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WO2011006040A2 - Procédés pour traiter une maladie rénale polykystique (pkd) ou d’autres maladies de formation de kystes - Google Patents

Procédés pour traiter une maladie rénale polykystique (pkd) ou d’autres maladies de formation de kystes Download PDF

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WO2011006040A2
WO2011006040A2 PCT/US2010/041481 US2010041481W WO2011006040A2 WO 2011006040 A2 WO2011006040 A2 WO 2011006040A2 US 2010041481 W US2010041481 W US 2010041481W WO 2011006040 A2 WO2011006040 A2 WO 2011006040A2
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hdac5
hdac
activator
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WO2011006040A3 (fr
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Rong Li
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Stowers Institute for Medical Research
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Stowers Institute for Medical Research
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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
    • A61K31/16—Amides, e.g. hydroxamic acids

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  • the present invention relates, inter alia, to methods for treating or ameliorating the effects of a polycystic disease, such as for example, polycystic kidney disease (PKD).
  • PPD polycystic kidney disease
  • sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. ⁇ 1.52(e)(5).
  • PTD Polycystic kidney disease
  • ADPKD Autosomal Dominant Polycystic Kidney Disease
  • Polycystin-1 and polycystin-2 are components of a receptor-calcium channel complex that has been proposed to play a mechanosensory role on the epithelial lumenal surface in mammalian kidneys.
  • loss of polycystin functions leads to development of polycystic kidneys.
  • cyst formation and the associated symptoms may be caused by loss of polycystin function.
  • nephron formation relies on coordinated regulation of cell proliferation, cell polarity, differentiation and apoptosis.
  • Epithelial cell organization is disrupted in ADPKD kidneys, where numerous, fluid-filled cysts develop as a result of aberrant cell proliferation, loss of planar cell polarity and transepithelial fluid secretion, and changes of epithelial cell polarity and cytoskeleton (Grantham, 2003; Wilson and Goilav, 2007).
  • PC-1 is a large G-protein-coupled receptor-like protein with a complex array of functions, and has been shown to bind polycystin-2 (PC-2), a TRP calcium channel, through a COOH-terminal coiled-coil region (Boletta and Germino, 2003).
  • PC-1 and PC-2 localize to a number of cellular compartments, such as the primary cilia, microtubule-based structures that extend from the apical surface of epithelial cells into the tubule lumen (Berbari et al., 2009; Eley et al., 2005 ), as well as other epithelial surfaces, and PC-2 associates prominently with the ER (reviewed in Wilson and Goilav, 2007).
  • the cilia localization has gained most attention as physical bending of the primary cilia or fluid flow across the apical surface of epithelial cells caused an increase in intracellular Ca 2+ (Praetorius and Spring, 2001 ). Later studies showed that polycystins are required for the fluid flow induced Ca 2+ influx, suggesting that these proteins, possibly through their association with cilia, function as mechanosensors (Grimm et al., 2002; Nauli et al., 2003). However, conditional inactivation of genes required for ciliogenesis in adult mice did not lead to rapid development of cysts despite the loss of primary cilia (Davenport et al., 2007; Patel et al., 2008). These findings cast doubts on whether the loss of mechanosensory function mediated by the primary cilia is responsible for cyst formation.
  • the present invention is directed, inter alia, to treating and/or ameliorating the effects of PKD and related conditions by regulating HDAC pathway modulators.
  • one embodiment of the present invention is a method of treating or ameliorating an effect of a polycystic disease. This method comprises administering to a patient in need thereof an amount of a modulator of a histone deacetylase (HDAC) pathway, which amount is sufficient to treat or ameliorate an effect of a polycystic disease.
  • HDAC histone deacetylase
  • Another embodiment of the present invention is a method of treating or ameliorating an effect of a polycystic kidney disease (PKD).
  • PPD polycystic kidney disease
  • This method comprises administering to a patient in need thereof an amount of an HDAC inhibitor (HDACi) that is sufficient to treat or ameliorate an effect of PKD.
  • HDACi HDAC inhibitor
  • a further embodiment of the present invention is a method of treating or ameliorating an effect of a polycystic kidney disease (PKD). This method comprises administering to a patient in need thereof an amount of an HDAC5 inhibitor that is sufficient to treat or ameliorate an effect of PKD.
  • PPD polycystic kidney disease
  • Figure 1 shows that fluid flow induced HDAC5 phosphorylation in MEK cells.
  • Figure 1A shows an immunoblot analysis of HDAC5 phosphorylation using a phospho-specific antibody against phosphorylated serine 489 of HDAC5 after P/cc/7 +/+ and Pk ⁇ T 1' MEK cells were stimulated with fluid flow at 0.2 ml/min for up to 4 hours.
  • Figure 1 B shows quantification of HDAC5 phosphorylation in the above experiment, normalized first against actin and then against the to values.
  • Figure 1 C shows an immunoblot analysis of HDAC5 phosphorylation in response to fluid flow for 4 hours in Pkd1 siRNA or control lentivirus transduced MEK cells.
  • Figure 1 D shows the effects of a PKC activator (12-myristate-13-acetate (PMA)), a PKC inhibitor (GO6983), a calcium ionophore (ionomycin (“iono”)) and a calcium channel blocker (GdCIs) on HDAC5 phosphorylation without or with fluid flow in MEK cells.
  • PMA Sigma Aldrich
  • GO6983 Sigma Aldrich
  • ionomycin was used at 1 ⁇ M
  • GdC ⁇ (Sigma Aldrich) was used at 20 ⁇ M.
  • Figure 2 shows that fluid flow induced HDAC5 nuclear export in MEK cells.
  • Figure 2A shows Pkd1 +/+ and PkdV 1' MEK cells co-transfected with FLAG- MEF2C (red) and HDAC5-GFP (green). These cells were stimulated with fluid flow at 0.2 ml/min for 30 min (no flow controls are also included). Then, the cells were fixed, stained and imaged using confocal microscopy. Scale bar: 16 ⁇ m.
  • Figure 2B shows time-lapse images of HDAC5-GFP translocation stimulated by fluid flow. Time (minutes) after flow initiation is indicated on each panel. The graph shows quantification of fluorescence ratio (nuclear/cytosolic) over time.
  • FIG. 2C shows the quantification of HDAC5-GFP distribution in the cytosol or nucleus in Pkd1 +/+ and PkdV 1' cells with or without fluid flow stimulation, in the absence or presence of GdCI 3 , Rab8 T22N transfection (blocking cilia assembly), and HDAC5 S250/S489A mutations as indicated. Percentages of cells (over total) with HDAC5-GFP in the nucleus or cytosol or in both compartments are shown.
  • Figure 2D shows quantification of HDAC5-GFP distribution in the cytosol or nucleus in cells treated with DMSO (solvent control), 100 nM PMA, 10 ⁇ M GO 6983 in the absence or presence of fluid flow as indicated.
  • DMSO solvent control
  • FIG. 3 shows that missing in metastasis (MIM) is a transcriptional target of MEF2C and HDAC5.
  • Figure 3A shows that a chromatin immunoprecipitation (ChIP) experiment demonstrating that MEF2C and HDAC5 bind to the promoter region of MIM.
  • FLAG-MEF2C or FLAG-HDAC5 were transfected into wild-type MEK cells, which were either resting (-flow) or stimulated with fluid flow for 0.5 hour (+flow), and an anti-FLAG antibody was used for ChIP.
  • PCR was performed using primers in the MIM promoter region as explained in Example 1 below.
  • Input is DNA template before ChIP.
  • Ctrl is ChIP using MEK cells transfected with the empty FLAG vector.
  • FIG. 3B shows that MEF2C is important for normal MIM expression in MEK cells. RNAi knock-down of MEF2C in Pkd1 +/+ MEK cells led to reduced MIM expression, quantified by qPCR. The results shown are averages of three experiments. Ctrl: cells transfected with control siRNA. Error bars indicate ⁇ s.d. Figure 3C shows the reconstitution of MIM reporter expression in Pk ⁇ T 1' MEK cells.
  • Luciferase activity averaged from three experiments are shown for Pk ⁇ V 1' cells tranfected with either empty vector (Ctrl), MEF2C alone, GATA6 alone, or MEF2C and GATA6 together. ** indicates P ⁇ 0.05 compared with control. Error bars indicate standard deviation (s.d.).
  • Figure 3D shows that HDAC5 negatively regulates MIM expression in MEK cells, lmmunoblot and quantification show that HDAC5 S250/489A -GFP overexpression reduced MIM expression in Pkd1 +/+ MEK cells.
