WO2013076501A2 - Procédé de criblage - Google Patents

Procédé de criblage Download PDF

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WO2013076501A2
WO2013076501A2 PCT/GB2012/052905 GB2012052905W WO2013076501A2 WO 2013076501 A2 WO2013076501 A2 WO 2013076501A2 GB 2012052905 W GB2012052905 W GB 2012052905W WO 2013076501 A2 WO2013076501 A2 WO 2013076501A2
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vps34
mice
insulin
pi3k
mutation
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WO2013076501A3 (fr
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Bart Vanhaesebroeck
Benoit BILANGES
Samira ALLIOUACHENE
Claire Chaussade
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Queen Mary And Westfield College University Of London
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present invention relates to methods for identifying agents useful in the treatment of diseases associated with insulin resistance and/or glucose intolerance, such as type II diabetes, non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases.
  • diseases associated with insulin resistance and/or glucose intolerance such as type II diabetes, non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases.
  • Insulin controls glucose and lipid homeostasis by modulating the functions of multiple organs and tissues, including liver, muscle and fat. In muscle and fat, insulin stimulates glucose uptake resulting in glucose clearance from circulation, and induces lipid synthesis. In the liver, insulin blocks glucose production and stimulates fatty acid synthesis. Any impairment in the insulin signalling pathway plays a key role in the development of insulin resistance.Insulin resistance initially manifests itself as the constellation of symptoms called "insulin resistance syndrome", which include glucose intolerance, obesity, and hypertension. Insulin resistance promotes the development of diseases such as type II diabetes andnon-alcoholic fatty liver (hepatic steatosis). Insulin resistance syndrome is increasing in prevalence with alarmingrapidity, affectingmore than 25% of adults in the United States. More than 50% of obese children have insulin resistance syndrome.
  • Type II diabetes and non-alcoholic fatty liver are the most prevalent and are associated with obesity.
  • Type II diabetes is characterised by insulin resistance associated with an insulin secretion defect.
  • Pancreatic beta cells initially compensate for this insulin resistance by increasing their insulin output. Over time, these cells become unable to produce enough insulin to maintain normal glucose levels, indicating progression to Type II diabetes.
  • NAFLD non-alcoholic fatty liverdisease
  • Januvia is another recently approved drug that increases blood levels of incretin hormones, which can increase insulin secretion, reduce glucagon secretion and have other less well characterized effects.
  • Januvia and other dipeptidyl peptidases IV inhibitors may also influence the tissue levels of other hormones and peptides, and the long-term consequences of this broader effect have not been fully investigated.
  • FIG. 1 Vps34 D761A/+ mice exhibit enhancedinsulin sensitivity and better glucose tolerance.
  • FIG. 3 Vps34 D761A/+ mice remain insulin-sensitive under obese condition.
  • FIG. 4 Reduced High Fat Diet-induced hepatic steatosis in Vps34 D761A/+- mice (A and
  • B is correlated with enhanced blood adiponectin level (C).
  • FIG. 5 Vps34 D761A/+ female mice also exhibit enhanced insulin sensitivity and better glucose tolerance.
  • FIG. 8 PI3K-C2 D1212A/D1212A mice remain insulin sensitive under obese condition and there is a decrease in accumulation of triglycerides in the liver (F and G).
  • Figure 9 A and B.Confocal analysis of mouse hepatocytesC. Comparing levels of P-S6K (T389) and P-S6 in WT and PI3K-C2p D1 12A/D1212A mice. D. No difference in Insulin receptor (IR) and increase in IKS levels in C2p D121 A/D1212A hepatocytes verswswild-type.
  • IR Insulin receptor
  • Figure 10 Comparing leptin and adiponectin levels in pi3K-C2 D,212A/D,212A and wild type mice.
  • Figure 11 Table showing IC50 values ( ⁇ ) for selected PI3K inhibitors against Lipid Kinases according to Knight et al. Cd ⁇ 125, 733-747 (2006), Kong et al, EJC 46 1 1 11 -1121 (2010) and Miller, et al. Science, 327, 1638-1642 (2010).
  • Figure 13 Vps34 inactivation protects against high-fat diet induced hepatic steatosis.
  • H&E staining is the classic staining used for basic cellular cytology.
  • the "H” turns acidic structures (such as DNA) blue and the “E” turns the proteins red.
