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WO2017208174A2 - Méthodes de traitement d'une maladie à l'aide d'inhibiteurs de pfkfb3 - Google Patents

Méthodes de traitement d'une maladie à l'aide d'inhibiteurs de pfkfb3 Download PDF

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Publication number
WO2017208174A2
WO2017208174A2 PCT/IB2017/053209 IB2017053209W WO2017208174A2 WO 2017208174 A2 WO2017208174 A2 WO 2017208174A2 IB 2017053209 W IB2017053209 W IB 2017053209W WO 2017208174 A2 WO2017208174 A2 WO 2017208174A2
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Prior art keywords
pfkfb3
cell
cells
inhibitor
subject
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WO2017208174A3 (fr
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Slavica TUDZAROVA-TRAJKOVSKA
Peter C. Butler
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides

Definitions

  • Embodiments are directed generally to biology and medicine. In certain aspects there are methods and compositions for treating protein misfolding disorders.
  • a protein misfolding disease refers to a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Often the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way (a gain of toxic function) or they can lose their normal function.
  • the diseases also known as proteinopathies, protein conformational disorders, or proteopathies
  • proteins include such diseases as Creutzfeldt-Jakob disease, Alzheimer's disease, Parkinson's disease, prion disease, amyloidosis, and a wide range of other disorders.
  • Protein aggregation diseases are not exclusive to the central nervous system; they can also appear in peripheral tissues.
  • amyloidogenic diseases include type 2 diabetes, inherited cataracts, some forms of atherosclerosis, hemodialysis-related disorders, and short- chain amyloidosis, among many others. All these diseases have in common the expression of a protein outside its normal context, leading to an irreversible change into a sticky conformation rich in beta sheets that make the protein molecules interact with each other.
  • the general pattern that emerges in all these diseases is an abnormal tendency of proteins to aggregate as a result of misfolding.
  • the aggregation can be caused by chance; by protein hyperphosphorylation (a condition where multiple phosphate groups are added to the protein), by prion self-catalytic conformational conversion, or by mutations that make the protein unstable. Aggregation can also be caused by an unregulated or pathological increase in the intracellular concentration of some of these proteins. Such imbalances in protein concentration can be a consequence of mutations such as duplications of the amyloidogenic gene or changes in the protein's amino acid sequence. Imbalances can also be caused by deficiencies in the proteasome, the cellular machinery involved in the degradation of aging proteins. Inhibition of autophagy (a process by which cells engulf themselves) also promotes amyloid aggregation. In addition, some evidence suggests that the severity of these diseases correlates with an increase in oxidative stress, mitochondrial dysfunction, alteration of cytoplasmic membrane permeability, and abnormal calcium concentration.
  • Protein misfolding can cause an acute stress response that may have beneficial survival-promoting effects in the short-term, but can be detrimental to cells experiencing chronic stresses, such as those involved in protein misfolding deases. While traditional therapeutic design has been to target the misfolded protein, there is a need in the art for therapies that attenuate the acute stress response resulting from the misfolded proteins, since this response can contribute to the pathology of the disease.
  • aspects of the disclosure relate to a method of treating a protein misfolding disease in a subject comprising administering a PFKFB3 inhibitor to the subject. Further aspects of the disclosure relate to a method of treating a protein misfolding disease in a subject comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the subject.
  • Further aspects relate to a method for treating or preventing protein misfolding- induced cell death in a cell, the method comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the cell.
  • the cell is in a subject.
  • Further aspects relate to a method for inhibiting or reducing ⁇ -cell death in a subject with Type 2 diabetes, the method comprising administering a PFKFB3 inhibitor to the subject. Further aspects relate to a method for inhibiting or reducing ⁇ -cell death in a subject with Type 2 diabetes, the method comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the subject.
  • Yet further aspects relate to a method for treating or preventing an acute stress response in a cell or subject in need thereof, the method comprising administering a PFKFB3 inhibitor and/or a K(ATP) channel opener to the cell or subject.
  • the cell is in vitro.
  • the cell is transplanted into the subject.
  • the subject is one that has or will receive transplanted cells.
  • the cell or transplanted cells are pancreatic islet cells.
  • the subject has a protein misfolding disease or the cells has pathogenic misfolded protein.
  • the misfolded protein is one described herein.
  • the protein misfolding disease is one known in the art or described herein.
  • the protein misfolding disease is Alzheimer's, Parkinsin's, Type 2 Diabetes, or prediabetes.
  • the protein misfolding disease is Type 2 diabetes.
  • the subject does not have cancer or is not diagnosed as having cancer.
  • the subject has been diagnosed with a protein misfolding disease.
  • the subject has previously been treated for a protein misfolding disease.
  • the PFKFB3 inhibitor or K(ATP) channel opener may be a protein inhibitor, a nucleic acid inhibitor, or a small molecule inhibitor.
  • the protein inhibitor is an antibody.
  • the antibody binds to a PFKFB3 protein and inhibits the activity of PFKFB3.
  • the inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor is 3-(3-Pyridinyl)-l-(4- pyridinyl)-2-propen-l-one (3-PO) or an analog thereof.
  • the analog is is 1- (4-pyridinyl)-3-(2-quinolinyl)-2-propen-l-one (PFK15).
  • the small molecule inhibitor is one known in the art, described herein, or incorporated by reference.
  • the PFKFB3 inhibitor is an antisense nucleic acid.
  • the antisense nucleic acid is an siRNA, a double stranded RNA, a short hairpin RNA.
  • the inhibitor or K(ATP) channel opener is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, intra-articularly, intrasynovially, intrathecally, orally, topically, through inhalation, or through a combination of two or more routes of administration.
  • the inhibitor is administered by a method described herein.
  • the inhibitor is administered to a specific cell type, such as a pancreatic cell, a bone cell, a neural cell, a blood cell, a breast cell, an epithelial cell, an adipocyte, a kidney cell, a muscle cell, a pancreatic islet, an alpha cell, a beta cell, a delta cell, a pancreatic polypeptide secreting cell, or an epsilon cell.
  • a specific cell type such as a pancreatic cell, a bone cell, a neural cell, a blood cell, a breast cell, an epithelial cell, an adipocyte, a kidney cell, a muscle cell, a pancreatic islet, an alpha cell, a beta cell, a delta cell, a pancreatic polypeptide secreting cell, or an epsilon cell.
  • a “subject,” “individual” or “patient” is used interchangeably herein and refers to a vertebrate, for example a primate, a mammal or preferably a human. Mammals include, but are not limited to equines, canines, bovines, ovines, murines, rats, simians, humans, farm animals, sport animals and pets. In some embodiments, the subject is a human subject.
  • compositions may be employed based on methods described herein. Use of one or more compositions may be employed in the preparation of medicaments for treatments according to the methods described herein. Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. The embodiments in the Example section are understood to be embodiments that are applicable to all aspects of the technology described herein.
  • treatment is an approach for obtaining beneficial or desired clinical results. This includes: reduce the alleviation of symptoms, the reduction of inflammation, the inhibition of cell death, and/or the restoration of cell function.
  • treatment refers to the inhibition or reduction of cell death, such as the rejction of ⁇ -cell death.
  • therapeutically effective amount refers to an amount of the drug that treats or inhibits disease in a subject. In some embodiments, the therapeutically effective amount inhibits at least or at most or exactly 100, 99, 98, 96, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 30, 20, or 10%, or any derivable range therein, of PFKFB3 activity.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIG. 1A-D hAPP and autoreactive cytokines induce ⁇ -cell mitochondrial fragmentation, a) scheme of ⁇ -cell synchronization by serum deprivation in combination with Aphidicolin treatment (5 ⁇ g/ml) for enrichment of cells in Gl/S.
  • CTRL Gl/S synchronized control
  • hIAPP cytokine mix treated INS 823/13 ⁇ -cells stained with Mitotracker Red
  • Islet-derived primary ⁇ -cells from wild- type (WT) or hIAPP (hTG) transgenic mouse show mitochondrial fragmentation after staining with Mitotracker Red (Mito Red) and insulin
  • FACS diagrams overlay showing the mitochondrial membrane potential of control (CTRL) and hIAPP expressing ⁇ -cells at Oh and 4h post-release from the synchronization block measured using TMRE in presence or absence of oligomycin (Oligo) or 2-deoxy glucose (DOG).