  • FIG. 4 shows that kidney specific knock-out of Mef2C resulted in epithelial tubule dilations/cysts.
  • Kidneys from 5-6 month old Mef2C loxp/loxP Sglt2-Cre mice and their congenic wild-type controls were fixed in 4% formaldehyde, sectioned (5 ⁇ m thick), and stained with hematoxylin and eosin ( Figures 4A-4G), tubule markers (Figure 4H) or Ki67 ( Figure 4I).
  • Figure 4A shows a representative section of wild-type kidney.
  • Figure 4B shows a representative kidney section from a Mef2C loxp/loxp Sglt2-Cre mouse.
  • Figure 4C shows a magnified region corresponding to the box in Figure 4A.
  • Figure 4D shows a magnified region corresponding to the box in Figure 4B, which reveals a broad distribution of dilated tubules (arrows).
  • Figure 4E shows a 4OX magnified cortex region in wild-type mouse kidney, showing normal epithelial tubules.
  • Figure 4F shows a 4OX magnified cortex region of a Mef2C loxp/loxP Sglt2-Cre kidney, revealing dilated tubules and small cysts with flat lining cells (arrows).
  • Figure 4G shows glomerular cysts (arrows) in Mef2C loxp/loxP Sglt2-Cre kidney.
  • Figure 4H shows staining of wild-type and Mef2C loxp/loxP Sglt2-Cre kidney sections against various tubule markers (green): Na + /K + ATPase a-1 (which indicates the location of distal tubule), LTA (which indicates the location of proximal tubule) and DBA (which indicates the location of collecting duct).
  • DAPI stain reveals the location of nuclei (blue). Arrows point to dilated tubules/cysts.
  • Figure 4I shows representative Ki67 staining of wild-type and Mef2C loxp/loxP Sglt2-Cre kidney sections, showing increased cell proliferation in the mutant kidney section. Scale bars: Figures 4A and 4B, 1000 ⁇ m; Figures 4C and 4D, 200 ⁇ m; Figures 4E, 4F, 4G, and 4I, 50 ⁇ m; Figure 4H, 100 ⁇ m.
  • Figure 5 shows that MIM is a transcriptional target of MEF2C and is required for normal renal epithelial organization.
  • Figure 5A shows the results of a ChIP experiment, which demonstrates that MEF2C binds to the promoter region of MIM that contains a MEF2C-binding site.
  • Flag-MEF2C was transfected into wild- type MEK cells, which were stimulated with or without fluid flow, and an anti-flag antibody was used for ChIP. PCR was performed using primers that flank the predicted MEF2C binding sites. Input, DNA template before ChIP. MEK cells transfected with empty flag vector were used as control (Ctrl).
  • Figure 5B shows that MEF2C is important for normal MIM expression in MEK cells.
  • the results shown are averages of three experiments. Cells transfected with control siRNA were used as control (Ctrl). Error bars indicate ⁇ standard deviation (s.d.)
  • Figure 5C shows reconstitution of MIM reporter expression in Pk ⁇ T 1' MEK cells. Luciferase activity averaged from three experiments are shown for PkdV 1' cells transfected with either empty vector (Ctrl), MEF2C alone, GATA6 alone, or MEF2C and GATA6 together. Double asterisks ⁇ ** ) indicate P ⁇ 0.05 compared with control. Error bars indicate ⁇ s.d.
  • Figures 5D and 5E show representative histology sections of wild-type (Figure 5D) and MM 1' (Figure 5E) kidneys from 5 month old mice.
  • Figure 5F shows a magnified image of the boxed region in Figure 5E.
  • Figure 5G shows a magnified image of cyst lining cells in the boxed region in Figure 5F.
  • Figures 5H and 51 show representative Ki67 staining of wild-type (Figure 5H) and M/Af A kidney sections ( Figure 51), indicating increased cell proliferation in the mutant kidney section.
  • Figure 6 shows the effects of Hdac ⁇ genetic or chemical inactivation on cyst formation in Pkd2 ⁇ / ⁇ embryonic kidneys.
  • Figures 6A-6E show representative embryonic kidney sections from E18.5 embryos of different genotypes.
  • Figure 6A shows a kidney section from a P/cc/2 +/+ Hdac5 +/+ embryo
  • Figure 6B shows a kidney section from a P/cc/2 +/+ Hdac ⁇ ' ⁇ embryo
  • Figure 6C shows a kidney section from a Pkd2 ⁇ ' ⁇ Hdac ⁇ '1' embryo
  • Figure 6D shows a kidney section from a P/cc/2 "A Hdac5 +/ ⁇ embryo
  • Figure 6E shows a kidney section from a Pkd2 ⁇ / ⁇ Hdac5 +/+ embryo.
  • Figures 6F-6H show representative histology sections of E18.5 Pkd2 +/+ and Pkd2 ⁇ ' ⁇ embryonic kidneys from pregnant mothers injected (from 10.5 dpc to 18.5 dpc) with TSA or control solvent (DMSO).
  • Figure 6F Pkd2 +/+ from TSA-treated mother
  • Figure 6G P/cc/2 "7" from TSA-treated mother
  • Figure 6H Pkd2 ⁇ ' ⁇ from DMSO-treated mother.
  • Figure 6I shows quantification of the percentage of cystic areas over total kidney section areas of different genotypes or treatment as indicated. The middle section of each kidney was quantified for all mice under each condition. Shown are mean and SEM of all sections quantified for each condition.
  • Figures 6J, 6K and 6L show magnified images of the box region in Figures 6F, 6G, and 6H respectively. Lining cells around a normal tubule (Figure 6J), small cyst (Figure 6K) and large cyst (Figure 6L) are indicated by arrows.
  • Figure 6M shows immonublots demonstrating reduced MIM and MEF2C expression in 18.5 dpc Pkd2 ⁇ / ⁇ embryonic kidneys.
  • Figure 6N shows that TSA stimulates MIM and MEF2C expression in Pkd2 ⁇ / ⁇ embryonic kidneys. Scale bars: 6A-6H, 200 ⁇ m; 6J-6L, 50 ⁇ m.
  • FIG. 7 shows a schematic diagram depicting a pathway that connects polycystins and calcium signaling to HDAC5 and MEF2C-based transcription activation in the suppression of epithelial cyst formation.
  • Fluid flow through lumens of renal tubules activates the polycystin-1 and 2 receptor-calcium channel complex.
  • the observed increase of HDAC5 and MEF2C transcript levels in the microarray analysis may be explained by feedback loops, depicted with dotted lines.
  • Figure 8 shows the confirmation of HDAC5, MIM and PKD1 antibody specificities.
  • Figure 8A is an immunoblot analysis of protein extracts from Hdac +/+ and Hdac ⁇ ' ⁇ adult mouse kidneys with the anti-HDAC5 antibody.
  • Figure 8B is an immunoblot analysis of protein extracts from M/M +/+ and MM 1' adult mouse kidneys with the anti-MIM antibody.
  • Figure 8C is an immunoblot analysis of protein extracts from PkdV 1' and PkdV 1' MEK cells with the antibody against PC-1.
  • Figure 9 shows the flow experiment setup.
  • Figure 9A shows that fluid flow was applied using a peristaltic pump at a pump rate of 0.2 ml/min across a 3.5 cm (diameter) dish or 6-well plate.
  • Figure 9B shows a diagram of computed flow field distribution based on the experiment setup. The smaller circle represents the coverslip on which cells were grown for imaging experiments. For all other experiments cells were grown directly on the culture dish.
  • Figure 9C shows that the cells were grown to confluency and then differentiated for 1 -2 days.
  • DAPI blue
  • phalloidin red
  • acetylated tubulin antibody (green) stainings show nuclei (blue), cell boundaries (F-actin, red) and cilia (green), respectively.
  • Scale bars 16 ⁇ m.
  • Figure 10 shows the quantification of intra-cellular calcium rise in response to fluid flow using the genetically coded calcium sensor GCAMP2.
  • GCAMP2 transfected MEK cells were stimulated with fluid flow at 20s after start of imaging (arrow in top panel). The first peak of GCAMP2 fluorescence occurs 1 -2 min after the start of fluid flow. The bottom panel shows a cell imaged long enough to demonstrate 3-4 calcium transients.
  • Figure 11 shows the results of an microarray analysis to identify genes induced by fluid flow in a PC-1 dependent manner.
  • Figure 11A is a heat map showing genes that were induced by flow in pkd1 +/+ but not in pkd1 " ⁇ MEK cells.
  • Figure 11 B shows functional categorization of the genes induced by fluid flow in a PC1 -dependent manner, as revealed by expression microarray analysis of Pkd1 +/+ and Pkd1 " ' " MEK cells.