  • Figure 14 Effect of long term High Fat Diet on WT and Vps34 D761A/+ body composition. The ratio of lean/fat tissue of wild type and Vps34 D761A+ mice which had been subjected to
  • Figure 15 Echocardiographic Measurements.
  • Echocardiography was used to measure LV dimensionswith M-mode and to trace endocardial area with B-mode at the level of the papillary muscles with the average of 3 cardiac transmitral flow measured by Doppler traces.
  • Figure 16 Metabolic parameters of high-fat-diet subjected mice.
  • FFA Plasma Free Fatty Acid
  • TG Plasma triglycerides
  • FIG. 17 PI3P levels are reduced in ⁇ 3 ⁇ 2 ⁇ primary hepatocytes
  • Akt signalling is enhanced in PIK3C2p. insulin responsive tissues and primary hepatocytes and not in the spleen.
  • FIG. 19 Enhanced Akt signalling is maintained in PfK3C2mice subjected to high fat diet.
  • PI3 -C2p and Vps34 are class II and III isoforms, respectively, of the phosphoinositide 3- kinase (PI3K) family, which are involved in endosomal trafficking and autophagy.
  • PI3K phosphoinositide 3- kinase
  • the present inventors have created Vps34 kinase-dead (KI) mice, in which the active site of Vps34 has been inactivatedgiving rise to Vps34 D761A.
  • KI Vps34 kinase-dead mice
  • the same approach has been employed to generate PI3K-C2p KI mice orPI3K-C2 D1212A/D12,2A mice.
  • heterozygous vps34 KI mice where 50% of vps34 activity is inactivated
  • homozygous and heterozygous ⁇ 3 ⁇ -02 ⁇ KI mice display improved glucose tolerance and enhanced insulin sensitivity.
  • heterozygous male Vps34 KI mice and homozygous PI3K-C2p KI mice are protected from High Fat Diet-induced fatty liver (also known as hepatic steatosis).
  • Vps34 and/or PI3K-C2 inhibitors will be useful to increase insulin sensitivity and/or improve glucose tolerance.
  • the present invention provides a method for identifying agents useful in the treatment and/or prevention of a disease associated with insulin resistance and/or glucose intolerance which comprises the step of investigating the capacity of a test agent to inhibit the Vps34 signalling pathway and/or the PI3K-C2 signalling pathway.
  • the method may comprise the step of investigating whether a test agent inhibits the kinase activity of Vps34 or PI3K-C2P or both.
  • the method may be conducted in vitro or in a cell in culture.
  • the method may be used to screen for compounds capable of increasing insulin sensitivity in a subject.
  • the method may be used to screen for compounds capable of improving glucose tolerance in a subject.
  • Adiponectin may be used as a biomarker for agents capable of inhibiting the Vps34 signalling pathway and/or the PI3K-C2p signalling pathway.
  • the disease associated with insulin resistance and/or glucose intolerance may, for example, be selected from the following group: type II diabetes, hepatic steatosis and non-alcoholic fatty liver disease (NAFLD).
  • the present invention provides a transgenic non-human animal which comprises a mutation in Vps34 or PI3K-C2psuch that the active site is inactivated.
  • the transgenic non-human animal may comprise a mutation in the DFG motif of the ATP- binding site.
  • the mutation may cause the motif to have the sequence AFG.
  • transgenic non-human animal comprises a mutation in PI3K-C2P it may comprise the mutation D1212A.
  • PI3K Phosphoinositide 3-kinases are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking.
  • PI3Ks are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol (Ptdlns).
  • the PI3 family is divided into three different classes: Class I, Class II, and Class III.
  • the classifications are based on primary structure, regulation, and in vitro lipid substrate specificity.
  • Class II comprises three catalytic isoforms (PI3K-C2a, PI3K-C2p, and PI3K-C2y) which, unlike Classes I and III, arenot constitutively associated with a regulatory subunit.
  • Class II PI3Ks catalyse the production of PI(3)P and PI(3,4)P 2 from the PI lipid.
  • PI3K-C2a and PI3K- C2p are expressed throughout the body, whereas expression of PI3K-C2y is limited to hepatocytes.
  • the distinct feature of Class II PI3Ks is the C-terminal C2 domain. This domain lacks critical Asp residues to coordinate binding of Ca 2+ , which suggests class II PBKs bind could bind lipids in a Ca 2+ -independent manner.