  • FIG. 2A-G Mitochondrial dysfunction leads to compensatory enhancement of aerobic glycolysis and PFKFB3 expression, a) Lactate rate in isolated islets from wild type (WT) and hIAPP transgenic (HIP) rats, b) Protein expression of PFKFB3 in islet whole cell extracts from 2 WT and 2 HIP rats at 2, 4 and 6 months (mo) of age as measured by immunoblotting.
  • WT wild type
  • HIP hIAPP transgenic
  • FIG. 3A-C PFKFB3 expression is enhanced in islets from prediabetic HIP rats and in islets from human obese (obese T2D) and lean (lean T2D) diabetes.
  • Confocal images (upper panel) showing PFKFB3 expression in a) WT compared to prediabetic HIP rat at 7 months of age.
  • b) (middle panel) showing PFKFB3 cytoplasmic enrichment in islets from obese type 2 (T2D) diabetes and c) (lower panel) PFKFB3 nuclear enrichment in lean type 2 (T2D) diabetes.
  • FIG. 4A-C PFKFB3 expression is enhanced in islets from prediabetic HIP rats and in islets from human obese (obese T2D) and lean (lean T2D) diabetes.
  • ⁇ -cell rest by suppressed glycolysis permits the mitochondrial network to withstand stress and enhances ⁇ -cell survival
  • cell cycle distribution map
  • FIG. 5A-C FACS histograms of GO INS 823/13 ⁇ -cells with indicated treatments to demonstrate rescue of hIAPP-induced cell death by PFKFB3 knockdown or K + ATP channel opener NN-414 and reversal to cell death after addition of Glibenclamide (Gli) (hi APP+PFKFB 3 si+Gli).
  • Glibenclamide Gli
  • ⁇ -cell rest by suppressed glycolysis through inhibition of PFKFB3 with small molecule inhibitor 3PO enhances ⁇ -cell survival in dose-dependent fashion and stimulates insulin secretion after high glucose challenge in prediabetic HIP rat islets, a) FACS histograms of GO INS 823/13 ⁇ -cells with indicated treatments to demonstrate partial rescue of hIAPP-induced cell death by inhibition of PFKFB3 with 10 ⁇ 3PO and prevention of cell death with 30 and 50 ⁇ 3PO.
  • DMSO and Ad-LacZ with DMSO in presence or absence of either 10 ⁇ or 50 ⁇ 3PO were used as controls, b) Confocal images of islets isolated from WT or HIP rats at 6 months of age showing enhanced nuclear staining for PFKFB3 in islets of HIP rats, c) Dynamic insulin secretion presented as concentration of measured insulin by spectrophotometry (AU* 1000) in perifused WT versus HIP islets at 6 months of age in presence or absence of PFKFB3 inhibitor - 3PO.
  • FIG. 6A-E ⁇ -cell mitochondrial fragmentation in T2D is reproduced in hIAPP model. Images of mitochondria in (A) human pancreatic sections from non-diabetic (ND) and (B) T2D subjects stained with Tom20 (mitochondria), insulin, and DAPI (nuclei) (C) Quantification of mitochondrial area per ⁇ -cell in ND and T2D subjects. (D) Images of mitochondria in control (CTRL, LacZ-AdV) and hIAPP-expressing (hIAPP-AdV) INS 832/13 cells synchronized at the Gl/S cell cycle stage, stained with the mitochondrial probe Mitotracker Red (MTR).
  • D Images of mitochondria in control (CTRL, LacZ-AdV) and hIAPP-expressing (hIAPP-AdV) INS 832/13 cells synchronized at the Gl/S
  • FIG. 7A-7D Mitochondrial respiration but not mitochondrial membrane potential is decreased by hIAPP.
  • OCR Oxygen consumption rate
  • B Quantification of OCR presented as a fold change of stimulated respiration versus WT as measured by Seahorse in FIG. 6A. Mitochondrial membrane potential measured by FACS after labelling with TMRE
  • C CTRL
  • FIG. 8A-8E hIAPP leads to upregulation of HIF 1 a-PFKFB3 stress pathway and increases aerobic glycolysis.
  • A Summary of the differentially expressed genes of interest after microarray analysis performed on RNA isolated from rat WT and HIP islets (4-6 months) presented as a -fold change over WT. * represents genes that were differentially expressed in one set
  • B LDHA and MCT1 mRNA levels in HIP vs. WT as measured by qRT-PCR
  • C Lactate production rate (-fold change) measured in isolated islets from HIP relative to WT.
  • D PFKFB3 protein expression assessed by immunoblotting in whole cell extracts from islets from 2 WT and 2 HIP rats from 2-6 months of age.
  • FIG. 9A-9D HIF 1 a PFKFB3 stress pathway is upregulated in ⁇ -cells from transgenic hIAPP rats and humans with T2D. Images of PFKFB3 immunostaining in (A) islets from HIP and WT rats and in (B) islets from non-diabetic (ND) and T2D subjects. (C) Quantification of frequency of PFKFB3 positive ⁇ -cells in HIP vs. WT rats, left, and T2D vs. ND subjects, right.
  • FIG. 10A-10B Silencing of PFKFB3 restores ⁇ -cell metabolome
  • A Heatmap of relative metabolite composition in CTRL and hIAPP overexpressing INS 832/13 cells in presence or absence of PFKFB3 siRNA.
  • PFKFB3 silencing restores Ca 2+ homeostasis, mitochondrial networks and reduces ⁇ -cell death.
  • A Images illustrating restoration of mitochondrial networks as visualized by Mitotracker red (MTR) and reduction of genotoxic stress illustrated by ⁇ 2 ⁇ . ⁇ staining in INS 832/13 b-cells treated with hIAPP and PFKFB3 siRNA and quantification of mitochondrial morphology.
  • B FACS histograms of GO INS 823/13 b-cells with indicated treatments demonstrate rescue from hIAPP induced cell death after PFKFB3 silencing and quantification of the percentage (%) of the subGl (apoptotic) cells as measured by FACS.
  • C Immunoblotting analysis of whole cell extracts from INS 832/13 cells demonstrating reduction of ⁇ 2 ⁇ . ⁇ , cleaved caspase 3 and truncated (p89) PARP-1 upon PFKFB3 silencing and quantification of WB signals using ImageJ analysis.
  • LacZ-AdV represents CTRL.
  • FIG. 12A-B Scheme and working model.
  • A Schematic presentation of the isotopologue distribution depicts a change in the metabolic pathway contribution after silencing of PFKFB3 in presence of hIAPP.
  • B Working model depicting hIAPP toxicity inducing mitochondrial adaptation to perturbed Ca 2+ homeostasis leading to increase in the aerobic glycolysis and PFKFB3 upregulation, thus resulting in loss of a metabolic-insulin secretion coupling and ⁇ -cell loss. This phenotype is partly reversed via inhibition of PFKFB3 by restoration of Ca 2+ homeostasis and mitochondrial networks.
  • FIG. 13A-B hIAPP induces cell death in synchronized INS 832/13 cells.
  • FIG. 14A-B hIAPP affects fusion by reducing MFN-2 levels but not mitochondrial fission.
  • A Immunoblotting of indicated dynamin-related proteins in untreated (UT), control (CTRL, LacZ) and hIAPP overexpressing (hIAPP) INS 832/13 cells.
  • B Images of CTRL-LacZ and hIAPP-AdV transduced INS 823/13 ⁇ -cells in presence or absence of the dominant negative DrplK48A mutant illustrate non-sustained fusion after hIAPP overexpression.
  • FIG. 15A-E hIAPP reduces the flux through TCA cycle (A-B) and hIAPP increases the flux through de novo purine synthesis and oxidized glutathione (C-E).
  • A Relative metabolite composition of hIAPP (30h) overexpressing INS 832/13 cells presented as a -fold change of control rIAPP overexpressing cells.