  • Figure 12 shows the quantification of HDAC5 phosphorylation against total HDAC5.
  • Figures 12A and 12B show immunoblots of total HDAC5 in response to fluid flow for 4 hours in Pkd1 +/+ ( Figure 12A) and Pkd1 " ⁇ ( Figure 12B).
  • Figure 12C shows quantification of HDAC5 phosphorylation (top band) against total HDAC5 over time.
  • Figure 13 shows the knockdown of PC-1 in MEK cells using Pkd1 siRNA lentivirus. Pkd1 siRNA reduced PC1 expression by 85% compared to PC-1 in control virus transduced cells.
  • Figure 14 shows HDAC5, MEF2C, GATA6 and MIM expression quantification.
  • Figure 14A shows that MIM expression was increased in Pkd1 +/+ MEK cells after fluid flow with microarray and Northern quantification.
  • Figures 14B and 14C show that compared with Pkd1 +/+ MEK cells, the expression of HDAC5, MEF2C, GATA6 and MIM in Pkd1 " ⁇ cells was reduced dramatically as demonstrated by microarray ( Figure 14B) and qPCR ( Figure 14C) quantification.
  • Figure 15 shows Sglt2-Cre expression in kidneys of new-born mice from the cross of Sglt2-Cre with the Gt(ROSA)26Sortm1 Sor reporter (Jackson laboratory).
  • Figure 15A shows frozen-section X-GaI staining of a Sglt2-Cre/R26R kidney.
  • Figure 15B shows LTA chromogen staining on a frozen-section X-GaI staining slide.
  • Figure 15C shows Na7K + ATPase a-1 chromogen staining on a frozen-section X-GaI staining slide.
  • Figure 15D shows DBA chromogen staining on a frozen-section X-GaI staining slide. Scale bars: 50 ⁇ m.
  • Figure 16 shows MIM knock-out mouse generation and MIM expression pattern in mouse kidney.
  • Figure 16A is a diagram showing genetrap disruption of the MIM gene. It was confirmed by RACE that the exon 1 of the trapped MIM gene abuts the splice acceptor sequence/Engrailed-2 (En2) exon after transcription and intron splicing.
  • Figures 16B-E show MIM expression in embryonic and adult kidneys of MIM +/" mice demonstrated using X-GaI staining.
  • FIG. 16B-16C Whole-mount X-GaI staining of E16.5 MIM +/" embryonic kidneys
  • Figures 16D-16E frozen- section X-GaI staining of MIM +/" and MIM +/+ adult kidneys
  • Figures 16C and 16E are images at a higher resolution than that in Figures 16B and 16D. These figures show that MIM is expressed in collecting ducts (CD) , tubules (T) and glomeruli (G) in E16.5 kidney (as shown in Figures 16B and 16C), and in glomeruli and tubules in adult kidney ( Figures 16D and 16E). Scale bars: B 1 C and E,
  • Figures 17A and 17B show that HDAC inhibitors inhibit cAMP-induced cysts in mouse embryonic kidney organ cultures.
  • FIG. 18 shows that GFP-Rab8 T22N blocked cilia formation in transfected MEK cells.
  • the location of GFP-Rab8 T22N is shown in green, the location of acetylated tubulin (cilia marker) is shown in red, and the location of the DAPI- stained nuclei are shown in blue. Note the presence of cilia in surrounding non- transfected cells. Scale bar: 16 ⁇ m.
  • Figure 19A shows an immunoblot analysis demonstrating decreased expression of HDAC5, MEF2C and MIM in human ADPKD cyst lining cells compared to normal human kidney epithelial cells (WT).
  • Figure 19B shows a quantitative analysis of the immunoblot shown in Figure 19A. Actin was used as the standard for normalization.
  • Figure 19C is an immunoblot analysis using a phospho-specific antibody against HDAC5 demonstrating fluid flow-stimulated phosphorylation of
  • HDAC5 in normal human kidney epithelial cells (WT) but not in ADPKD cyst lining cells.
  • Figure 19D shows the amount of HDAC5 under control and flow conditions.
  • the quantifications are based on the immunoblot shown in Figure 9C, after normalization using actin as the standard.
  • Figure 20 shows a quantitative analysis of the immunoblot shown in
  • FIG. 21 shows that MIM " ' " mice are viable but exhibit polycystic kidneys by 5 months.
  • Figure 22 shows that animals treated with SAHA had significantly lower blood urea nitrogen (BUN) levels, compared to vehicle treated animals, indicating improved kidney function.
  • BUN blood urea nitrogen
  • SAHA blood urea nitrogen
  • Figure 23 shows that SAHA treatment of pkd2 ws25/" adult mice drastically improved renal tubular epithelial morphology.
  • top to bottom show images of a representative SAHA-treated ( Figures 23A, 23C, and 23E) or control (Ctrl, Figures 23B, 23D, and 23F) mouse kidney with increasing magnification. Small cysts or tubule dilations are widely observed in the control images whereas SAHA-treated images show well organized, nearly normal epithelial morphology.
  • One embodiment of the present invention is a method of treating or ameliorating an effect of a polycystic disease.
  • This method comprises administering to a patient in need thereof an amount of a modulator of a histone deacetylase (HDAC) pathway, which amount is sufficient to treat or ameliorate an effect of a polycystic disease.
  • HDAC histone deacetylase
  • Treatment means preventing, suppressing, repressing, or completely eliminating a disease, such as, e.g., a polycystic disease.
  • Preventing a disease involves administering a composition of the present invention to a patient, such as a mammal, preferably a human, prior to onset of the disease.
  • Suppressing a disease involves administering a composition of the present invention to a patient after induction of a disease but before its clinical appearance.
  • Repressing a disease involves administering a composition of the present invention to a patient after clinical appearance of the disease.
  • “Ameliorating" an effect of a disease means that at least one symptom of the disease is eliminated or decreased.
  • Non-limiting examples of a polycystic disease according to the present invention include polycystic kidney disease (PKD), polycystic liver disease (PLD), polycystic ovary syndrome (PCOS), pancreatic cysts, cancer, or combinations thereof.
  • PPD polycystic kidney disease
  • PLD polycystic liver disease
  • PCOS polycystic ovary syndrome
  • pancreatic cysts cancer, or combinations thereof.
  • the polycystic disease is autosomal dominant polycystic kidney disease (ADPKD) or autosomal recessive polycystic kidney disease (ARPKD).
  • ADPKD autosomal dominant polycystic kidney disease
  • ARPKD autosomal recessive polycystic kidney disease
  • carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma) esophagus, stomach, gall bladder, cervix, thyroid, renal, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B- cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma;
  • a "modulator of an HDAC pathway” means any agent that regulates the activity of any member of the HDAC pathway, which results in, e.g., decreased expression of HDAC, increased nuclear export of HDAC, increased retention of HDAC in the cytoplasm, or activation of transcription factors such as MEF2.
  • a modulator of the HDAC pathway may act upstream or downstream of HDAC in the HDAC pathway.
  • members of an HDAC pathway include heterothmehc G proteins, phospholipase C, protein kinase C, protein kinase D, inositol 1 , 4, 5 triphosphate receptor (IP3R), calcium calmodulin kinase II, salt inducible kinase 1 (Sik1 ), 14-3-3 proteins, MEF2, GATA, and MIM.
  • heterothmehc G proteins include heterothmehc G proteins, phospholipase C, protein kinase C, protein kinase D, inositol 1 , 4, 5 triphosphate receptor (IP3R), calcium calmodulin kinase II, salt inducible kinase 1 (Sik1 ), 14-3-3 proteins, MEF2, GATA, and MIM.
  • heterothmehc G-proteins which are composed of an alpha subunit, a beta subunit, and a gamma subunit, are activated by receptors that are associated with them.
  • the G- proteins activate phospholipase C, which cleaves phosphatidylinositol 4,5- bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1 ,4,5-triphosphate (IP 3 ).
  • IP3 actives receptors such as IP3R.
  • IP3R One of the effects of activating IP3R is an increase in calcium level, which activates calcium calmodulin kinase II.
  • Calcium calmodulin kinase Il is one of the kinases that phosphorylate HDAC5 at positions which result in dissociation of HDAC5 from MEF2C and translocation of HDAC5 from the nucleus to the cytosol.
  • Other kinases that are able to effect translocation of HDAC5 include Sik1 and protein kinase D. Protein kinase D may be activated by protein kinase C, and DAG can activate both protein kinase C and protein kinase D.
  • HDAC5 Once HDAC5 is in the cytosol, it binds to 14-3-3 proteins.