  • Vps34 There is only one known class III PI 3 -kinase, Vps34, which is also the only PI3 expressed in all eukaryotic cells. In humans it is encoded by the PIK3C3 gene. In human cells Vps34 associates with a regulatory subunit, pl50.
  • the class III kinase produces PI(3)P from PI.
  • the capacity of a test agent to inhibit the Vps34 and/or PI3K-C2 signalling pathway is investigated.
  • the agent may directly inhibit Vps34 and/or PI3K-C2P, for example by inhibiting the kinase activity of Vps34 and/or PI3K-C2 .
  • the agent may downregulate Vps34 and/or PI3K-C2 expression, or downregulate one or more binding partner(s) of Vps34 and/or PI3K-C2 .
  • the present invention relates to a method for identifying agents useful in the treatment and/or prevention of a disease.
  • Agents are identified on the basis of their capacity to inhibit the Vps34 signalling pathway or the PI3K-C2 signalling pathway.
  • An in vitro screen may be conducted for agents such as for small molecule inhibitors that inhibit the in vitro kinase activity of vps34 or PI3K- C2p or both.
  • Agents such as RNAi, capable ofdownregulating binding partners of Vps34, may interfere with Vps34 stability, and thus indirectly with its function.
  • the method may be a cell-based assay, looking for agentswhichinterfere with the biology and signaling of these PD s.
  • the assay may use autophagy, a biological phenomenon in which this kinase is involved in cells.
  • Such assays which are based on monitoring intracellular vesicular traffic, are known in the art.
  • Alternative cell-based assays include those based on forced over/inducible expression of Vps34 in cells, which may lead to cell death. Agentswhich inhibit the Vps34 signalling pathway would then rescue this cell death.
  • cell-based assays may be based on potentiating insulin signaling in cells.
  • GTTs Glucose tolerance tests
  • ITTs insulin tolerance tests
  • tissue levels of the Vps34 lipid product PtdIns3P may be assayed in primary tissues in vivo. This provides a direct readout of target inhibition in vivo.
  • Vps34- or PI3K-C2p-dependent phosphorylation response/biomarkers may be used to investigate the effect of test agents on Vps34-dependent orPI3K-C2p-dependent cellular phosphorylation.
  • Characteristics associated with ⁇ 3 ⁇ -02 ⁇ or Vps34 kinase-dead mice may be used as markers for ⁇ 3 ⁇ -02 ⁇ or Vps34 inhibition.
  • adiponectin an adipokine known to reduce hepatic and serum triglyceride levels and to protect from nonalcoholic hepatic steatosis
  • Leptin may be used as a biomarker for agents capable of inhibiting PI3K-C2p.
  • the test agent may be based on one of the known class H/III PI3K inhibitors.
  • Vps34 and PI3K-C2p activity can be inhibited by pan-PI3K inhibitors such as wortmannin and 2-(4- mo holinyl)-8-phen lchromone (LY294002).
  • PI3K-C2p is also inhibited by these compounds, but at higher doses.
  • 3-methyladenine (3 -MA) has often been used and at very high concentration (e.g., 10 mM for 3 -MA) to inhibit Vps34.
  • 3-MA has been used to test whether protein degradation processes are autophagy-dependent in cells.
  • other general PI3K inliibitors such as wortmannin also inliibit autophagy suggesting that these agents have additional off-target effects (see table 1) that may confound interpretations in certain contexts.
  • test agent may be based on one of the PI3K inhibitors shown in Figure 1 1.
  • test agent may have a similar chemical structure to one of the PI3K inhibitors shown in Figure 1 1 , with one or more minor variation(s), such as to side chain or ring substiruents to increase the selectivity of the molecule for class II or III PDKs.
  • the test agent may alternatively have previously unreported chemical structure.
  • test agent is capable of inhibiting the expression of Vps34 or ⁇ 3 ⁇ - €2 ⁇ , a binding partner, or another component involved in the Vps34 or ⁇ 3 ⁇ -02 ⁇ signalling pathways, it may be a nucleic acid-based molecule, such as an antisense sequence or ansiRNA.
  • the disease may be any disease or medical condition associated with insulin resistance and/or glucose intolerance.
  • the disease may, for example, be type II diabetes ornon-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • Diabetes mellitus type II also known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes, is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. Diabetes is often initially managed by increasing exercise and dietary modification. The classic symptoms of diabetes are polyuria (frequent urination), polydipsia (increased thirst), polyphagia (increased hunger), and fatigue.