  • (B) Mass isotopomer distribution (MID) of the TCA intermediates derived from culturing INS 832/13 with [U- 13 C 6 ] glucose; Glucose - Glc; Aspartate - Asp; Citrate - Cit; a— ketoglutarate - a— KG; Succinate - Sue; Fumarate - Fum; Malate -Mai; Glutamate - Glu. Data are presented as mean ⁇ SEM, n 3. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005.
  • Inositol monophosphat - IMP Adenosin diphosphate - ADP; Adenosin triphosphate - ATP; Cytidin diphosphate -CDP; Cytidin triphosphate - CTP; Uridin diphosphate - UDP; Uridin triphosphate - UTP
  • D reduced Glutathione -GSH; oxidized Glutathione - GSSG.
  • FIG. 16A-F hIAPP induced apoptosis is linked to PFKFB3.
  • A PFKFB3 protein levels in GO synchronized INS 832/13 after transduction with hIAPP-AdV for 36h (upper panel) with or without PFKFB3 siRNA and after transduction with rIAPP- or hlAPP- AdV for 30h (lower panel) as assessed by immunoblotting of whole cell extracts.
  • B Relative PFKFB3 mRNA levels in indicated treatments of INS 832/13 cells as measured by qRT-PCR and presented as -fold change to CTRL.
  • C PFKFB3 immunostaining of INS 832/13 cells transfected with hIAPP or LacZ (CTRL) adenoviruses.
  • FIG. 17A-B Silencing of PFKFB3 stimulates the PPP flux.
  • MID Mass isotopomer distribution
  • FIG. 18A-D NN-414 K(ATP) opener prevents ⁇ -cell death by restoring the cell cycle distribution and mitochondrial networks similar to PFKFB3 silencing.
  • ⁇ -cells In health, ⁇ -cells (collectively referred to as ⁇ -cell mass) regulate glucose metabolism by secretion of insulin in a glucose dependent manner that constrains hepatic glucose release.
  • the mitochondrial network is critical for ⁇ -cell function and viability.
  • the prevailing glucose concentration is sensed by the rate of flux through glycolysis regulated by glucokinase, with tight linkage of acetyl CoA generated by glycolysis to ATP synthesis by mitochondria.
  • the ATP acts to close K +( ATP) sensitive channels and thus opening of voltage gated Ca 2+ channels to elicit insulin secretion in response to glucose.
  • the mitochondrial network is highly fused and interconnected, a form that favors oxidative phosphorylation.
  • the inventors of the application have now, for the first time, investigated the mitochondrial network in replicating ⁇ -cells and confirmed its departing from highly fused to a fragmented form prior to mitosis, switching from aerobic to a high rate of aerobic glycolysis during Gl-to-S phase transition, an adaptation synchronized by signaling cross talk between regulators of cell cycle and the mitochondrial network. These changes permit synthesis of nucleotides required for new DNA and elimination of compromised mitochondria (mitophagy) prior to sorting the remainder to daughter cells.
  • Mitochondrial morphology is highly disrupted in islet amyloid pancreatic peptide (IAPP)-expressing-cells and in ⁇ -cells isolated from the islets of IAPP -transgenic rodents, being represented by shortened spheroid mitochondria with reduced motility and reflecting mitochondrial dysfunction in type 2 diabetes (T2D) in vivo.
  • Mitochondrial fragmentation, and a switch to aerobic glycolysis is also an adaptive defense by cells in response to hypoxia and related stress.
  • this metabolic pattern is also present in ⁇ -cells in infancy that are characterized by high rates of aerobic glycolysis, and therefore minimal glucose responsiveness. This adaptation likely is to permit the high rate of ⁇ -cell replication in infancy.
  • PFKFB3/PFK-2 phospho-fructokinase 2,6-biphosphatase
  • the inventors have demonstrated that initial ⁇ -cell stressors converge on the mitochondrial network, which by adapting a defensive fragmented form, disrupts the coupling of glycolysis to ATP production, and thus insulin secretion. It was further found that the fragmented mitochondrial network leads to aberrant signal cross talk between the mitochondria and cell cycle regulatory network, recruits ⁇ -cells into cell cycle but that under stress conditions, this leads to ⁇ -cell loss rather than ⁇ -cell regeneration.
  • the inventors identified a strategy based on targeting PFKFB3 from the aerobic glycolysis that maintains a fused mitochondrial network, during entry into cell cycle, and in the face of these initial stressors sustains ⁇ -cell survival.
  • the studies described in the examples support a concept of benefit by ⁇ -cell rest, as achieving protection by attenuating sustained high glycolytic flux induced by activation of PFKFB3 and its action to fragment the mitochondrial network, at least in part by sustained activation of K + ATP channels and Ca 2+ activation of calcineurin.
  • This strategy confers prevention of ⁇ -cell death and promotion of ⁇ - cell survival.
  • This is also a novel and original approach which attempts to support ⁇ -cell survival and stimulate ⁇ -cell renewal after restoring the mitochondrial architecture, function and related physiological metabolic profile which then secures restoration of ⁇ -cell function by orderly insulin secretion.
  • PFKFB3 is also referred to as 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3, 6PF-2-K/Fru-2,6-P2ase Brain/Placenta- Type Isozyme, renal carcinoma antigen NY-REN-56, 6PF-2-K/Fru-2,6-P2ase 3, PFK/FBPase 3, IPFK-2, Inducible 6- Phosphofoicto-2-Kinase/Fructose-2,6-Bisphosphatase 3, Fructose-6-Phosphate,2- Kinase/Fructose-2, 6-Bisphosphatase 3, 6-Phosphofructo-2-Kinase/ Fructose-2,6- Bisphosphatase 3, IPFK2, and PFK2.
  • the protein encoded by this gene belongs to a family of bifunctional proteins that are involved in both the synthesis and degradation of fructose-2,6-bisphosphate, a regulatory molecule that controls glycolysis in eukaryotes.
  • the encoded protein has a 6-phosphofructo- 2-kinase activity that catalyzes the synthesis of fructose-2,6-bisphosphate (F2,6BP), and a fructose-2,6-biphosphatase activity that catalyzes the degradation of F2,6BP.
  • F2,6BP fructose-2,6-bisphosphate
  • This protein is required for cell cycle progression and prevention of apoptosis. It functions as a regulator of cyclin-dependent kinase 1, linking glucose metabolism to cell proliferation and survival in tumor cells.
  • Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.
  • a PFKFB3 inhibitor may refer to any member of the class of compound or agents having an IC50 of 100 ⁇ or lower concentration for a PFKFB3 activity, for example, at least or at most or about 200, 100, 80, 50, 40, 20, 10, 5, 1 ⁇ , 100, 10, 1 nM or lower concentration (or any range or value derivable therefrom) or any compound or agent that inhibits the expression of PFKFB3.
  • PFKFB3 activity or function may include, but not be limited to, regulation of glycolysis, kinase activity, regulation of CDK1, 6- phosphofructo-2-kinase activity, fructose-2,6-bisphosphate 2-phosphatase activity, ATP binding activity, and enzyme catalysis activity.
  • the inhibition can be a decrease as compared with a control level or sample.
  • functional assay such as MTT assay, cell proliferation assay, Ki67 immunofluoresence, apoptosis assay, or glycolysis assay may be used to test the PFKFB3 inhibitors.
  • the above protein and mRNA sequence represents one isoform (isoform 2) of the gene, but other isoforms are known in the art.
  • isoform 2 isoform 2
  • Genbank numbers below represent additional isoforms. The sequences associated with these Genbank numbers are incorporated by reference for all purposes.
  • Inhibitory nucleic acids or any ways of inhibiting gene expression of PFKFB3 known in the art are contemplated in certain embodiments.
  • Examples of an inhibitory nucleic acid include but are not limited to antisense nucleic acids such as: siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, an any other antisense oligonucleotide. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein.
  • An inhibitory nucleic acid may inhibit the transcription of a gene or prevent the translation of a gene transcript in a cell.
  • An inhibitory nucleic acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long.
  • the nucleic acid may have nucleotides of at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 or any range derivable therefrom.
  • isolated means altered or removed from the natural state through human intervention.
  • an siRNA naturally present in a living animal is not “isolated,” but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered.
  • Inhibitory nucleic acids are well known in the art.