  • the dissociation and translocation of HDAC5 allow for MEF2C and its co-factors, such as GATA6, to transcribe target genes such as MIM.
  • the modulator modulates a class Il
  • modulating means altering the function of or expression level of a protein, e.g., a protein in the HDAC pathway, including but not limited to lowering or increasing the expression level of a protein (either at the transcription stage or the translation stage), altering the sequence of such a protein (by, e.g., mutation, pre-translational or post-translational modification or otherwise), or inhibiting or activating such a protein (by, e.g., binding, phosphorylation, glycosylation, translocation or otherwise).
  • modulation may be achieved genetically or pharmacologically.
  • a "Class Il HDAC” means the phylogenetic class of HDACs that share domains with similarity to the yeast deacetylase HDA1.
  • Representative non-limiting members of the HDAC class Il family include HDAC4, 5, 6, 7, 9, and 10.
  • the class Il HDAC is HDAC5.
  • the modulator inhibits HDAC5.
  • “inhibit” and “inhibiting” and like terms when used with respect to HDAC5, means a decrease of the function of HDAC5 as a repressor of gene transcription.
  • Non-limiting examples of how the function of HDAC5 may be decreased include decreasing expression of HDAC5, increasing nuclear export of HDAC5, and increasing retention of HDAC5 in the cytoplasm.
  • the modulator of the HDAC pathway is an HDAC inhibitor (HDACi).
  • HDACi is selected from the group consisting of nucleic acids, polypeptides, polysaccharides, small organic or inorganic molecules, and combinations thereof.
  • HDACi HDACi according to the present invention
  • Entinostat Bossen, Germany
  • KD-5170 Kalypsys, San Diego, California
  • KD-5150 Kalypsys, San Diego, California
  • KLYP-278 Kalypsys, San Diego, California
  • KLYP-298 Kalypsys, San Diego, California
  • KLYP-319 Kalypsys, San Diego, California
  • KLYP-722 Kalypsys, San Diego, California
  • CG- 200745 CrystalGenomics, Inc., Seoul, South Korea
  • Avugane TopicoTarget AS, K ⁇ benhavn, Denmark
  • SB-939 S * BIO, Singapore
  • ARQ-700RP ArQuIe, Woburn, Massachusetts
  • KA-001 Kearus Therapeutics, Chilworth, Hampshire, United Kingdom
  • MG-3290 MethodhylGene, Montreal, Quebec, Canada
  • PXD-118490 LEO- 80140
  • the HDACi is selected from the group consisting of hydroxamic acids, short chain fatty acids, cyclic tetrapeptides/epoxides, benzamides, electrophilic ketone derivatives, and combinations thereof.
  • a “hydroxamic acid” means a chemical compound sharing the same functional group represented by the formula -CO-NH-OH.
  • hydroxamic acids according to the present invention also contain a phenyl group, and such hydroxamic acids may be represented by the following general structure:
  • Non-limiting examples of hydroxamic acids according to the present invention include trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), pyroxamide (suberoyl-3-aminopyhdineamide hydroxamic acid), azelaic-1- hydroxamate-9-anilide (AAHA), compound 13, compound 14, CRA-024781 (Pharmacyclics, Sunnyvale, California), bombesin-2 (BB2) receptor antagonist, JNJ- 16241199 (Johnson & Johnson, Langhorne, Pennsylvania), Oxamflatin ((2E)-5-[3- [(phenylsufonyl) aminol phenyl]-pent-2-en-4-ynohydroxamic acid), CG-1521 (Errant Gene Therapeutics, LLC, Chicago, Illinois), CG-1255 (Errant Gene Therapeutics, LLC, Chicago, Illinois), SK-7068 (ln2Gen/SK Chemical Co., Suweon, Korea),
  • Pyroxamide may be synthesized according to Butler et al., "Inhibition of
  • BB2 receptor antagonists are described in, for example, Ashwood et al., "The first high affinity non-peptide gastrin-releasing peptide (BB2) receptor antagonist,” Bioorg Med Chem Lett, 8(18): 2589-94 (1998).
  • Oxamflatin and Scriptaid are commercially available from, e.g., Enzo Life Sciences, Inc. (Plymouth Meeting, PA).
  • CBHA is commercially available from, e.g., Cayman Chemical Co. (Ann Arbor, Ml).
  • the structures of compounds 13, 14, 48, 49, 50, 51 , and 70 are as follows:
  • a "short chain fatty acid” means a chemical compound containing a carboxylic acid and containing an aliphatic chain of less than 20 carbons, preferably eight or fewer carbons, for example, 2-6 carbons.
  • a "carboxylic acid” means a chemical compound containing a functional group represented by the formula -COOH.
  • An "aliphatic chain” means a functional group containing only carbon and hydrogen atoms that is not an aromatic. Aromatic functional groups (and other functional groups), however, may be present in addition to the aliphatic chain.
  • the aliphatic chain may be branched or unbranched, and it may be saturated or unsaturated.
  • Short chain fatty acids may be represented by the following general structure:
  • Non-limiting examples of short chain fatty acids according to the present invention include butyrate, phenylbutyrate, valporic acid (VPA), PivanexTM (Titan Pharmaceuticals, Inc.), AN-1 (Titan Pharmaceuticals, Inc.), tributyrin, compound G1 , pivaloyloxymethyl butyrate (AN-9, Titan Pharmaceuticals, Inc.), hyaluronic acid butyric acid ester (HA-But), pharmaceutically acceptable salts thereof, and combinations thereof.
  • VPA valporic acid
  • PivanexTM Tian Pharmaceuticals, Inc.
  • AN-1 Tian Pharmaceuticals, Inc.
  • tributyrin compound G1
  • pivaloyloxymethyl butyrate AN-9, Titan Pharmaceuticals, Inc.
  • HA-But hyaluronic acid butyric acid ester
  • Butyrate and phenylbutyrate are commercially available from, e.g.,
  • Tributyrin is commercially available from, e.g., Colonial Scientific (Richmond, VA).
  • HA-But may be obtained by the esterification of butyric acid (BA), the smallest HDAC inhibitor, with hyaluronic acid (HA) and may be prepared as disclosed in Speranza et al., "Hyaluronic acid butyric esters in cancer therapy” Anticancer Drugs. 16(4):373-9. (2005).
  • the structure of compound G1 is as follows:
  • Compound G1 was disclosed in Balakin et al., "Histone Deacetylase Inhibitors in Cancer Therapy: Latest Developments, Trends and Medicinal Chemistry Perspective” Anti-Cancer Agents in Medicinal Chemistry, 7:576-592 (2007).
  • a cyclic tetrapeptide means a cyclic compound containing four amino acids, which may be natural, synthetic, or a modification, or a combination of natural and synthetic amino acids, joined by peptide bonds. Cyclic tetrapeptides may be represented by the following general structure:
  • An "epoxide” means a compound containing a cyclic ether with three ring atoms, or a compound which may be prepared from such a cyclic ether.
  • a compound containing a cyclic ether with three ring atoms may be represented by the following general structure:
  • Non-limiting examples of a cyclic tetrapeptide/epoxide according to the present invention include Apicidine (Merck & Co., Inc., Whitehouse Station, NJ), Trapoxin-A (cyclo((S)-phenylalanyl-(S)-phenylalanyl-(R)-pipecolinyl-(2S,9S)-2- amino-8-oxo-9,10-epoxydecanoyl), Trapoxin-B (cyclo[(S)-phenylalanyl-(S)- phenylalanyl-(R)-prolyl-2- amino-8-oxo-9,10-epoxydecanoyl-]), cyclic hydroxamic acid-containing peptide 1 (CHAP-1 ), CHAP-31 , CHAP-15, chlamidocin, HC-toxin, WF-27082B (Fujisawa Pharmaceutical Company, Ltd., Osaka, Japan), Romidep
  • Trapoxins A and B may be isolated, e.g., from the culture broth of
  • CHAP-1 and CHAP-15 may be synthesized, e.g., as disclosed in Furumai et al., "Potent histone deacetylase inhibitors built from thchostatin A and cyclic tetrapeptide antibiotics including trapoxin,” Proc. Natl. Acad. Sci., 98:87-92 (2001 ).
  • CHAP-31 may be synthesized, e.g., as disclosed in Komatsu et al., "Cyclic Hydroxamic-acid- containing Peptide 31 , a Potent Synthetic Histone Deacetylase Inhibitor with Antitumor Activity.”
  • HC-toxin and Depudesin are commercially available from, e.g., Enzo Life Sciences, Inc.
