FS (Fig. 5B). In addition, analysis of cardiac structure revealed a decrease in LVPWd and IVSTd in STA-treated mice as compared to vehicle-treated obese mice suggesting that PIKfyve inhibition prevents cardiac hypertrophy (Fig. 5C-D). The anti-hypertrophic effect of STA was further confirmed by measuring the cardiomyocytes cross-sectional area in the heart sections (Fig. 5E), the heart weight to body weight ratio (HW/BW, Fig. 5F) and myocardial expression of hypertrophic markers MHC and BNP (Fig. 5 G-H). Importantly, STA-dependent prevention of cardiac hypertrophy was accompanied by a reduction in myocardial fibrosis (Fig. 51). Moreover, chronic treatment with STA improved glucose tolerance as compared to vehicle-treated mice (data not shown), without significant changes in body weight (44.8g +/-3.3 in control vs. 51.7g +/-2.8 in STA-treated mice). In addition, anti-glycemic activity of STA was associated with reduced levels of cardiac triglycerides (data not shown).

PIKfyve inhibition reduces cardiac oxidative stress and apoptosis linked to obesity. Based on our in vitro data, we next asked whether PIKfyve inhibition was able to preserve mitochondrial integrity in vivo. Electron microscopy analysis revealed in HFD-fed mice the presence of fragmented rounded interfibrillar mitochondria (data not shown), a typical hallmark of cardiac injury (Ong, S.B. et al. Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury. Circulation 121, 2012-2022 (2010)). In contrast, chronic treatment with STA preserved mitochondrial ultrastructure (data not shown) and mitochondrial size (Fig. 6A). Defects in mitochondrial architecture are hallmarks for respiratory chain damage and ROS production. Therefore, we analyzed the expression profile of key complexes of the mitochondrial respiratory chain (OXPHOS complexes). Strikingly, STA treatment increased the expression of the OXPHOS I, II, III, IV and V complexes in cardiac tissue (Fig. 6B). This change in cardiac mitochondrial respiratory chain complexes may suggest an improved respiratory efficiency and therefore a decrease in mitochondrial ROS production. Indeed, we observed reduced mitochondrial O2" levels (Fig. 6C) and lipid peroxidation (Fig. 6D) in cardiac tissue from STA-treated HFD-fed mice. Finally, chronic PIKfyve inhibition culminated in the reduction of apoptotic cell death (Fig. 6E) and down-regulation of the pro-apoptotic factor Bax (Fig. 6F).

STA has been initially characterized based on its anti-inflammatory properties (Burakoff, R. et al. A phase 1/2A trial of STA 5326, an oral interleukin- 12/23 inhibitor, in patients with active moderate to severe Crohn's disease. Inflamm Bowel Dis 12, 558-565 (2006)) (Wada, Y. et al. Selective abrogation of Thl response by STA-5326, a potent IL-12/IL-23 inhibitor. Blood 109, 1156-1164 (2007)). In order to investigate whether this could account for its anti-oxidant and anti-apoptotic properties, we analyzed the cardiac inflammatory profile of STA-treated mice. STA-treatment reduced the infiltration of CD68-positive macrophages in cardiac tissue of obese mice (data not shown). However, STA-treatment did not alter cardiac mRNA level of IL-Ιβ, IL-12, IL-23, IL-6, TNF-a and MCP1 (data not shown), suggesting that its anti-inflammatory properties do not account for the observed effects.

PIKfyve inactivation drives cardiac SIRT3 pathways in obesity-related cardiometabolic phenotype. We next studied the activation status of SIRT3 in STA-treated mice. It has been recently shown that phosphorylation of SIRT3 on Ser/Thr residues led to increased enzymatic activity in mitochondria (Liu, R. et al. CDK1 -Mediated SIRT3 Activation Enhances Mitochondrial Function and Tumor Radioresistance. Mol Cancer Ther

14, 2090-2102 (2015)). Therefore, we performed immunoprecipitation of phosphorylated proteins on serine residues in heart extracts from control or STA-treated mice followed by immunoblot of SIRT3. STA treatment increased significantly the amount of phosphorylated SIRT3 (data not shown). Moreover, we investigated the acetylation status of two mitochondrial targets of SIRT3 involved in redox homeostasis, the superoxide dismutase 2 (SOD2) (Tao, R. et al. Sirt3 -mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress. Mol Cell 40, 893-904 (2010)) and the isocitrate dehydrogenase 2 (IDH2) (Yu, W., Dittenhafer-Reed, K.E. & Denu, J.M. SIRT3 protein deacetylates isocitrate dehydrogenase 2 (IDH2) and regulates mitochondrial redox status. J Biol Chem 287, 14078-14086 (2012)). STA treatment significantly reduced the amount of acetylated cardiac IDH2 and SOD2 (data not shown). Altogether, these data suggest that PIKfyve inhibition led to increased SIRT3 activity in hearts from obese mice.