  • NIDDM non-insulin-dependent diabetes mellitus
  • adult-onset diabetes is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. Diabetes is often initially managed by increasing exercise and dietary modification. The classic symptoms of diabetes are polyuria (frequent urination), polydipsia (increased thirst), polyphagia (increased hunger), and fatigue.
  • Hepatic steatosis is an abnormal fat accumulation in the liver that can have detrimental consequences on liver functions and in some cases can lead to hepatocarcinomas.
  • Non-alcoholic fatty liver disease is one cause of a fatty liver, occurring when fat is deposited (steatosis) in the liver not due to excessive alcohol use. It is related to insulin resistance and the metabolic syndrome and may respond to treatments originally developed for other insulin-resistant states (e.g. diabetes mellitus type ⁇ ) such as weight loss, metformin and thiazolidinediones.
  • the second aspect of the invention relates to a transgenic non-human animal in which the active site of Vps34 or PI3K-C2 is inactivated.
  • the non-human animal may be a mammal.
  • the non-human animal may be, for example, a rodent, such as a mouse or rat.
  • the transgenic animal may comprise an inactivating mutation in Vps34 or PI3K-C2p.
  • the mutation may be in the active site of the enzyme.
  • the mutation may be in the DFG motif of the ATP-binding site of the enzyme.
  • the mutation may mean that the encoded amino acid sequence has an AFG sequence instead of a DFG motif.
  • the mutation may be D761A.
  • the mutation may be in the position equivalent to D761 in the mouse Vps34 sequence.
  • the mutation may be D1212A.
  • the mutation may be in the position equivalent to D1212A in the mouse PI3K-C2p sequence.
  • Knock-In (KI) vps34 mice were generated by introducing a germline inactivating mutation (D761A) in the ATP-binding site (DFG motif) of the PIK3C3 gene that encodesvps34 (Fig. 1A).
  • the resulting mutant allele hereafter designated as vps34 D7S1A ), encodes vps34 protein in a constitutively inactive form (also called kinase dead).
  • the Vps34 D7fiIA allele was subsequently maintained into the C57BL/6J mouse genetic background through backcrossing.
  • the heterozygous vps34 D761A + (+ indicates the wild-type (WT) vps34 allele) mice are fertile, phenotypically comparable to the vps34 +/+ controls and can live at least up to 54 months with no apparent behavioural defects during this period.
  • no viable homozygous (vps34 D761A/D7 1A ) mice were obtained from any heterozygous intercrosses suggesting that homozygosity for vps34 mutation leads to embryonic lethality.
  • Vps34 was ubiquitously expressed in murine adult tissues with variable expression levels across the different organs (the highest being in the brain). Importantly, vps34 expression was unchanged in the vps34 D761A/+ mice (and Mouse Embryonic Fibroblasts (MEFs)) and was similar to WT levels (Fig. IB). As expected, tissues isolated from vps34 hetero2ygous mice display a 50% reduction of vps34 kinase activity (Fig. 1C) confirming that heterozygous mice carrying the D761A mutation have a 50% loss of function of vps34 activity.
  • mice Males and females werefound to be reduced in heterozygous mice compared to WT mice, correlating with the enhanced insulin sensitivity observed in mutant mice (Fig. 2C). Altogether, these data show that 50% reduction of vps34 activity strongly affects glucose homeostasis and insulin sensitivity in mice.
  • the present inventors therefore subjected WT and vps34 D761A/+ male mice to 16 weeks of HFD (45% fat) and monitored weekly their body weight. Comparison of HFD-fed WT and vps34 D761A/+ miceshowed that these animals gained similar body mass, with a mild tendency for the heterozygous mice to be leaner (area under the curve (AUC) p ⁇ 0.0577) (Fig. 3A). No significant differences between vps34 'and WT mice regarding food/water intake (data not shown) and leptin levels (Fig. 4C) were found.
  • HFD-fed vps34 D761A/+ mice continued to develop a mild fasting hypoglycaemia whereas no significant differences were apparent in fed states (Fig. 3B).
  • HFD-fed vps34 D761A/+ mice remained insulin sensitive as assessed during an insulin tolerance test (Fig. 3C, left panel).