  • siRNA and double-stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.
  • an inhibitory nucleic acid may be capable of decreasing the expression of PFKFB3 by at least 10%, 20%, 30%, or 40%, more particularly by at least 50%, 60%, or 70%, and most particularly by at least 75%, 80%, 90%, 95% or more or any range or value in between the foregoing.
  • an inhibitor may be between 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature PFKFB3 mRNA.
  • an inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein.
  • an inhibitor molecule has a sequence (from 5' to 3') that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5' to 3' sequence of a mature PFKFB3 mRNA, particularly a mature, naturally occurring mRNA.
  • One of skill in the art could use a portion of the probe sequence that is complementary to the sequence of a mature mRNA as the sequence for an mRNA inhibitor. Moreover, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of a mature mRNA.
  • Inhibitor nucleic acids for PFKFB3 are also commercially available.
  • the following miRNAs may inhibit PFKFB3 : hsa-mir-26b-5p (MIRT028775), hsa-mir-330- 3p (MIRT043840), hsa-mir-6779-5p (MIRT454747), hsa-mir-6780a-5p (MIRT454748), hsa- mir-3689c (MIRT454749), hsa-mir-3689b-3p (MIRT454750), hsa-mir-3689a-3p (MIRT454751), hsa-mir-30b-3p (MIRT454752), hsa-mir-1273h-5p (MIRT454753), hsa-mir- 6778-5p (MIRT454754), hsa-mir-1233-5p (MIRT454755), hsa-mir-6799-5p (MIRT4545
  • an antibody or a fragment thereof that binds to at least a portion of PFKFB3protein and inhibits PFKFB 3 activity and/or function is used in the methods and compositions described herein.
  • the PFKFB3 inhibitor polypeptide is a PFKFB3 antibody.
  • the anti- PFKFB3 antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a chimeric antibody, an affinity matured antibody, a humanized antibody, or a human antibody.
  • the antibody is an antibody fragment.
  • the antibody fragment comprises a Fab, Fab', Fab'-SH, F(ab')2, or scFv.
  • the antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous non-human, human or humanized sequence (e.g., framework and/or constant domain sequences).
  • the non-human donor is a mouse.
  • an antigen binding sequence is synthetic, e.g., obtained by mutagenesis (e.g., phage display screening, etc.).
  • a chimeric antibody has murine V regions and human C region.
  • the murine light chain V region is fused to a human kappa light chain or a human IgGl C region.
  • antibody fragments include, without limitation: (i) the Fab fragment, consisting of VL, VH, CL and CHI domains; (ii) the "Fd” fragment consisting of the VH and CHI domains; (iii) the "Fv” fragment consisting of the VL and VH domains of a single antibody; (iv) the "dAb” fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules ("scFv”), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form a binding domain; (viii) bi-specific single chain Fv dimers (see U.S.
  • a monoclonal antibody is a single species of antibody wherein every antibody molecule recognizes the same epitope because all antibody producing cells are derived from a single B-lymphocyte cell line.
  • Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with a PFKFB3 antigen with an immortal myeloma cell (usually mouse myeloma).
  • This technology provides a method to propagate a single antibody-producing cell for an indefinite number of generations, such that unlimited quantities of structurally identical antibodies having the same antigen or epitope specificity (monoclonal antibodies) may be produced.
  • a goal of hybridoma technology is to reduce the immune reaction in humans that may result from administration of monoclonal antibodies generated by the non-human (e.g., mouse) hybridoma cell line.
  • a hybridoma or other cell producing an antibody may also be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced by the hybridoma.
  • polyclonal or monoclonal antibodies, binding fragments and binding domains and CDRs may be created that are specific to PFKFB3 protein, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.
  • Antibodies may be produced from any animal source, including birds and mammals. Particularly, the antibodies may be ovine, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken.
  • newer technology permits the development of and screening for human antibodies from human combinatorial antibody libraries.
  • bacteriophage antibody expression technology allows specific antibodies to be produced in the absence of animal immunization, as described in U.S. Pat. No. 6,946,546, which is incorporated herein by this reference. These techniques are further described in: Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).
  • antibodies to PFKFB3 will have the ability to neutralize or counteract the effects of the PFKFB3 regardless of the animal species, monoclonal cell line or other source of the antibody.
  • Certain animal species may be less preferable for generating therapeutic antibodies because they may be more likely to cause allergic response due to activation of the complement system through the "Fc" portion of the antibody.
  • whole antibodies may be enzymatically digested into "Fc" (complement binding) fragment, and into binding fragments having the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen binding fragment will elicit an undesirable immunological response and, thus, antibodies without Fc may be particularly useful for prophylactic or therapeutic treatments.
  • antibodies may also be constructed so as to be chimeric, partially or fully human, so as to reduce or eliminate the adverse immunological consequences resulting from administering to an animal an antibody that has been produced in, or has sequences from, other species.
  • a "small molecule” refers to an organic compound that is either synthesized via conventional organic chemistry methods (e.g., in a laboratory) or found in nature. Typically, a small molecule is characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than about 1500 grams/mole. In certain embodiments, small molecules are less than about 1000 grams/mole. In certain embodiments, small molecules are less than about 550 grams/mole. In certain embodiments, small molecules are between about 200 and about 550 grams/mole. In certain embodiments, small molecules exclude peptides (e.g., compounds comprising 2 or more amino acids joined by a peptidyl bond). In certain embodiments, small molecules exclude nucleic acids.
  • a small molecule PFKFB3 inhibitory may be any small molecules that is determined to inhibit PFKFB3 function or activity. Such small molecules may be determined based on functional assays in vitro or in vivo.
  • PFKFB3 inhibitory molecules are known in the art and described in, for example, U.S. Patent publications 20130059879, 20120177749, 20100267815, 20100267815, and 20090074884, which are herein incorporated by reference.
  • Exemplary inhibitory compounds include: (lH-Benzo[g]indol-2-yl)-phenyl- methanone; (3H-Benzo[e]indol-2-yl)-phenyl-methanone; (3H-Benzo[e]indol-2-yl)-(4- methoxy-phenyl)-methanone; (3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanone; HC1 salt of (3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanone; (3H-Benzo[e]indol-2-yl)-(3-methoxy- phenyl)-methanone; (3H-Benzo[e]indol-2-yl)-pyridin-3-yl-methanone; (3H-Benzo[e]indol-2-yl)-(2-methoxy-pheny
  • inhibitory compounds include: l-Pyridin-4-yl-3-quinolin-4-yl- propenone; l-Pyridin-4-yl-3-quinolin-3-yl-propenone; l-Pyridin-3-yl-3-quinolin-2-yl- propenone; l-Pyridin-3-yl-3-quinolin-4-yl-propenone; l-Pyridin-3-yl-3-quinolin-3-yl- propenone; l-Naphthalen-2-yl-3-quinolin-2-yl-propenone; l-Naphthalen-2-yl-3-quinolin-3- yl-propenone; l-Pyridin-4-yl-3-quinolin-3-yl-propenone; 3-(4-Hydroxy-quinolin-2-yl)-l- pyridin-4-yl-propenone; 3 -(8-Hydroxy-quinol
  • inhibitory compounds include: PFK15 (l-(4-pyridinyl)-3-(2- quinolinyl)-2-propen-l-one); (2S)-N-[4-[[3-Cyano-l-(2-methylpropyl)-lH-indol-5- yl]oxy]phenyl]-2-pyrrolidinecarboxamide 3PO (3-(3-Pyridinyl)-l-(4-pyridinyl)-2-propen-l- one); (2S)-N-[4-[[3-Cyano-l-[(3,5-dimethyl-4-isoxazolyl)methyl]-lH-indol-5- yl]oxy]phenyl]-2-pyrrolidinecarboxamide; and Ethyl 7-hydroxy-2-oxo-2H-l-benzopyran-3- carboxylate.
  • THERAPEUTIC METHODS The methods described herein may be used to treat or prevent protein misfolding disorders by inhibition of PFKFB3.
  • the diseases amenable for treatment include, but are not limited to those tabulated below with their major aggregating protein.