  • Spiruchostatin A may be synthesized, e.g., according to Yurek-George et al., "Total synthesis of spiruchostatin A, a potent histone deacetylase inhibitor” Journal of the American Chemical Society, 126(4): 1030-1031 (2004).
  • the cyclostellettamines may be isolated as described in Fusetani et al., "Three new cyclostellettamines, which inhibit histone deacetylase, from a marine sponge of the genus Xestospongia.” Bioorg Med Chem Lett. 14(10):2617-20 (2004).
  • the structure of compound D1 is as follows:
  • benzamide means a compound containing the following functional group:
  • Preferred benzamides according to the present invention may be represented by the following general structure:
  • Non-limiting examples of benzamides include MS-27-275 (Schering
  • Tacedinaline N-acetyldinaline
  • ITF-2357 Italfarmaco, Cinisello Balsamo, Italy
  • HDAC-42 N-hydroxy-4-(3- methyl-2-phenyl-butyrylamino)benzamide
  • MGCD-0103 MethodhylGene Inc., Montreal, Quebec, Canada
  • PX- 117794 TopicoTarget AS, K ⁇ benhavn, Denmark
  • compound 37 Belinostat (TopoTarget AS, K ⁇ benhavn, Denmark)
  • Compound 39 sulfonamide hydroxamic acid, pharmaceutically acceptable salts thereof, and combinations thereof.
  • Tacedinaline is commercially available from, e.g., Medical Isotopes,
  • HDAC-42 may be synthesized, e.g., according to Lu et al., "Structure-based optimization of phenylbutyrate-derived histone deacetylase inhibitors" J Med Chem. 48(17):5530-5 (2005).
  • the structures of compounds 27, 29, 30, 37, and 39 are as follows:
  • an "electrophilic ketone derivative” means a compound containing a ketone group that may be an acceptor of electron pairs in a chemical reaction.
  • electrophilic ketone derivatives include, e.g., a trifluoromethyl ketone or an alpha-keto amide.
  • a "trifluoromethyl ketone” means a compound containing a functional group represented by the formula -COCF 3 .
  • An alpha-keto amide means a compound containing a functional group represented by the following structure:
  • the HDACi is selected from the group consisting of TSA,
  • the modulator regulates the activity of a myocyte enhancer factor 2 (MEF2) protein, an heterotrimehc G protein, a phospholipase C (PLC), a protein kinase C (PKC), a protein kinase D (PKD), an inositol 1 , 4, 5 triphosphate receptor (IP3R), a calcium calmodulin kinase Il (CaMKII), a salt inducible kinase 1 (Sik1 ), or a 14-3-3 polypeptide.
  • MEF2 protein is a MEF2C protein.
  • the modulator is a MIM activator, a
  • GATA activator a MEF2 activator, an heterotrimeric G protein activator, a PLC activator, a PKC activator, a PKD activator, an IP3R activator, a CaMKII activator, a
  • the GATA activator is a GATA6 activator.
  • An "activator" of a polypeptide according to the present invention increases the function of the polypeptide by, e.g., increasing the expression of the polypeptide or by binding to the polypeptide.
  • an heterothmehc G protein activator include mastoparan, fluoroaluminate (AIF4 " ), guanosine 5'-O-(3-thiotriphosphate), G-protein bg (beta gamma) binding peptide mSIRK, MAS 7, Pasteurella multocida toxin, and combinations thereof.
  • Fluoroaluminate may be prepared, e.g., according to Jeschke et al., "Fluoroaluminate Induces Activation and Association of Src and Pyk2 Tyrosine Kinases in Osteoblastic MC3T3-E1 Cells" J. Biol. Chem., 273 (18):11354-11361 (1998).
  • Mastoparan, guanosine 5'-O-(3-thiothphosphate), G-protein bg (beta gamma) binding peptide mSIRK, MAS 7, and Pasteurella multocida toxin are commercially available from, e.g., Merck Chemical Ltd. (Nottingham, United Kingdom).
  • a non-limiting example of a PLC activator is m-3M3FBS, which is commercially available from, e.g., Sigma (St. Louis, MO).
  • Non-limiting examples of a PKC activator include 12-myristate 13- acetate (PMA), phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-didecanoate (PDD), farnesyl thiothazole, ingenol 3,20-dibenzoate, (-)-7-octylindolactam V, n-heptyl-5- chloronaphthalene-1 -sulfonamide, mezerein, ingenol mebutate (Peplin, Emeryville, California), KAI-1455 (KAI Pharmaceuticals, South San Francisco, California), KAI- 9706 (KAI Pharmaceuticals, South San Francisco, California), bryostatin-1 nanosome (Aphios, Woburn, Massachusetts), bryostatin-1 , Sapintoxin A, 8-octyl- benzolactam-V9, 1 -hexylindolactam-V10, phorbol 12-myristate 13
  • PMA, PDBu, PDD, farnesyl thiothazole, ingenol 3,20-dibenzoate, (-)-7- octylindolactam V, mezerein, bryostatin-1 , Sapintoxin A, and farnesyl thiotriazole are commercially available from, e.g., Enzo Life Sciences, Inc.
  • 8-octyl-benzolactam-V9 may be synthesized, e.g., as disclosed by Nakagawa et al., "Design and synthesis of 8-octyl-benzolactam-V9, a selective activator for protein kinase C epsilon and eta" J Med Chem. 49(9):2681-8 (2006).
  • n-heptyl-5-chloronaphthalene-i -sulfonamide is commercially available from, e.g., Sigma.
  • 1-hexylindolactam-V10 may be made, e.g., as disclosed by Yanagita et al., "Synthesis, Conformational Analysis, and Biological Evaluation of 1 -Hexylindolactam-V10 as a Selective Activator for Novel Protein Kinase C Isozymes" J Med Chem. 51 (1 ):46-56 (2008).
  • Phorbol 12-myristate 13-acetate is commercially available from, e.g., Calbiochem (San Diego, CA).
  • Cholesterol sulfate is commercially available from, e.g., Axxora LLC (San Diego, CA).
  • Daphnoretin may be isolated according to Ko et al., "Daphnoretin, a new protein kinase C activator isolated from Wikstroemia indica CA. Mey” Biochem J. 295 (Pt 1 ):321 -7 (1993).
  • DiC8 is commercially available from, e.g., Echelon Biosciences Incorporated (Salt Lake City, UT).
  • IP3R activator is adenophostin, which is commercially available from A.G. Scientific, Inc. (San Diego, CA).
  • Another embodiment of the present invention is a method of treating or ameliorating an effect of a polycystic kidney disease (PKD).
  • PPD polycystic kidney disease
  • This method comprises administering to a patient in need thereof an amount of an HDAC inhibitor (HDACi) that is sufficient to treat or ameliorate an effect of PKD.
  • HDACi HDAC inhibitor
  • the HDACi is selected from the group consisting of hydroxamic acids, short chain fatty acids, cyclic tetrapeptides/epoxides, benzamides, electrophilic ketone derivatives, and combinations thereof. Hydroxamic acids, short chain fatty acids, cyclic tetrapeptides/epoxides, benzamides, and electrophilic ketone derivatives are as described above.
  • the HDACi is selected from the group consisting of Entinostat (Bayer AG, Leverkusen, Germany), KD-5170 (Kalypsys, San Diego, California), KD-5150 (Kalypsys, San Diego, California), KLYP- 278 (Kalypsys, San Diego, California), KLYP-298 (Kalypsys, San Diego, California), KLYP-319 (Kalypsys, San Diego, California), KLYP-722 (Kalypsys, San Diego, California), CG-200745 (CrystalGenomics, Inc., Seoul, South Korea), Avugane (TopoTarget AS, K ⁇ benhavn, Denmark), SB-939 (S * BIO, Singapore), ARQ-700RP (ArQuIe, Woburn, Massachusetts), KA-001 (Karus Therapeutics, Chilworth, Hampshire, United Kingdom), MG-3290 (MethylGene, Montreal, Quebec, Canada), PXD-118490
  • the HDACi inhibits a class Il
  • the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD) or autosomal recessive polycystic kidney disease (ARPKD).
  • ADPKD autosomal dominant polycystic kidney disease
  • ARPKD autosomal recessive polycystic kidney disease
  • a further embodiment of the present invention is a method of treating or ameliorating an effect of a polycystic kidney disease (PKD).
  • PPD polycystic kidney disease
  • This method comprises administering to a patient in need thereof an amount of an HDAC5 inhibitor that is sufficient to treat or ameliorate an effect of PKD.
  • the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD) or autosomal recessive polycystic kidney disease (ARPKD).
  • HDAC5 inhibitors are as disclosed herein.