  • no differences were observed during a glucose tolerance test when compared to HFD-fed WT mice, indicating that HFD-fed vps34 D761A/+ mice lost their ability to clear glucose better under HFD conditions (Fig. 3C, right panel).
  • HFD-fed vps34 mice did not show any effect on hepatic insulin signalling (Fig. 3D).
  • NAFLD nonalcoholic fatty liver disease
  • hepatic steatosis also known as fatty liver
  • hepatic steatosis also known as fatty liver
  • vps34 D7 ⁇ 51A/+ mice showed a markedly reduced liver lipid accumulation (correlated with steatosis) when compared to controls as assessed by Oil Red O staining of liver sections (Fig. 4B, lower panel).
  • adiponectin an adipokine known to reduce hepatic and serum triglyceride levels and protects from nonalcoholic hepatic steatosis(Polyzos et al, Diabetes, Obesity and Metabolism (2010)).
  • adiponectin an adipokine known to reduce hepatic and serum triglyceride levels and protects from nonalcoholic hepatic steatosis(Polyzos et al, Diabetes, Obesity and Metabolism (2010)).
  • Fig. 4C blood levels of adiponectin were strongly increased in the HFD-fed vps34 D7ilA/+ mice. This finding correlates with the steatosis protection observed in the mutant mice as adiponectin is often inversely correlated with body fat levels.
  • Example 5 Effect of long term High Fat Diet on WT and Vps34 D7MA/+ body composition
  • Wild typeandVps34 D761A/+ mice were subjected to 45% High Fat Diet, using the methodology described in Example 3, for 7 months.
  • Body composition measurements were performed with a BrukerBioSpec 70/30 7 Tesla magnetic resonance imaging (MRI) scanner (BrukerBiospin, Ettlingen, Germany).
  • the ratio of lean/fat tissue was calculated from the nuclear magnetic resonance (NMR) data and expressed as % ( Figure 14).
  • a long term High Fat Diet did not have a further deleterious impact on body composition of Vps34 D761A/+ compared to WT control animals.
  • Vps34 D761A + mice showed a normal cardiac function, wall thickness, mass (representing hypertrophy) and cardiac dimension (for dilatation).
  • Example 7 Metabolic parameters of high-fat-diet subjected mice
  • the Plasma Free Fatty Acid (FFA) levels, Plasma triglycerides (TG) levels and Plasma Insulin levels were compared between Vps34 D?61A + mice and control animals under normal chow (NC) or High-Fat_Diet (HFD). The results are shown in Figure 16.
  • ⁇ 3 ⁇ -02 ⁇ In order to study the organismal role of ⁇ 3 ⁇ -02 ⁇ , we created a germline knockin mouse line in which the DNA encoding the ATP-binding DFG motif of PIK3C2B, the gene encoding PI3K-C2P, is mutated to encode the AFG sequence instead, resulting in a kinase-dead protein, further referred to as ⁇ 3 ⁇ - €2 ⁇ D1212A (Fig. 6A). Homozygous mice for the mutated PIK3CB allele (hereafter called ⁇ 3 ⁇ -02 ⁇ ⁇ 1212 ⁇ ⁇ ) were bom at the expected Mendelian ratios.
  • ⁇ -02 ⁇ protein is highly expressed in some tissues including brain, white adipose tissue (WAT), spleen, pancreas, lung and prostate (Fig. 6B). Expression of the mutant PI3K-C2 protein and other PI3 isofonns was unaffected, both in Mouse embryonic fibroblasts (MEFs) derived from E13.5 embryos and in WAT from adult mice (Fig. 6C). PI3K activity in PI3K- 02 ⁇ ' ⁇ 3 ⁇ 65 was completely abolished from pi 3 K-C2 D1212A D12,2A brain and liver and no compensatory alterations in the activities of ⁇ 3 ⁇ -02 ⁇ (Fig. 6D). Despite considerable effort, the main physiological lipid products produced by ⁇ 3 ⁇ -02 ⁇ class II PI3Ks in general are still unknown. The present invention provides a relevant physiological model to address this question.
  • Phosphoinositide levels were measured in wt and ⁇ 3 ⁇ -02 ⁇ mutant MEFs in unstimulated conditions or after 10 minutes insulin stimulation. MEFs were labelled with 3 P- orthophosphate and the relative levels of each phosphoinositide species PIP 3 , PI3,4P 2 , PI3,5P 2, PI4P and PI3P were quantified. A small but significantincrease in the levels of PDP and PI4P production were observed after 10 min insulin stimulation (Fig6D). In contrast, the levels of PEP3,5P2, PI3,4P2 and PIP3in WT MEFs were not significantly changed upon insulin stimulation.