  • Embodiments include methods for treating cancer with compositions comprising a PFKFB3 inhibitor.
  • Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral, or intravenous injection.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, or about 25% to about 70%.
  • compositions are administered orally.
  • compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying.
  • the quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a pharmaceutical composition are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like.
  • the dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject. [0085] In many instances, it will be desirable to have multiple administrations of at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the administrations may range from 2 day to twelve week intervals, more usually from one to two week intervals. The course of the administrations may be followed by assays for PFKFB3 activity.
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.
  • the PFKFB3 inhibitors can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intradermal, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • the composition is administered by intravenous injection.
  • the preparation of an aqueous composition that contains an active ingredient will be known to those of skill in the art in light of the current disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • 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 vegetable oils.
  • 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, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active ingredients in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • An effective amount of therapeutic or prophylactic composition is determined based on the intended goal.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • a subject is administered about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,
  • a dose may be administered on an as needed basis or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or any range derivable therein) or 1, 2, 3, 4, 5, 6, 7, 8, 9, or times per day (or any range derivable therein).
  • a dose may be first administered before or after signs of a condition.
  • the patient is administered a first dose of a regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range derivable therein) or 1, 2, 3, 4, or 5 days after the patient experiences or exhibits signs or symptoms of the condition (or any range derivable therein).
  • the patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therein) or until symptoms of an the condition have disappeared or been reduced or after 6, 12, 18, or 24 hours or 1, 2, 3, 4, or 5 days after symptoms of an infection have disappeared or been reduced.
  • compositions and related methods may also be used in combination with the administration of conventional therapies, such as those known in the art and/or described below.
  • conventional therapies such as those known in the art and/or described below.
  • the current methods and compositions may be used in combination with tradition therapies for treating a protein misfolding disease such as type 2 diabetes.
  • sulfonylureas such as glyburide, glipizide, and glimepiride (Amaryl)
  • meglitinides such as repaglinide and nateglinide
  • thiazolidinediones such as rosiglitazone and pioglitazone
  • DPP-4 inhibitors such as sitagliptin, saxagliptin, and linagliptin
  • GLP-1 receptor agonists such as exenatide and liraglutide
  • SGLT2 inhibitors such as canagliflozin and dapagliflozin
  • insulin therapy such insulin glulisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, and insulin isophane
  • insulin therapy such as insulin glulisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, and insulin isophane
  • insulin therapy such as insulin glu
  • dosage levels of the active ingredients in the methods of this disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors, including the activity of the chemotherapeutic agent selected, the route of administration, the time of administration, the rate of excretion of the therapeutic agent, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular therapeutic agent, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • PFKFB3 inhibitor is "A” and the traditional therapy (or a combination of such therapies) given as part of a treatment for a protein misfolding disorder, is "B": A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B/B
  • kits containing compositions described herein or compositions to implement methods described herein are provided.
  • kits are envisioned containing therapeutic agents and/or other therapeutic and delivery agents.
  • a kit for preparing and/or administering a therapy described herein may be provided.
  • the kit may comprise one or more sealed vials containing any of the pharmaceutical compositions, therapeutic agents and/or other therapeutic and delivery agents.
  • the kits comprise lipid delivery systems.
  • the lipid is in one vial, and the therapeutic agent is in a separate vial.
  • the kit may include, for example, at least one inhibitor of PFKFB3 expression/activity, one or more lipid component, as well as reagents to prepare, formulate, and/or administer the components described herein or perform one or more steps of the methods.
  • the kit may also comprise a suitable container means, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
  • a suitable container means which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
  • the container may be made from sterilizable materials such as plastic or glass.
  • the kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill.
  • the instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent.
  • kits may be provided to evaluate the expression of PFKFB3 or related molecules.
  • kits can be prepared from readily available materials and reagents.
  • such kits can comprise any one or more of the following materials: enzymes, reaction tubes, buffers, detergent, primers and probes, nucleic acid amplification, and/or hybridization agents.
  • these kits allow a practitioner to obtain samples in blood, tears, semen, saliva, urine, tissue, serum, stool, colon, rectum, sputum, cerebrospinal fluid and supernatant from cell lysate.
  • these kits include the needed apparatus for performing RNA extraction, RT-PCR, and gel electrophoresis.
  • Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.
  • the components may include probes, primers, antibodies, arrays, negative and/or positive controls.
  • Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, 10x, or 20x or more.
  • the kit can further comprise reagents for labeling PFKFB3 in the sample.
  • the kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer.
  • Labeling reagents can include an amine-reactive dye or any dye known in the art.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquotted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits may also include a means for containing the nucleic acids, antibodies or any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • labeling dyes are provided as a dried power.
  • kits in certain aspects.
  • the dye may then be resuspended in any suitable solvent, such as DMSO.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated.
  • kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • the kits may include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • kits may also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
  • Type 2 diabetes is characterized by a progressive defect in insulin secretion in the setting of relative insulin resistance (Cantley and Ashcroft, 2015).
  • the mechanisms that initiate ⁇ -cell dysfunction in T2D remain unclear, partly because there is no means to access the human pancreas preceding diabetes.
  • Pathological studies in individuals that have T2D reveal a partial ⁇ -cell deficit with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein co-expressed and secreted with insulin by ⁇ -cells (Butler et al., 2003; Clark et al., 1987; Clark et al., 1988; Junker et al., 1977).
  • IAPP islet amyloid polypeptide
  • IAPP aggregates In common with other protein misfolding diseases, the most toxic forms of IAPP aggregates are small membrane permeant oligomers (Gurlo et al., 2010; Janson et al., 1999; Kegulian et al., 2015). ⁇ -cell dysfunction in T2D shares characteristics of affected cells in other protein misfolding diseases, for example Alzheimer's disease and Parkinson's disease.
  • ROS reactive oxygen species
  • the autophagy-lysosome pathway and ubiquitin-proteasome pathway are altered in both ⁇ -cells in T2D and affected cells in neurodegenerative diseases, consistent with the accumulation of misfolded proteins (Cook et al., 2012; Costes et al., 2014; Costes et al., 2011; Rivera et al., 2011).
  • the inventors employed a variety of tools to investigate the mechanisms subserving the toxic effects of human IAPP in ⁇ -cells of HIP rats, a transgenic model of type 2 diabetes. It is established that hIAPP induces major metabolic and mitochondrial network changes through the activation of the HIFla/PFKFB3 stress pathway that, whereas in the short term are aimed to preserve ⁇ -cell survival, longer term result in cell death mediated by cytosolic Ca2+ accumulation.
  • the inventors further identify novel strategies to protect ⁇ -cells from IAPP toxicity by either constraining the HIFla/PFKFB3 induced metabolic changes or by inhibiting the cytosolic accumulation of Ca2+ consequent to the metabolic changes.
  • hIAPP toxic oligomers induce mitochondrial network fragmentation with reduced mitochondrial function
  • ⁇ -cells in T2D are characterized by a fragmented mitochondrial network, mitochondrial dysfunction and disrupted Ca2+ dynamics (Anello et al., 2005; Gurlo et al., 2016a; Gurlo et al., 2010; Lu et al., 2010).
  • the inventors visualized ⁇ -cell mitochondria with Tom20 immunostaining in pancreatic sections from human T2D donors (FIG. 6A and B) and found that they were more fragmented and less dense (p ⁇ 0.005) compared to ⁇ -cell mitochondria from non-diabetic (ND) donors (FIG.
  • hIAPP induces significant mitochondrial network fragmentation (Gurlo et al., 2010; Jung and Lee, 2010; Lu et al., 2010; Ma et al., 2012).
  • the inventors next investigated the impact of hIAPP overexpression on regulators of mitochondrial fission (dynamin related protein 1 ((Drpl)) and fusion (mitofusins 1/2 (MFN1/2) and optical atrophy related 1 (Opal)).
  • the inventors measured the oxygen consumption rate (OCR) of islets isolated from 5-6 month old prediabetic HIP rats versus those from WT. The inventors observed a 30% decrease in OCR in response to 20 mM glucose in HIP rat islets (p ⁇ 0.01) (FIG. 7A and B). To investigate if the hIAPP-induced decrease in mitochondrial function was mediated by a loss of mitochondrial membrane potential, the inventors treated INS 832/13 cells synchronized at Gl/S and S (Oh and 4h post-release from aphidicolin block) with tetramethylrhodamine, ethyl ester (TMRE) and performed FACS analysis.