  • nucleic acid or "oligonucleotide” or “polynucleotide” used herein mean at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions.
  • a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequences.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids may be synthesized as a single stranded molecule or expressed in a cell (in vitro or in vivo) using a synthetic gene. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
  • the nucleic acid may also be a RNA such as a mRNA, tRNA, shRNA, siRNA, Piwi-interacting RNA, pri-miRNA, pre-miRNA, miRNA, or anti-miRNA, as described, e.g., in U.S. Patent Application Nos. 11/429,720, 11/384,049, 11/418,870, and 11/429,720 and Published International Application Nos. WO 2005/116250 and WO 2006/126040.
  • a RNA such as a mRNA, tRNA, shRNA, siRNA, Piwi-interacting RNA, pri-miRNA, pre-miRNA, miRNA, or anti-miRNA
  • the nucleic acid may also be an aptamer, an intramer, or a spiegelmer.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), disclosed in U.S. Pat. No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions may be double- stranded or single-stranded, and may include deoxyhbonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups ⁇ e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • intramer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-
  • RNA or other left-handed nucleotide derivatives or nucleotide-like molecules.
  • Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • a nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages.
  • Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those disclosed in U.S. Pat. Nos. 5,235,033 and 5,034,506. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within the definition of nucleic acid.
  • the modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule.
  • Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g.
  • the 2'-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is CrC 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as disclosed in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Application Publication No. 20050107325.
  • Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as disclosed in U.S. Patent Application Publication No. 20020115080. Additional modified nucleotides and nucleic acids are disclosed in U.S. Patent Application Publication No. 20050182005. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • LNA locked nucleic acids
  • Additional modified nucleotides and nucleic acids are disclosed in U.S. Patent Application Publication No. 20050182005. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g.
  • peptide means a linked sequence of amino acids, which may be natural, synthetic, or a modification, or combination of natural and synthetic.
  • the term includes antibodies, antibody mimetics, domain antibodies, lipocalins, targeted proteases, and polypeptide mimetics.
  • the term also includes vaccines containing a peptide or peptide fragment intended to raise antibodies against the peptide or peptide fragment.
  • Antibody as used herein includes an antibody of classes IgG, IgM,
  • the antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.
  • the antibody may also be a chimeric antibody.
  • the antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art.
  • the antibody may be conjugated with a chemical moiety.
  • the antibody may be a human or humanized antibody.
  • antibody-like molecules are also within the scope of the present invention.
  • Such antibody-like molecules include, e.g., receptor traps (such as entanercept), antibody mimetics (such as adnectins, fibronectin based "addressable” therapeutic binding molecules from, e.g., Compound Therapeutics, Inc.), domain antibodies (the smallest functional fragment of a naturally occurring single-domain antibody (such as, e.g., nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr 15;64(8):2853-7)).
  • receptor traps such as entanercept
  • antibody mimetics such as adnectins, fibronectin based "addressable” therapeutic binding molecules from, e.g., Compound Therapeutics, Inc.
  • domain antibodies the smallest functional fragment of a naturally occurring single-domain antibody (such as, e.g., nanobodies; see, e.g., Cortez
  • Suitable antibody mimetics generally can be used as surrogates for the antibodies and antibody fragments described herein.
  • Such antibody mimetics may be associated with advantageous properties ⁇ e.g., they may be water soluble, resistant to proteolysis, and/or be nonimmunogenic).
  • peptides comprising a synthetic beta-loop structure that mimics the second complementarity- determining region (CDR) of monoclonal antibodies have been proposed and generated. See, e.g., Saragovi et al., Science. Aug. 16, 1991 ;253(5021 ):792-5.
  • Peptide antibody mimetics also have been generated by use of peptide mapping to determine "active" antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, so as to design a peptide mimic containing antigen contact residues from multiple CDRs. See, e.g., Cassett et al., Biochem Biophys Res Commun. JuI. 18, 2003;307(1 ):198-205. Additional discussion of related principles, methods, etc., that may be applicable in the context of this invention are provided in, e.g., Fassina, Immunomethods. October 1994;5(2):121 -9.
  • peptide includes targeted proteases, which are capable of, e.g., substrate-targeted inhibition of post-translational modification such as disclosed in, e.g., U.S. Patent Application Publication No. 20060275823.
  • peptide further includes anticalins.
  • Anticalins can be screened for specific binding to a modulator of the HDAC pathway such as HDAC5, fragments of a modulator of the HDAC pathway, or variants of a modulator of the HDAC pathway such as HDAC5.
  • Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001 ) J. Biotechnol. 74:257- 275).
  • the protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the amino acid substitutions can be made using methods known in the art, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
  • a polypeptide mimetic is a molecule that mimics the biological activity of a polypeptide, but that is not peptidic in chemical nature. While, in certain embodiments, a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids), the term peptidomimetic may include molecules that are not completely peptidic in character, such as pseudo-peptides, semi-peptides, and peptoids. Examples of some peptidomimetics by the broader definition (e.g., where part of a polypeptide is replaced by a structure lacking peptide bonds) are described below.
  • peptidomimetics may provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in a polypeptide. As a result of this similar active-site geometry, the peptidomimetic may exhibit biological effects that are similar to the biological activity of a polypeptide.
  • polypeptides may exhibit two undesirable attributes, i.e., poor bioavailability and short duration of action.
  • Peptidomimetics are often small enough to be both orally active and to have a long duration of action.
  • stability, storage and immunoreactivity for polypeptides may be reduced with peptidomimetics.
  • Polypeptides having a desired biological activity can be used in the development of peptidomimetics with similar biological activities.
  • Techniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure, shape or reactivity.
  • the development of peptidomimetics can be aided by determining the tertiary structure of the original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling.
  • “Derivative,” as used with respect to a peptide or polypeptide, means a peptide or polypeptide different other than in primary structure (amino acids and amino acid analogs).
  • derivatives may differ by being glycosylated, one form of post-translational modification.
  • peptides or polypeptides may exhibit glycosylation patterns due to expression in heterologous systems. If at least one biological activity is retained, then these peptides or polypeptides are derivatives according to the invention.
  • derivatives may include fusion peptides or fusion polypeptides having a covalently modified N- or C- terminus, PEGylated peptides or polypeptides, peptides or polypeptides associated with lipid moieties, alkylated peptides or polypeptides, peptides or polypeptides linked via an amino acid side-chain functional group to other peptides, polypeptides or chemicals, and additional modifications as would be understood in the art.
  • Fragment may include fusion peptides or fusion polypeptides having a covalently modified N- or C- terminus, PEGylated peptides or polypeptides, peptides or polypeptides associated with lipid moieties, alkylated peptides or polypeptides, peptides or polypeptides linked via an amino acid side-chain functional group to other peptides, polypeptides or chemicals, and additional modifications as would be understood in the art.
  • Fragment may include
  • fragment when used in the context of a peptide or polypeptide, may mean a peptide of from about 5 to about 150, about 6 to about 100, or about 8 to about 50 amino acids in length. e. Small organic or inorganic molecules
  • small organic or inorganic molecule includes any chemical or other moiety, other than polysaccharides, polypeptides, and nucleic acids, that can act to affect biological processes.
  • Small molecules can include any number of therapeutic agents presently known and used, or can be synthesized in a library of such molecules for the purpose of screening for biological function(s).
  • Small molecules are distinguished from macromolecules by size.
  • the small molecules of this invention usually have a molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1 ,000 Da, most preferably less than about 500 Da.
  • Organic compounds include without limitation organic compounds, peptidomimetics and conjugates thereof.
  • organic compound refers to any carbon-based compound other than macromolecules such as nucleic acids and polypeptides.
  • organic compounds may contain calcium, chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements.
  • An organic compound may be in an aromatic or aliphatic form.
  • Non-limiting examples of organic compounds include acetones, alcohols, anilines, carbohydrates, mono-sacchahdes, di-saccharides, amino acids, nucleosides, nucleotides, lipids, retinoids, steroids, proteoglycans, ketones, aldehydes, saturated, unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters, ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds, imidizoles, and phenols.
  • An organic compound as used herein also includes nitrated organic compounds and halogenated (e.g., chlorinated) organic compounds.
  • Preferred small molecules are relatively easier and less expensively manufactured, formulated or otherwise prepared. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules may be placed in tight association with macromolecules to form molecules that are biologically active and that have improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are favorable to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy characteristics of the chemical entity. f. Polysaccharides
  • polysaccharides means polymeric carbohydrate structures, formed of repeating units (either mono- or di-saccharides) joined together by glycosidic bonds.
  • the units of mono- or di-saccharides may be the same or different.