  • Example 9 Increasedglucose tolerance and insulin sensitivity in PI3K-C2 D1 12A/D1212A mice
  • PI3K-C2p D1212A/D1212A mice are viable and fertile, with no apparent defects. No body weight difference was observed either in postnatal or at the embryonic stage in the mutant mice compared to WT mice (Fig.7A and data not shown). The levels of blood glucose in overnight- fasted or randomly-fed states are unaffected in 12 week old pi3 -C2p D,2I AD, 212A mice (Fig. 7B).
  • Example 10 - PI3K-C2 "' ⁇ " ⁇ ' ⁇ mice are protected against HFD-induced liver steatosis
  • the pi3K_c2p D12,2AD12I2A mice were challenged with a high fat diet for 16 weeks. Firstly, body weight was monitored over the HFD weeks. No difference in weight gain was observed between the WT and PI 3 K _ C 2p DI212AyDm2A mice (Fig.8A). The levels of blood glucose and plasma insulin (Fig. 8B) are unaffected in the PI3K- C2 D1212A/D1212A mice, with both genotypes displaying increased in glycaemia. Surprisingly no difference in glucose tolerance (Fig.
  • Example 11 - PI3K-C2p inactivation may affect endocytic trafficking
  • FIG. 9A An immunofluorescence staining was perfonned using EEA1 as an endocytic marker or using the PI3P-binding protein GST-2X FYVE ms transfected in hepatocytes. Confocal microscopic analysis revealed bigger and distorted endosomes in the pi3K-C2p D1212A/m212A hepatocytes (Fig. 9A).Fig 9B shows a decrease of the POP levels in the ⁇ 3 ⁇ -02 ⁇ DI212AyD1212A hepatocytes after starvation.
  • Example 12 Investigating PI3P levels in ⁇ 3 €2 ⁇ primary hepatocytes
  • mice were maintained at 22°C with a 12-hour dark, 12-hour light schedule with free access to water and housed in individually-ventilated cages and cared for according to United Kingdom Animals (Scientific Procedures) Act (1986).
  • the mice used were on mixed C57BL/6- C57BL/6NT background. Animals were fed either a normal chow diet (20% protein, 75% carbohydrate, 5% fat) or high fat diet (D 12492; 20% protein, 20% carbohydrate and 60% fat) for 16 weeks.
  • Serum levels of insulin, leptin, adiponectin and free fatty acids were measured using ELISA kits (CrystaChem Inc., for insulin and Millipore for the others).
  • Glucose tolerance tests were performed by ip injection of 2g glucose/kg after an overnight fast and 4h fasting for the HFD fed mice.
  • Insulin tolerance tests were performed by ip injection of 0.75U/kg of recombinant human insulin after an overnight fast and 4h fasting for the HFD fed mice.
  • the pyruvate challenge (PTT) was performed by injecting 2 g/kg of pyruvate (Sigma-Aldrich) ip after an 18 h fast. Mice have been injected with insulin (lU/kg) by intraperitoneal injection.
  • MEFs were labeled with 0.6 mCi/ml [ 32 P]orthophosphate during 8h in a phosphate-free N-2- hydroxyethylpiperazine-N -2-ethanesulfonic acid-Tyrode buffer (pH 6.5) at 37°C.
  • ImM insulin stimulation
  • reaction was stopped by addition of chloroform/methanol (vol/vol) containing 0.6 N HC1, and lipids were immediately extracted and analyzed by a combination of thin-layer chromatography and high-performance liquid chromatography (HPLC) as described previously (Gratacap MP J Biol Chem. 1998 Sep 18;273(38):24314-21.
  • Protein extract was resolved by SDS-PAGE before transfer onto PVDF membrane and incubation over-night at 4°C with the following antibodies : anti- phospho-ser 473 Akt antibody (Cell SignalingTechnology).
  • PDK-C2a pl70 and ⁇ 3 ⁇ -02 ⁇ antibodies was from BD Biosciences (BD #61 1046 and 61 1342, respectively); anti ⁇ ⁇ ⁇ and pl l06were from Santa Cruz (SC #602 and #7176, respectively); anti pi 1 Oct and vps34 antibody were from Cell Signalling Technology (CST #4249 and #9145, respectively).