  • OCR oxygen consumption rate
  • hIAPP toxicity increases aerobic glycolysis disengaged from mitochondrial oxidative phosphorylation.
  • the inventors investigated the metabolic changes induced by hIAPP toxicity.
  • Analysis of the microarray data (referred to also in Schludi et al., JCI Insight in press, GEO Accession number GSE90779) from WT and HIP transgenic rats showed upregulation of a subset of genes involved in aerobic glycolysis (lactate dehydrogenase A and C, LDHA and LDHC; phosphofructokinase L, PFKL; pyruvate kinase M2, PKM2 and 6- phosphofructo-2-kinase fructose 2,6 biphosphatase, PFKFB3) concomitant with the downregulation of genes involved in the TCA cycle (pyruvate carboxylase, PC; malate dehydrogenase, MDH; fumarate hydratase, FH; succinate dehydrogenase, SDH (FI
  • hIAPP alters cellular metabolism
  • hIAPP did not alter the glucose labeling pattern as there was no difference in the M6 fraction compared to control rIAPP overexpressing cells (FIG. 15B).
  • PFKFB3 6-phosphofructo-2-kinase 2,6- fructose biphosphatase
  • PFKFB3 6-phosphofructo-2-kinase 2,6- fructose biphosphatase
  • PFKl glycolysis - phosphofructokinase 1
  • ⁇ - cell glucokinase activator of ⁇ - cell glucokinase
  • PFKFB3 protein levels were increased in isolated islets from HIP versus WT rats especially prior to diabetes onset at 6 months of age (FIG. 8D) consistent with a role of hIAPP in inducing glycolysis through enhanced PFKFB3 expression.
  • PFKFB3 protein levels were also increased in islets from hIAPP transgenic mice before the onset of hyperglycemia (2.6- and 2-fold vs WT, respectively) (not shown).
  • PFKFB3 was also upregulated in hIAPP overexpressing INS 832/13 cells as shown by western blot (1.7-fold) and confirmed by immunocytochemistry (FIG. 16A-C).
  • the inventors observed increase in PFKFB3 immunoreactivity in the nuclei of ⁇ - cells of humans with T2D compared to non-diabetic subjects, similar to what was observed in HIP rats (FIG. 9A-C) (p ⁇ 0.05 vs. WT and p ⁇ 0.005 vs ND).
  • PFKFB3 hypoxia inducible factor-la
  • PFKFB3 Downregulation of PFKFB3 restores control metabolite composition and TCA flux via acetyl-CoA and stimulates PPP from lower isotopologue fractions.
  • PFKFB3 silencing restored most metabolites to their control levels (FIG. 10A).
  • ATP levels and ADP/ATP and AMP/ ATP ratios were also normalized, as well as lactate and palmitate levels, and precursors of nucleotide synthesis and metabolites of the thiopyruvate (homocysteine) pathway (FIG. 10A).
  • PFKFB3 silencing led to a decrease of pyruvate anaplerosis via OAA as demonstrated by a relative decrease in the M3 fractions of Mai, Asp, and Fum (FIG. 10B and FIG. 17A).
  • the conversion of acetyl-CoA to Cit, a-KG and Fum via the generation of M2 isotopologues was increased, implying re-engagement of glycolysis with the TCA cycle (FIG. 10B).
  • the M6 isotopologues of F16BP and G6P-F6P were suppressed by PFKFB3 silencing in cells overexpressing hIAPP (not shown).
  • M2 to M5- of G6P-F6P, M3 of ribulose-5-phosphate (R5P) and Ml to M6 isotopologues of sedoheptulose-7-phosphate (S7P) were increased in hIAPP overexpressing cells when PFKFB3 was silenced, indicating that the PPP was favored during ⁇ -cell rescue (FIG. 10B).
  • the inventors concluded that inhibition of PFKFB3 promotes a partial re- engagement of glycolysis with the mitochondrial TCA cycle in ⁇ -cells overexpressing hIAPP by reinforcing metabolic flux through the acetyl-CoA while permitting further PPP.
  • PFKFB3 suppression reduced cell death in INS 832/13 cells overexpressing hIAPP, as demonstrated by the reduction of the subGl peak in the flow cytometry histogram (46.3 ⁇ 2.5% vs 20.7 ⁇ 0.1%, p ⁇ 0.005) (FIG. 1 IB).
  • the levels of cleaved caspase 3 and the caspase 3 derived PARP-1 fragment (89 KDa) were reduced upon PFKFB3 silencing (FIG. 11C).
  • PFKFB3 silencing restores normal cytosolic levels of calcium. While it is known from previous work in the inventors' laboratory that hIAPP induces calpain hyperactivation, which is also a characteristic of ⁇ -cells in humans with T2D, the subcellular compartment(s) that exhibit aberrant Ca2+-induced calpain hyperactivation are unknown. One possibility is that enhanced glycolysis due to hIAPP leads to unregulated (e.g. independent of extracellular glucose) K(ATP) channel closure and therefore unregulated, sustained Ca2+ entry in the cell through voltage-gated Ca2+ channels.
  • the main adaptive metabolic response is the disengagement of glycolysis from the mitochondrial TCA cycle along with fragmentation of the mitochondrial network.
  • the inventors also establish that in common with some neurodegenerative diseases, this metabolic adaptation is mediated at least in part by the activation of the HIFla/PFKFB3 stress pathway.
  • the HIFla stress response provides short term survival benefit in response to acute stress such as a hypoxic event.
  • this adaptive change predictably causes impairment of ⁇ -cell function with relatively high insulin secretion at baseline glucose values (because of ATP generated by unrestrained glycolysis) but a deficient response to glucose stimulation, both characteristics of ⁇ -cells in T2D.
  • ⁇ -cells exposed to hIAPP induced stress adopt a metabolic pattern that mimics the so called Warburg effect reported in cancer cells (Vander Heiden et al., 2009).
  • the inventors sought to test the postulate that suppression of accelerated glycolysis (by silencing PFKFB3) or by suppressing cytosolic Ca2+ uptake by inhibiting K(ATP) channel mediated membrane depolarization would protect ⁇ -cells against hIAPP toxicity.
  • Silencing of PFKFB3 was indeed beneficial to both ⁇ -cell viability and function in the context of hIAPP toxicity.
  • Silencing of PFKFB3 suppressed hIAPP-increased glycolytic flux, restored pyruvate incorporation into TCA to generate Ac-CoA, while still permitting the adaptive increase in the PPP pathway.
  • INS 832/13 was provided by Dr. Christopher Newgard (Duke University, Durham, NC).
  • INS 832/13 cells were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 100 IU/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), 10% heat- -inactivated fetal calf serum (FCS) (Gemini Bio—Products, West Sacramento, CA, USA), and 50 ⁇ ⁇ -mercaptoethanol (Sigma, St. Louis, MO, USA) at 37 °C in a humidified 5% C0 2 atmosphere.
  • FCS heat- -inactivated fetal calf serum
  • INS 832/13 cells were plated in culture medium with 10% FCS for 24h. Medium was then replaced with fresh medium containing 0.1% FCS for 56h to allow cells to reach the GO out-of-cycle state. Synchronization of cells in Gl/S, S and G2/M stages of cell cycle was carried out as follows: after 24h in medium containing 10% FCS, cells were maintained in culture medium + 0.1% FCS for 56h. Medium was replaced with fresh medium + 10% FCS and, 12h later, aphidicolin was added. After 12h treatment with aphidicolin (Sigma A0781, St.
  • Adenoviruses Cells were transduced with rodent IAPP (rIAPP) or human IAPP (hIAPP) adenoviruses (Huang et al 2010) (100 MOI [multiplicity of infection]) for 24h or 36h.
  • Small interfering RNA PFKFB3 small interfering RNAs (siRNAs) (L--095107-02- -0005) were purchased from Dharmacon, Lafayette, CO, USA.