  • Non-limiting examples of polysaccharides include starch, glycogen, cellulose, and chitin.
  • a modulator of an HDAC pathway according to the present invention may be administered to treat a polycystic or other related disease in a number of different formulations.
  • a soluble modulator of an HDAC pathway may be administered in the form of a composition comprising purified protein in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers may be nontoxic to recipients at the dosages and concentrations employed.
  • compositions may entail combining the modulator of an HDAC pathway with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients.
  • antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins
  • chelating agents such as EDTA, glutathione and other stabilizers and excipients.
  • Neutral buffered saline or saline mixed with nonspecific serum albumin may be appropriate diluents.
  • a modulator of an HDAC pathway may be formulated as a lyophilizate using appropriate excipient solutions ⁇ e.g., sucrose) as diluents.
  • a modulator of an HDAC pathway according to the present invention may be formulated as a tablet or lozenge in a conventional manner.
  • tablet or lozenge capsules may contain conventional excipients such as a binding compound, filler, lubricant, disintegrant or wetting compound.
  • the binding compound may be syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone.
  • the filler may be lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, or sorbitol.
  • the lubricant may be magnesium stearate, stearic acid, talc, polyethylene glycol, or silica.
  • the disintegrant may be potato starch or sodium starch glycollate.
  • the wetting compound may be sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
  • a modulator of an HDAC pathway according to the present invention may also be a liquid formulation which may be an aqueous or oily suspension, solution, emulsion, syrup, or elixir.
  • a modulator of an HDAC pathway according to the present invention may also be formulated as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain an additive such as a suspending compound, emulsifying compound, nonaqueous vehicle or preservative.
  • the suspending compound may be a sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, or a hydrogenated edible fat.
  • the emulsifying compound may be lecithin, sorbitan monooleate, or acacia.
  • the nonaqueous vehicle may be an edible oil, almond oil, fractionated coconut oil, oily ester, propylene glycol, or ethyl alcohol.
  • the preservative may be methyl or propyl p-hydroxybenzoate, or sorbic acid.
  • a modulator of an HDAC pathway according to the present invention may also be formulated as a suppository, which may contain suppository bases which may be cocoa butter or glycerides.
  • a modulator of an HDAC pathway according to the present invention may also be formulated for inhalation, which may be in a form such as a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane.
  • a modulator of an HDAC pathway according to the present invention may also be a transdermal formulation comprising an aqueous or nonaqueous vehicle which may be a cream, ointment, lotion, paste, medicated plaster, patch, or membrane.
  • a modulator of an HDAC pathway according to the present invention may also be formulated for parenteral administration, which may be by injection or continuous infusion.
  • Formulations for injection may be in the form of a suspension, solution, or emulsion in oily or aqueous vehicles, and may contain a formulation compound such as a suspending, stabilizing, or dispersing compound.
  • a modulator of an HDAC pathway according to the present invention may also be provided in a powder form for reconstitution with a suitable vehicle such as sterile, pyrogen-free water.
  • a modulator of an HDAC pathway according to the present invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
  • a modulator of an HDAC pathway according to the present invention may be formulated with a suitable polymeric or hydrophobic material (as an emulsion in an acceptable oil, for example), ion exchange resin, or as a sparingly soluble derivative (as a sparingly soluble salt, for example).
  • a modulator of an HDAC pathway according to the present invention may also be formulated as a liposome preparation.
  • the liposome preparation may comprise a liposome which penetrates the cells of interest or the stratum corneum, and fuses with the cell membrane, resulting in delivery of the contents of the liposome into the cell.
  • the liposome may be as disclosed in U.S. Patent No. 5,077,211 of Yarosh, U.S. Patent No. 4,621 ,023 of Redziniak et al., or U.S. Patent No. 4,508,703 of Redziniak et al.
  • Other suitable formulations may employ niosomes.
  • Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.
  • a modulator of an HDAC pathway according to the present invention may be administered orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, or combinations thereof.
  • Parenteral administration may be intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, or intraarticular.
  • a modulator of an HDAC pathway according to the present invention may also be administered in the form of an implant, which allows slow release of the agent as well as a slow controlled i.v. infusion.
  • subcutaneous injection may be used because many modulators of an HDAC pathway are destroyed by the digestive process or otherwise ineffective if ingested. 4. Dosage of A Modulator of an HDAC Pathway
  • An amount sufficient to treat or ameliorate an effect of a polycystic or other related disease may vary with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and ultimately may be determined by the attendant physician.
  • the amount or dose of a modulator of an HDAC pathway according to the present invention that may be administered to a patient may also vary depending on a variety of factors known in the art ⁇ e.g., species, sex, age, weight, condition of the patient, desired response, nature of the condition, metabolism, severity of disease, side-effects).
  • the desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more subdoses per day. Multiple doses often are desired, or required.
  • a number of factors may lead to the modulator of an HDAC pathway being administered at a wide range of dosages.
  • the dosage of the modulator of an HDAC pathway of the present invention may be given at a relatively lower dosage.
  • the use of a targeting substituent may allow the necessary dosage to be relatively low.
  • a modulator of an HDAC pathway according to the present invention may be administered at a relatively high dosage, which may be due to a factor such as low toxicity, high clearance, or low rates of processing.
  • the dosage of a modulator of an HDAC pathway according to the present invention may be from about 1 ng/kg to about 1000 mg/kg.
  • doses employed for adult human treatment typically may be in the range of 0.0001 mg/kg/day to 0.0010 mg/kg/day, 0.0010 mg/kg/day to 0.010 mg/kg/day, 0.010 mg/kg/day to 0.10 mg/kg/day, 0.10 mg/kg/day to 1.0 mg/kg/day, 1.00 mg/kg/day to about 200 mg/kg/day.
  • the dosage may be about 1 mg/kg/day to about 100 mg/kg/day, such as, e.g., 2-10 mg/kg/day, 10-50 mg/kg/day, or 50-100 mg/kg/day.
  • the dosage of the modulator of an HDAC pathway also may be about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg.
  • the disclosed method of treating or ameliorating the effect of a polycystic disease with a modulator of an HDAC pathway according to the present invention may be based upon murine models.
  • those skilled in the art study human PKD using a PKD loss of function mutant mouse because various strains of mutant mice exhibit disease symptoms that parallel both ADPKD and ARPKD. See, e.g., "Polycystic Kidney Disease," retrieved from the National Institute of Diabetes and Digestive and Kidney Diseases website.
  • Mouse models for polycystic diseases may be possible, in part, because the molecular pathways underlying the pathology of PKD cyst formation are shared by both mice and humans.
  • Polycystin-1 and polycystin-2 which are products of the PKD-1 and PKD-2 genes, respectively, may exhibit the same kidney cell localization in mice and humans (Cano et al., Development, 2004;131 :3457-67).
  • PKD mouse mutants aim at understanding the genetic and nongenetic mechanisms involved in cyst formation, and at discovering candidate compounds that inhibit cyst formation in PKD mutants.
  • a modulator of an HDAC pathway such as for example, an HDACi, may be used to inhibit cyst formation associated with PKD.
  • HDAC5 Phospho-Ser498 human antibody was obtained from
  • Confluent mouse embryonic kidney (MEK) cells were cultured for 1-2 additional days in the absence of interferons to induce optimal differentiation (Nauli et al., 2003).
  • Hanks' Balanced Salt Solution with 20.0 mM HEPES buffer (pH 7.4) and 1 % bovine serum albumin was used for calcium imaging and cell culture media for the other experiments.
  • Fluid flow was applied at 0.2 ml/min using a peristaltic pump in 35 mm dishes or 6-well plates, with tubings connected through two 18 Gauge needles at opposing sides of a well (Figure 9A).
  • Time-lapse imaging was performed on an Axiovert 200M inverted microscope (Karl Zeiss Microimaging Inc., Thornwood, NY) equipped with a 20x/0.8 NA Plan-Apochromat objective, a Pecon XL-3 environmental chamber (PeCon GmbH, Erbach, Germany) and an AxioCam HSm r1.1 digital monochrome camera (Karl Zeiss).
  • PMA (12- myhstate 13-acetate) (Sigma-Aldhch, St.
  • Biotinylated cRNA was prepared from 5 ⁇ g total RNA using the standard Affymetrix one-cycle target labeling protocol (Affymetrix, Santa Clara, CA). Samples were assayed using Affymetrix GeneChip Mouse Genome 430 2.0 arrays consisting of probe sets representing over 39,000 transcripts based on the Unigene database (Build 107 from June 2002). Data was analyzed using the R statistical environment. Affymetrix CEL files were processed and normalized using RMA (Irizarry et al., 2003). The linear modeling package Limma (Azzam et al., 2004) was used to derive gene expression coefficients using genotype and presence or absence of fluid flow as factors, and to identify differentially expressed genes. The data was also examined and characterized with GCOS (Affymethx) and Partek (St. Louis, MO) ANOVA.