  • Aiiti- phospho-Thr389 p70S6K and anti phospho-ser240/244 S6 antibodies were from CST. Antigen-specific binding of antibodies was visualised using an ECL detection system.
  • hematoxylin and eosin (H&E) staining was performed on 5 um paraffin sections of tissues fixed overnight in 4% phosphate-buffered paraformaldehyde at 4 °C.
  • H&E hematoxylin and eosin staining
  • cryosections were fixed with 4% PFA in PBS at room temperature for 15 min andwashed again with PBS and stained with Oil Red O (0.5% w/isopropanol, diluted 3 :2 in PBS) for 1 h at room temperature. Stained sections were rinsed in 60% isopropanol, followed by deionized water and mounted in Vectashield.
  • each adipocyte was measured in at least 300 cells of representative sections per fat pad (epididymal WAT) per mouse (4 per genotype) was determined using Image J software (NIH). Then mean value was designated as in index of the cell size.
  • PI3K assays using Ptdlns as a lipid substrate were performed as previously described (Chaussade et al.Biochem. J. (2007) 404 (449 ⁇ -58).
  • Vps34 activity is analysed/?? vitroby measuring the transfer of the gamma phosphate of 32 P- labelled ATP to the phosphatidyinositol (Ptdlns or PI) lipid followed by binding of the product to nitrocellulose membranes or thin layer chromatography plates. The capacity of test agents to modulate this activity is analysed/?? vitro.
  • High-throughput fluorescence polarization protocol is also used to test the effect of test agents on Vps34 kinase activity.
  • Vps34 inactivation impacts on endocytosis and autophagy, which can be monitored by commercially cell-based imaging assays (for example from Biotek and Millipore). The capacity of test agents to modulate endocytosis and/or autophagy is analysed using such a cell- based assay.
  • Vps34 inhibitors may then be screened for their capacity to rescue cell lethality.
  • Vps34 Stable cell lines that are homozygous for kinase-dead Vps34 are also created, in order to probe for cellular off-target effects of Vps34-selective inhibitors.
  • Assays to probe cell-based functions of class I and III PI3 isoforms are known in the art.
  • Example 16 Rodent-based assays for Vps34 inhibitors.
  • the time and dose-dependent impact of the vps34 inhibitors is tested on blood glucose levels under fasting conditions in mice. This is complemented by glucose and insulin tolerance assays (GTT and ITT), followed by metabolic analysis (measurement of lipoproteins and adipokines - leptin, adiponectin, resistin- and analysis of the glycaemia control with a euglycaemic-hyperinsulinaemic clamps) to further assess improved insulin sensitivity and determine potential side-effects.
  • GTT and ITT glucose and insulin tolerance assays
  • metabolic analysis measurement of lipoproteins and adipokines - leptin, adiponectin, resistin- and analysis of the glycaemia control with a euglycaemic-hyperinsulinaemic clamps
  • a mass assay is set up to determine the tissue levels of the Vps34 lipid product PtdIns3P in primary tissues in vivo. This provides a direct readout of target inhibition in vivo.
  • P-proteomics technology allows label-free quantitation of phosphorylation in primary tissues. Primary hepatocytes and liver may be to develop robust in vivo vps34-dependent phosphorylation response/biomarkers. The effect of test agents on vps34-dependent cellular phosphorylation may then be assessed.
  • Vps34 inhibition is expected to reduce blood glucose levels under starvation and under high-fat diet conditions, and alter various disease markers.
  • the impact of vps34 inhibition is also tested in more advanced models of insulin-resistance related disease in rodents, including (1) protection from high fat-diet- induced liver steatosis; (2) improvement of metabolic disease parameters in insulin-resistant mouse models, such as ob/ob or db/db mice, andlipodistrophy mouse models.
  • Characterisation involves measurement of key hormones and blood parameters (such as lipoproteins and adipokines (leptin, adiponectin, resistin) and analysis of the glycaemia control with a euglycaemic-hyperinsulinaemic clamps.
  • hormones and blood parameters such as lipoproteins and adipokines (leptin, adiponectin, resistin) and analysis of the glycaemia control with a euglycaemic-hyperinsulinaemic clamps.