STA loses its anti-hypertrophic properties in SIRT3.KO mice. In order to confirm the implication of SIRT3 in the anti-hypertrophic response induced by STA-treatment, we evaluated the effects of PIKfyve inhibition on isoproterenol- (ISO) induced hypertrophy in WT and SIRT3.KO mice (data not shown). Treatment with ISO induced cardiac hypertrophy as measured by increased IVSTd both in WT and KO mice (data not shown). Interestingly, treatment with STA significantly reduced the hypertrophic response in WT mice, but no longer in SIRT3 KO mice (data not shown). This was further confirmed by analysing the cross sectional area of cardiomyocytes. While STA decreased cardiomyocytes hypertrophy following ISO-treatment in WT mice, the compound had no effect in SIRT3.KO mice (data not shown). Altogether, these data suggest that SIRT3 is required for the anti-hypertrophic properties of STA.

TABLE 1. Metabolic parameters of mice under ND or HFD feeding

Parameters ND HFD

Body weight (g) 42.8 ± 2.1 51.7 ± 1.8 **

Fat mass (%) 9.6 ± 0.8 19.6 ± 2.2 ***

Glucose

10.2 ± 0.4 12.8 ± 0.8 **

(mmol/L)

AUCglucose

Body weight, fat mass, plasma glucose, area under the curve of intraperitoneal glucose tolerance test (IGTT AUCglucose) were evaluated in male C57BL/6J mice after 12 months HFD or ND feeding. n=6-14 per group. Data are means ± SEM; *p <0.05, **p <0.01 and ***p <0.001 vs ND-fed group.

TABLE 2. Cardiac function in ND or HFD-fed mice after chronic STA treatment

ND HFD

Parameters Vehicle STA

47.6 ± 0.6

FS (%) 54.7 ± 1.8 56.9 ± 1.0 m

EF (%) 89.8 ± 1.3 84.9 ± 0.5 ** 91 .4 1: 0.6 $ss

1.30 ± 0.04 1.02 ± 0.05

LVPWd (mm) 0.96 ± 0.05

^ ^ ^ $$$

IVSTs (mm) 1.72 ±0.06 1.79 ±0.03 1.68 ±0.04

Heart rate, fractional shortening (FS), ejection fraction (EF), left ventricular posterior wall thickness (LVPW), interventricular septal thickness (IVST) and left ventricle internal diameter (LVID) at end diastole (d) or systole (s) were evaluated in male C57BL/6J mice after Vehicle or STA treatment. n=4-8 mice per group. Data are shown as means ± SEM; **p <0.01 and ***p <0.001 vs ND-fed group; $$$p <0.001 vs Vehicle-HFD-fed group;

Discussion

Obesity is closely associated with cardiovascular and metabolic complications (Battiprolu, P.K. et al. Diabetic cardiomyopathy and metabolic remodeling of the heart. Life Sci 92, 609-615 (2012)). Increasing evidence suggests that abnormal mitochondrial ROS production and mitochondrial defects are at the center of the pathophysiology of the failing heart and metabolic disorders (Bournat, J.C. & Brown, C.W. Mitochondrial dysfunction in obesity. Curr Opin Endocrinol Diabetes Obes 17, 446-452 (2010)) (Tsutsui, H., Kinugawa, S. & Matsushima, S. Oxidative stress and heart failure. Am J Physiol Heart Circ Physiol 301, H2181-2190 (2011)). The loss of mitochondrial integrity inevitably disturbs cell functions, sensitizes cells to stress and may trigger cell death, with potentially dramatic irreversible pathological consequences. In the present study, we unravel a critical role for the phosphoinositide kinase PIKfyve in the control of stress-induced mitochondrial damage, ROS generation, apoptosis, and ventricular dysfunction in obesity-induced phenotype. Long-term HFD-induced obesity increases the heart workload, causes left ventricular hypertrophy, and impairs cardiac function (Fuentes-Antras, J. et al. Updating experimental models of diabetic cardiomyopathy. J Diabetes Res 2015, 656795 (2015)). Our in vitro results demonstrate that inhibition of PIKfyve attenuated stress-induced hypertrophic responses in cardiomyoblasts. In addition, in a mouse model of obesity-induced phenotype we show that chronic treatment with STA decreased ventricular hypertrophy, a major predictor of cardiovascular events, and improves left ventricular contractility, suggesting a tight association between myocardial PIKfyve activity and cardiac function in the setting of obesity.