  • Drpl K48A plasmid containing a dominant negative mutation in Drpl gene was kindly provided by Dr. Takehiro Yasukawa (University College London, London, UK).
  • the potassium channel opener, NN— 414 (6— chloro— 3— (1- -methylcyclopropyl)amino-4H ⁇ thieno[3,2 ⁇ e] ⁇ l,2,4 ⁇ thiadiazine 1,1—dioxide) (Sigma SML0553, St. Louis, MO, USA), the sulfonylurea derivate, Glybenclamide (Sigma G0639, St. Louis, MO, USA) were dissolved in dimethylsulfoxide (DMSO) to prepare 3 mM and 25 mM stock solutions. The working concentrations were 3 ⁇ for NN414 and 100 ⁇ for Glybenclamide. Oligomycin (5 mM) (Sigma 04876, St.
  • Mitochondrial membrane potential Cells synchronized in Gl/S or S phase of cell cycle were washed with PBS and trypsinized. One million cells from each sample were incubated for 15 min at 37 °C with tetramethylrhodamine ethyl ester (TMRE) (10 nM, Sigma 87917, St. Louis, MO, USA). Afterwards cells were centrifuged at 2000 g for 2 min, TMRE solution was removed and cells were resuspended in fresh culture medium. Mitochondrial membrane potential was measured using NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA). Data were analyzed by NovoExpress software.
  • TMRE tetramethylrhodamine ethyl ester
  • Mitochondrial network INS 832/13 cells were grown on coverslips and incubated with the cell—permeant mitochondria—specific red fluorescent probe MitoTracker Red CMXRos (MTR) (Cell Signaling Technology 9082P, Danvers, MA, USA,) at a final concentration of 50 nM at 37 °C for the last 30 min in culture. Cells were then washed with PBS and fixed in 100% methanol at -20 °C for 20 min. Images were taken under a 63X objective with the AxioImager.M2a fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with the optical sectioning system ApoTome.2 and software ZEN2.
  • MTR MitoTracker Red CMXRos
  • Mitochondrial morphology was classified as fused ⁇ to ⁇ intermediate if fused mitochondria occupied >50% of the mitochondrial area and fragmented if fragmented mitochondria were present in >50% of the mitochondrial area. Mitochondrial morphology was independently scored by two observers (CM. and K. V ).
  • CM. and K. V Two observers.
  • Calcium measurements Cells were seeded on glass coverslips and synchronized in GO. For cytosolic free calcium measurements, cells were loaded with 2.5 ⁇ fura 2- AM for 45 min in medium containing 11 mM glucose. For mitochondrial and ER free calcium measurements, cells were transduced with adenovirus expressing specific probes.
  • Excitation (x) or emission (m) filters were maintained in combination with a FF444/521/608- -DiOldichroic (Semrock, Lake Forest, IL, USA) as follows: fura2, 340/10x and 380/10x, 535/30m (R340x/380x -535m);; D4ER, 430/24x, 470/24m and 535/30m (430x - R535m/470m);; Mito—Pericam, 480/410.
  • Fluorescence emission was collected with a QuantEM:512SC camera (PhotoMetrics, Arlington, AZ, USA) or an ORCA-1 camera (Hamamatsu, Skokie, IL, USA) at 0.125-0.2 Hz. 80-130 cells were analyzed per/sample and data were analyzed using Metafluor software (Molecular Devices, Sunnyvale, CA, USA).
  • PI staining cells were incubated with 0.5 ⁇ propidium iodide (PI, Molecular Probes, Eugene, OR, USA) for 20 min at 37 °C as previously described (Huang et al., 2010) and then fixed with 4% PFA. Coverslips or slides were mounted using Vectashield with DAPI (Vector Laboratories, H-1200, Burlingame, CA, USA). The frequency of cell death was evaluated after staining with PI or with MTR and antibody against ⁇ 2 ⁇ . ⁇ . 25 fields per section were imaged using a Leica DM6000 fluorescent microscope (Wetzlar, Germany) with a 20X objective equipped with a OpenLab 5.5 software (Improvision, Coventry, UK).
  • PI propidium iodide
  • HBSS Hanks' balanced salt solution
  • pancreas was then removed and transferred into a glass vial containing ice-cold liberase solution, digested for 20 min at 37 °C, and dispersed by shaking for 30 s. Islets were manually picked and cultured in RPMI 1640 medium (11 mM glucose) supplemented with
  • Lactate measurements Medium from cultured rodent islets was sampled every hour within 4 hours and lactate was analyzed using an enzymatic assay (Trinity Biotech 732- 10, Bray, Ireland) according to the manufacturer ' s instructions. Islets were collected for protein extraction. Lactate production (per ⁇ g protein) was expressed as the hourly change in the accumulated amount of lactate.
  • Oxygen consumption rate was determined using the Seahorse XF Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). After an overnight recovery, isolated islets from WT and HIP rats were seeded (25—50 islets per well) into the V7 plate (Seahorse Bioscience, North Billerica, MA, USA). To assess mitochondrial function, OCR was measured at the basal state and after stimulation with 20 mM glucose and sequential injection of oligomycin (ATP synthase inhibitor), carbonyl cyanide-p trifluoromethoxyphenylhydrazone (FCCP; uncoupler), and rotenone (complex I inhibitor).
  • OCR Oxygen consumption rate
  • Pancreata Human subjects. Pancreata were procured from brain dead organ donors by the JDRF Network for Pancreatic Organ Donors with Diabetes (nPOD), administered by the University of Florida, Gainesville, Florida. All procedures were in accordance with federal guidelines for organ donation and the University of Florida Institutional Review Board. Three pancreata from individuals with type 2 diabetes (T2D) (6186, 6275, 6255) and 3 from nondiabetic (ND) (6104, 6288, 6020) controls matched by age, sex and BMI were examined in this study.
  • T2D type 2 diabetes
  • ND nondiabetic
  • Human pancreatic islets were from the Islet Cell Resource Consortium. They were derived from 1 T2D brain—dead organ donor from the University of Pennsylvania, 1 T2D donors from Southern California Islet Cell Resources Center (City of Hope) (see table— HI128), 1 T2D from the University of Wisconsin (see table— HI130), 1 non diabetic brain— dead organ donor from Southern California Islet Cell Resources Center (City of Hope) (see table— HI126) and 1 non diabetic brain— dead organ donor from the University of Pennsylvania (see table— HI131). Dithizone staining was performed to assess the islet purity that was 90-95%. The donors, aged 25-60 years, were heart— beating cadaver organ donors.
  • Islets were cultured in RPMI 1640 medium (5.5 mM glucose) containing 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) for one day and then processed for western blotting analysis.
  • RPMI 1640 medium 5.5 mM glucose
  • penicillin 100 units/ml
  • streptomycin 100 g/ml
  • 10% fetal bovine serum Invitrogen, Carlsbad, CA, USA
  • Antibodies The following antibodies were used: anti-PFKFB3 (Abeam 181861, Cambridge, UK, 1 :200 for IF, 1 : 1000 for WB) anti-yH2A.X (Cell Signaling Technology 2577S, Danvers, MA, USA, 1 :200 for IF, 1 : 1000 for WB in human tissue), anti-yH2A.X (Abeam 26350, Cambridge, UK, 1 :200 for IF, 1 : 1000 for WB in rodent tissue), anti-Tom20 (Santa Cruz Biotechnology sc-11415, Dallas, TX, USA, 1 :200 for IF), anti-HIFla (NOVUS Biologicals NB100-105, Littleton, CO, USA, 1 : 1000 for WB), anti-MFN2 (Cell Signaling Technology 9482S, Danvers, MA, USA, 1 : 1000 for WB), anti- -Opal (BD [0173] Transduction 612606, San Diego, CA
  • qPCR Real-time quantitative polymerase chain reaction
  • ABI7900HT Applied BiosystemsTM, Foster City, CA, USA
  • Each qPCR reaction contained 1 ⁇ Fast SYBR® Green Master Mix (Applied BiosystemsTM, Foster City, CA, USA), 1 ⁇ of each primer, and 400 ng cDNA.