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Abstract

La présente invention concerne un procédé permettant d'identifier des agents utiles dans le traitement et/ou la prévention d'une maladie associée à la résistance à l'insuline et/ou à l'intolérance au glucose, ledit procédé de criblage comprenant une étape qui consiste à rechercher l'aptitude d'un agent test à inhiber la voie de signalisation Vps34 et/ou la voie de signalisation ΡΙ3Κ-02β . La présente invention porte également sur un animal non humain transgénique qui comprend une mutation dans le gène codant Vps34 ou ΡΙ3Κ-C2β, de sorte que le site actif soit inactivé.
PCT/GB2012/052905 2011-11-24 2012-11-23 Procédé de criblage WO2013076501A2 (fr)

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WO2017140841A1 (fr) 2016-02-19 2017-08-24 Sprint Bioscience Ab Composés 6-aryl-4-morpholin-1-yl-pyridone utilisés pour le traitement du cancer et du diabète
WO2017140843A1 (fr) 2016-02-19 2017-08-24 Sprint Bioscience Ab Composés 6-hétérocyclyl-4-morpholin-4-ylpyridine-2-one utilisés pour le traitement du cancer et du diabète
WO2019038384A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés d'azaindolylpyridone et de diazaindolylpyridone
WO2019038389A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés pyridinamine-pyridone et pyrimidinamine-pyridone
WO2019038387A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés de pyridylpyridone
WO2019038390A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés de morpholinylpyridone

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KR102401450B1 (ko) 2013-06-17 2022-05-24 도이체스크레브스포르슝스젠트룸스티프퉁데스외펜트리헨레크츠 전사 인자 tsc22d4의 억제제를 통한 인슐린 저항성의 치료
KR20160021106A (ko) * 2013-06-17 2016-02-24 도이체스크레브스포르슝스젠트룸스티프퉁데스외펜트리헨레크츠 전사 인자 tsc22d4의 억제제를 통한 인슐린 저항성의 치료
EP2816356A1 (fr) * 2013-06-17 2014-12-24 Ruprecht-Karls-Universität Heidelberg Traitement de la résistance à l'insuline par des inhibiteurs du facteur de transcription TSC22D4
US9879258B2 (en) 2013-06-17 2018-01-30 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Treatment of insulin resistance through inhibitors of transcription factor TSC22D4
WO2014202602A1 (fr) * 2013-06-17 2014-12-24 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Traitement de la résistance à l'insuline par des inhibiteurs du facteur de transcription tsc22d4
JP2016532098A (ja) * 2013-06-17 2016-10-13 ドイチェス クレープスフォルシュングスツェントルム シュティフトゥング デス エッフェントリッヒェン レヒツ 転写因子ｔｓｃ２２ｄ４の阻害剤によるインスリン耐性の治療
EP3072969A1 (fr) 2015-03-23 2016-09-28 DKFZ Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts Facteur de transcription tsc22d4 ciblant des séquences d'oligonucléotides pour le traitement d'une résistance à l'insuline
WO2017140841A1 (fr) 2016-02-19 2017-08-24 Sprint Bioscience Ab Composés 6-aryl-4-morpholin-1-yl-pyridone utilisés pour le traitement du cancer et du diabète
WO2017140843A1 (fr) 2016-02-19 2017-08-24 Sprint Bioscience Ab Composés 6-hétérocyclyl-4-morpholin-4-ylpyridine-2-one utilisés pour le traitement du cancer et du diabète
WO2019038387A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés de pyridylpyridone
WO2019038389A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés pyridinamine-pyridone et pyrimidinamine-pyridone
WO2019038390A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés de morpholinylpyridone
WO2019038384A1 (fr) 2017-08-23 2019-02-28 Sprint Bioscience Ab Composés d'azaindolylpyridone et de diazaindolylpyridone
EP4001278A1 (fr) 2017-08-23 2022-05-25 Sprint Bioscience AB Composés diazaindolylpyridone azaindolylpyridone et
EP4056556A1 (fr) 2017-08-23 2022-09-14 Sprint Bioscience AB Composés pyridylpyridone
EP4056569A1 (fr) 2017-08-23 2022-09-14 Sprint Bioscience AB Composés de morpholinylpyridone
EP4169581A1 (fr) 2017-08-23 2023-04-26 Sprint Bioscience AB Composés pyridinamine-pyridone et pyrimidinamine-pyridone

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