Obesity is associated with metabolic disorders leading to the installation of type 2 diabetes. The present study is the first report that demonstrates the efficacy of pharmacological inhibition of PIKfyve on glycemic status in obesity-induced type 2 diabetes. If the total knockout of PIKfyve in mice is lethal at embryonic stage (Ikonomov, O.C. et al.

The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice. J Biol Chem 286, 13404-13413 (2011)), the generation of tissue-specific PIKfyve knockout mice has given some insights in the in vivo functions of the lipid kinase. Indeed, muscle specific PIKfyve knockout mice are glucose intolerant and insulin resistant (Ikonomov, O.C. et al. Muscle-specific Pikfyve gene disruption causes glucose intolerance, insulin resistance, adiposity, and hyperinsulinemia but not muscle fiber-type switching. Am J Physiol Endocrinol Metab 305, El 19-131 (2013)). The key difference between the genetic and pharmacological inactivation of PIKfyve, is that in KO mice, the protein is totally absent, preventing both the kinase activity and any scaffolding/docking function of PIKfyve. The use of pharmacological inhibitors allows a more detailed dissection of these two characteristics. However, generating a conditional or heart-specific knockout for PIKfyve would be of great interest to further this study and decipher the molecular mechanisms implicated. Probably of greater interest yet, the generation of a kinase-dead knock-in mutant would help to distinguish between the requirement for PIKfyve kinase activity or scaffolding/docking functions.

Our study provides the first evidence that PIKfyve controls the structural mitochondrial integrity and ROS production in cardiac cells through a SIRT3 -dependent pathway. PIKfyve is a cytosolic protein localized on endosomes through its FYVE domain (Sbrissa, D., Ikonomov, O.C. & Shisheva, A. Phosphatidylinositol 3 -phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization. J Biol Chem 277, 6073-6079 (2002)) and therefore its potential regulation of SIRT3 in the mitochondrial compartment is unlikely. An alternative possibility could involve some lipid transfer leading to PI5P accumulation in mitochondrial membranes. The capacity of phospholipids to alter membrane dynamics is widely recognized (van Meer, G., Voelker,

D.R. & Feigenson, G.W. Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9, 112-124 (2008)), and interestingly, membrane fluidity has been shown to be a key factor in the respiratory chain efficiency (Waczulikova, I. et al. Mitochondrial membrane fluidity, potential, and calcium transients in the myocardium from acute diabetic rats. Can J Physiol Pharmacol 85, 372-381 (2007)). Although a direct transfer between two distant lipid bilayers is very unlikely, there are examples of lipid exchange through protein carriers between two organelles, e.g. endoplasmic reticulum to Golgi apparatus (Moser von Filseck, J., Vanni, S., Mesmin, B., Antonny, B. & Drin, G. A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes. Nat Commun 6, 6671) or to plasma membrane (Stefan, C.J., Manford, A.G. & Emr, S.D. ER-PM connections: sites of information transfer and inter-organelle communication. Curr Opin Cell Biol 25, 434-442 (2013)). Such a transfer mechanism could exist between the endosomal system and the mitochondria. Alternatively, we could not exclude that PIKfyve effects on mitochondria involve its protein kinase activity. Indeed, it has been suggested that PIKfyve is able to phosphorylate several protein substrates (Ikonomov, O.C. et al. Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport factor Rab9 effector p40. J Biol Chem 278, 50863-50871 (2003)), although the regulation of this protein kinase activity remains poorly documented. It is tempting to speculate that PIKfyve directly phosphorylates SIRT3 to control the redox status of the cell. However, given the fact that PIKfyve inhibition by STA increased SIRT3 phosphorylation, and that only activating phosphorylations have been described for SIRT3 (Liu, R. et al. CDK1 -Mediated SIRT3 Activation Enhances Mitochondrial Function and Tumor Radioresistance. Mol Cancer Ther 14, 2090-2102 (2015)), one has to admit that PIKfyve controls SIRT3 activity through a different mechanism. One possibility would be that PIKfyve phosphorylates SIRT3 on a different site through an inhibitory phosphorylation. In any case, our results clearly demonstrate that PIKfyve is activated upon metabolic and oxidative stress, adding new activation pathways to the already described osmotic shock (Sbrissa, D., Ikonomov, O.C., Deeb, R. & Shisheva, A. Phosphatidylinositol 5-phosphate biosynthesis is linked to PIKfyve and is involved in osmotic response pathway in mammalian cells. J Biol Chem 277, 47276-47284 (2002)). Taken together, these observations clearly identify PIKfyve as a stress sensor which is able to orchestrate the cell response.