  • Relative mRNA expression of target gene was determined using the comparative cycle threshold (Ct) method, where the amount of target cDNA was normalized to the internal control, GAPDH cDNA.
  • the primers used were : PFKFB3 (fwd: CACGGCGAGAATGAGTACAA (SEQ ID NO:3), rev: TTCAGCTGACTGGTCCACAC (SEQ ID NO:4)) (Arden et al., 2008); LDHA (fwd: TGC TGG AGC CAC TGT CG (SEQ ID NO:5), rev: CTG GGT TTG AGA CGA TGA GC (SEQ ID NO:6)) (Laybutt et al., 2003); MCT1 (fwd: ATG TAT GCC GGA GGT CCT ATC (SEQ ID NO:7), rev: CCA ATG GTC GCT TCT TGT AGA (SEQ ID NO:8)) (Smith and Drewes, 2006) and GAPDH (fwd: ATG ACT CTA CCC ACG GCA AG (SEQ ID NO:9), rev: CTG GAA GAT GGT GAT GGG TT (SEQ ID NO: 10)).
  • Tissue immunostaining and morphometrical analysis 4- ⁇ m paraffin tissue sections from human or rodent samples were exposed to toluene for 10 min and, then, to 100% ethanol for other 10 min, 95% ethanol and 70% ethanol for 5 min each, and water. Sections were transferred in heat— induced antigen retrieval solution in citrate buffer at pH 6.0, using microwave and then cooled to room temperature for 1 hour, then soaked in Soaking Buffer (TBS, 0.4% TX100) for 30 minutes on ice, and washed once with TBS.
  • TBS Soaking Buffer
  • CR6-interacting factor 1 is a key regulator in Abeta-induced mitochondrial disruption and pathogenesis of Alzheimer's disease. Cell Death Differ 22, 959-973.
  • Islet amyloid formed from diabetes-associated peptide may be pathogenic in type-2 diabetes. Lancet 2, 231-234. Clark, A., Wells, C.A., Buley, I D., Cruickshank, J.K., Vanhegan, R.I., Matthews,
  • Islet amyloid increased A-cells, reduced B-cells and exocrine fibrosis: quantitative changes in the pancreas in type 2 diabetes. Diabetes Res 9, 151-159.
  • UCHL1 deficiency exacerbates human islet amyloid polypeptide toxicity in beta-cells: evidence of interplay between the ubiquitin/proteasome system and autophagy. Autophagy 10, 1004-1014.
  • Gurlo T., Costes, S., Hoang, J.D., Rivera, J.F., Butler, A.E., and Butler, P.C. (2016a). beta Cell-specific increased expression of calpastatin prevents diabetes induced by islet amyloid polypeptide toxicity. JCI Insight 1, e89590. Gurlo, T., Rivera, J.F., Butler, A.E., Cory, M., Hoang, J., Costes, S., and Butler, P.C.
  • Glycolytic ATP fuels the plasma membrane calcium pump critical for pancreatic cancer cell survival.
  • hypoxia-inducible factor HIF
  • citric acid cycle intermediates possible links between cell metabolism and stabilization of HIF. J Biol Chem 282, 4524-4532. Lu, H., Koshkin, V., ister, E.M., Gyulkhandanyan, A.V., and Wheeler, M B.
  • Diabetes reduces beta-cell mitochondria and induces distinct morphological abnormalities, which are reproducible by high glucose in vitro with attendant dysfunction. Islets 4, 233-242.
  • Amyloid beta resistance in nerve cell lines is mediated by the Warburg effect.
  • Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic beta-cells: protective role of p62-positive cytoplasmic inclusions.
  • SIRT3 deacetylates and activates OPAl to regulate mitochondrial dynamics during stress. Mol Cell Biol 34, 807-819.
  • Islet amyloid-associated diabetes in obese A(vy)/a mice expressing human islet amyloid polypeptide Diabetes 47, 743-750.
  • Drp-1 -dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis. Mol Cell 16, 59- 68. Tarasov, A.I., Semplici, F., Li, D., Rizzuto, R., Ravier, M.A., Gilon, P., and Rutter,
  • SLP-2 is required for stress-induced mitochondrial hyperfusion.
  • Tornovsky-Babeay S., Dadon, D., Ziv, O., Tzipilevich, E., Kadosh, T., Schyr-Ben Haroush, R., Hija, A., Stolovich-Rain, M., Furth-Lavi, J., Granot, Z., et al. (2014).
  • Type 2 diabetes and congenital hyperinsulinism cause DNA double-strand breaks and p53 activity in beta cells. Cell Metab 19, 109-121. Vander Heiden, M.G., Cantley, L.C., and Thompson, C.B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029-1033.
  • Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell 19, 17-30.
  • hypoxia-inducible factor 1 alpha (HIF-lalpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation.
  • HIF-lalpha Hypoxia-inducible factor 1 alpha

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Abstract

L'invention concerne des procédés de traitement d'une maladie liée au mauvais repliement des protéines chez un sujet, les procédés comprenant l'administration d'au sujet un inhibiteur de PFKFB3. D'autres aspects concernent un procédé de traitement ou de prévention de la mort cellulaire induite par le mauvais repliement des protéines dans une cellule, le procédé comprenant l'administration d'un inhibiteur de PFKFB3 à la cellule. L'invention concerne également des procédés d'inhibition ou de réduction de la mort de cellules β chez un sujet souffrant de diabète de type 2, le procédé comprenant l'administration d'un inhibiteur de PFKFB3 au sujet.
PCT/IB2017/053209 2016-05-31 2017-05-31 Méthodes de traitement d'une maladie à l'aide d'inhibiteurs de pfkfb3 Ceased WO2017208174A2 (fr)

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Cited By (7)

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WO2022008574A1 (fr) * 2020-07-10 2022-01-13 Société des Produits Nestlé S.A. Composition nutritionnelle à base de mir-3184
WO2022040161A1 (fr) * 2020-08-18 2022-02-24 The Regents Of The University Of California Méthodes et compositions pour le traitement du diabète et la régénération de cellules bêta
EP3867226A4 (fr) * 2018-10-15 2022-11-23 Gero Pte. Ltd. Inhibiteurs de pfkfb3 et leurs utilisations
WO2022245698A1 (fr) * 2021-05-16 2022-11-24 Metanoia Bio Inc. Procédés et compositions pour traiter une maladie pancréatique et hépatique
WO2022245702A1 (fr) * 2021-05-16 2022-11-24 Metanoia Bio Inc. Méthodes et compositions pour le traitement d'une maladie cardiovasculaire
WO2022245711A1 (fr) * 2021-05-16 2022-11-24 Metanoia Bio Inc. Procédés et compositions pour le traitement d'états neurologiques
CN115851793A (zh) * 2022-09-23 2023-03-28 龙岩学院 一种高效表达猪pfkfb3基因的真核表达载体及其构建方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8088385B2 (en) * 2007-06-18 2012-01-03 University Of Louisville Research Foundation Inc. PFKB3 inhibitor for the treatment of a proliferative cancer

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3867226A4 (fr) * 2018-10-15 2022-11-23 Gero Pte. Ltd. Inhibiteurs de pfkfb3 et leurs utilisations
WO2022008574A1 (fr) * 2020-07-10 2022-01-13 Société des Produits Nestlé S.A. Composition nutritionnelle à base de mir-3184
WO2022040161A1 (fr) * 2020-08-18 2022-02-24 The Regents Of The University Of California Méthodes et compositions pour le traitement du diabète et la régénération de cellules bêta
WO2022245698A1 (fr) * 2021-05-16 2022-11-24 Metanoia Bio Inc. Procédés et compositions pour traiter une maladie pancréatique et hépatique
WO2022245702A1 (fr) * 2021-05-16 2022-11-24 Metanoia Bio Inc. Méthodes et compositions pour le traitement d'une maladie cardiovasculaire
WO2022245711A1 (fr) * 2021-05-16 2022-11-24 Metanoia Bio Inc. Procédés et compositions pour le traitement d'états neurologiques
CN115851793A (zh) * 2022-09-23 2023-03-28 龙岩学院 一种高效表达猪pfkfb3基因的真核表达载体及其构建方法

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