A line of evidence suggests that obesity-induced cardiac dysfunction is linked to excessive mitochondrial ROS production, oxidative stress and massive loss of cardiac cells (Sawyer, D.B. et al. Role of oxidative stress in myocardial hypertrophy and failure. J Mol Cell Cardiol 34, 379-388 (2002)) (Aurigemma, G.P., de Simone, G. & Fitzgibbons, T.P. Cardiac remodeling in obesity. Circ Cardiovasc Imaging 6, 142-152 (2013)). Both in vitro and in vivo, we show here that STA was able to reduce mitochondrial ROS generation, oxidative stress and apoptosis. Interestingly, anti-hypertrophic activity of STA was associated with activation of SIRT-3 pathways in the heart. SIRT3 has been shown to be a negative regulator of cardiac hypertrophy (Sundaresan, N.R. et al. Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice. J Clin Invest 119, 2758-2771 (2009)), ROS production (Qiu, X., Brown, K., Hirschey, M.D., Verdin, E. & Chen, D. Calorie restriction reduces oxidative stress by SIRT3 -mediated SOD2 activation. Cell Metab 12, 662-667 (2010)), apoptotic cell death (Sundaresan, N.R., Samant, S.A., Pillai, V.B., Rajamohan, S.B. & Gupta, M.P. SIRT3 is a stress-responsive deacetylase in cardiomyocytes that protects cells from stress-mediated cell death by deacetylation of Ku70. Mol Cell Biol 28, 6384-6401 (2008)), metabolism (Alfarano, C. et al. Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin. Int J Obes (Lond) 39, 312-320 (2014)) and ageing (McDonnell, E., Peterson, B.S., Bomze, H.M. & Hirschey, M.D. SIRT3 regulates progression and development of diseases of aging.

Trends Endocrinol Metab (2015)). Despite its fundamental role, little is known on how SIRT3 is translocated into the mitochondrial matrix (Schwer, B., North, B.J., Frye, R.A., Ott, M. & Verdin, E. The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase. J Cell Biol 158, 647-657 (2002).). Here, we show that PIKfyve inhibition induced the translocation of SIRT3 to the mitochondria, independently of stress. We postulate that such a regulation would prime the cell for a quick response under stress and ultimately protecting it from ROS overproduction and cell death. In that regard, deciphering the molecular mechanisms involved in STA-dependent SIRT3 translocation and activation would help to better understand the regulation of SIRT3, and would pave the way to new therapies for diseases associated with SIRT3 deficiency.

Currently there are no specific guidelines for the treatment of heart failure in patients with obesity/type 2 diabetes. No major trials have addressed this question specifically and choices must be made based on clinical experience, understanding of mechanisms of obesity-related heart failure, patient comorbidities and side effects. Therefore there is an urgent need to develop novel pharmacological treatment strategies, which target mechanisms underlying cardiac and metabolic remodelling processes. Traditional beta blockers have been shown to increase insulin resistance and predispose patients to diabetes, increasing weight gain, insulin-resistance and triglycerides levels (Elliott, W.J. & Meyer, P.M. Incident diabetes in clinical trials of antihypertensive drugs: a network meta-analysis. Lancet 369, 201-207 (2007)) (Lithell, H.O. Effect of antihypertensive drugs on insulin, glucose, and lipid metabolism. Diabetes Care 14, 203-209 (1991)). On the same line, ACE inhibitors, although widely used in clinic, have many undesirable adverse effects, including, hypotension, cough, hyperkalemia, headache, dizziness, fatigue, nausea, and renal impairment (Fein, A. ACE inhibitors worsen inflammatory pain. Med Hypotheses 72, 757

(2009)) (Sidorenkov, G. & Navis, G. Safety of ACE inhibitor therapies in patients with chronic kidney disease. Expert Opin Drug Saf 13, 1383-1395 (2014)).

In this context, our study highlights the therapeutic potential of PIKfyve pharmacological inhibition to limit mitochondrial damage and to improve cardiometabolic phenotype in obese patients.

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.