  • GCOS Affymethx
  • Partek St. Louis, MO
  • FLAG-MEF2C (SEQ ID NO: 1 ) was made by inserting the Mef2C coding sequence with stop codon (TAA) (SEQ ID NO: 2) into p3XFLAG-Myc-CMVTM- 24 (Sigma-Aldrich) cut by Kpnl and BamHI.
  • HDAC5-GFP (SEQ ID NO: 3) was made by inserting the HDAC5 coding sequence (without stop codon) (SEQ ID NO: 4) into pAcGFP1-N1 (Clonetech, Mountain View, CA), cut by Xhol and Hindlll.
  • Chromatin was prepared from FLAG-MEF2C or FLAG-HDAC5- expressing cells as described in Upstate protocol (MCPROTO407, available from Milipore's TECH LIBRARY, Milipore, Billerica, MA), with a cross-linking time of 15 min at 25°C and sonication to an average length of 200-700 bp. ChIP was performed using anti-Flag M2 monoclonal antibody (Sigma). Samples were analyzed by PCR.
  • Primers for MIM forward, 5'- CCAGCCAGGGTTGCCATAGCCAC -3' (SEQ ID NO: 5) and reverse, 5 1 - TGACTCTTGCAAACGGTTCAATTAGGTGC - 3 1 (SEQ ID NO:6), which flank a 1 kb region in the MIM promoter that harbor a potential MEF2 binding motif.
  • MEF2C siRNA was introduced into the wild-type MEK cells.
  • Four different MEF2C RNAi sequences were pooled (SEQ ID NOs: 7-10).
  • the primers and the probes used for qPCR are set forth below in Table 1.
  • a luciferase assay was carried out 54 hours post-transfection using a commercial kit (Promega Corp., Madison, Wl) and an Orin microplate luminometer
  • oligonucleotides were transfected by using the DharmaFECT siRNA transfection reagent (Dharmacon Inc..
  • VIRHD/P/siPKD13297 VIRHD/P/siPKD13297
  • control lentivirus Battini et a/., 2008
  • ES cells Boygenomics, Los Angeles, CA
  • ES cells carrying a trap in intron 1 of the MIM gene, were injected into the inner cell mass of C57BL6 blastocysts.
  • Chimeric mice with a high (65%) contribution to coat color from 129 were bred for germline transmission.
  • Mef2c loxP/loxP mice originally provided by John Schwarz (Albany
  • the duration of the calcium peaks was in the range of 10-20 seconds, similar to that from single-cell measurements in a recent study using a Fura-2 calcium sensor in Dolichos biflorus agglutinin (DBA)-positive MEK cells (Li et al., 2007).
  • HDAC5 histone deacetylase-5
  • MEF2C myocyte enhancer factor-2C
  • a well-known post-translational mechanism for stress-induced MEF2C activation in myocardial cells involves phosphorylation and nuclear export of HDAC5, which represses MEF2-dependent transcripts in the resting state (McKinsey et al., 2000).
  • Kinases activated by stress-induced Ca 2+ increase, such as CaM kinase and protein kinase C or D, phosphorylate HDAC5 at two 14-3-3 binding sites, an event that leads to disruption of HDAC5-MEF2C interaction and translocation of HDAC5 from the nucleus to the cytosol (McKinsey et al., 2002).
  • HDAC5 dissociation from MEF2C frees a binding site for the histone acetyl transferase p300/CBP, an activator of MEF2C.
  • Mutually exclusive interaction of MEF2C with HDAC5 or p300/CBP generates a binary switch for activation of MEF2C target genes (McKinsey et al., 2001 ).
  • HDAC5 phosphorylation was determined using a phospho-specific antibody against HDAC5 Ser489 site (equivalent of Ser498 of human HDAC5) (Bossuyt et al., 2008). While HDAC5 phosphorylation level was low prior to fluid flow onset, an increase in phosphorylation was observed 30 minutes after the start of fluid flow and maintained for several hours thereafter ( Figures 1A and 1 B). The increase in HDAC5 phosphorylation was accompanied by an increase in the level of total HDAC5 ( Figures 12A, C), consistent with the flow-induced gene expression of HDAC5 observed in the microarray analysis (Table 2).
  • Fluid flow-induced HDAC5 phosphorylation and increase in protein level were absent in Pk ⁇ T 1' MEK cells ( Figures 1A, 1 B, 12B, and 12C) and blocked in wild-type MEK cells by Pkd1 siRNA (Battini et al., 2008) ( Figure 1C), which reduced PC-1 expression by 85% ( Figure 13), suggesting a direct role for PC-1 in the observed response.
  • HDAC5 phosphorylation was also blocked by gadolinium (GdCIs), an inhibitor of stretch- activated cation channels previously shown to inhibit fluid flow-induced calcium response (Praetorius et al., 2003).
  • HDAC5 phosphorylation was induced by the calcium ionophore, ionomycin, in the absence of fluid flow ( Figure 1 D, see also Figure 20 for the quantification of HDAC5 phosphorylation levels under various conditions).
  • PMA an activator of protein kinase C (PKC)
  • PKC protein kinase C
  • FIGs 1 D and 20 show that PC-1 , calcium channel activity and a PKC isoform are required for fluid-flow induced HDAC5 phosphorylation.
  • HDAC5 S250/489A HDAC5 S250/489A
  • Figure 2C HDAC5 nuclear export
  • PKC inhibitor G06983 PKC inhibitor
  • HDAC5 nuclear export was unaffected in cells transfected with Rab8 T22N following fluid flow stimulation (Figure 2C), suggesting that the observed phenomenon, while being PC-1 -dependent, does not require cilia.
  • Figure 2C fluid flow stimulation
  • MIM Missing in Metastasis
  • MIM is a multifunctional regulator of actin cytoskeletal dynamics (Machesky and Johnston, 2007) and was previously implicated in Sonic hedgehog (Shh) signaling (Callahan et a/., 2004).
  • Sh Sonic hedgehog
  • MIM was also among a list of potential targets of MEF2 in the heart, although its function in cardiac myocytes has not been characterized (van Oort et a/., 2006).
  • chromatin immunoprecipitation ChIP was carried out using an anti-FLAG antibody in MEK cells transfected with FLAG-MEF2C with or without fluid flow stimulation.
  • MEF2C siRNA was introduced into wild-type MEK cells.
  • a 2 kb promoter fragment of MIM was cloned upstream of the luciferase reporter gene.
  • the luciferase reporter construct was introduced into PkdV 1' MEK cells, which showed a drastically reduced level of endogenous MEF2C and MIM expression compared to those in Pkd1 +/+ cells, as shown by microarray analysis and qPCR ( Figures 13B- 13C).
  • Transfection of a MEF2C-expressing plasmid into the PkdV 1' cells only minimally stimulated MIM reporter expression.
  • MEF2 members have been shown to function together with cell-type specific GATA transcription factors, which recruit MEF2 proteins to promoters through direct interactions between these proteins (Morin et al., 2000).
  • GATA6 also a flow- induced gene, that showed reduced expression in Pk ⁇ T 1' MEK cells (Table 2 and Figures 13B-13C).
  • GATA6 also a flow- induced gene, that showed reduced expression in Pk ⁇ T 1' MEK cells.
  • Several GATA sites are present within 500 base pairs of the MEF2 binding motif in the MIM promoter.
  • Mef2C-based transcription is important for kidney epithelial organization and proliferation
  • a previously established Mef2C conditional knock-out mouse line was obtained (Vong et al., 2005), because conventional Mef2C knockout resulted in early embryonic lethality (Lin et al., 1997).
  • Kidney-specific disruption of Mef2C was accomplished using a Sglt2 promoter-driven Cre (Rubera et al., 2004), which is expressed in renal tubules and glomeruli ( Figure 15).
  • MIM genetrap embryonic stem cell line was used to generate MIM-deficient mice ( Figure 16A).
  • the genetrap was inserted at intron 1 , truncating MIM after exon 1.
  • X-gal staining of MM 1"7" embryos showed that MIM is expressed abundantly in embryonic heart, spinal cord, liver and the limb bud (data not shown).
  • MIM is expressed in branching collecting ducts, tubules and glomeruli ( Figures 16B-16C).
  • MIM is significantly expressed in renal cortex and weakly expressed in renal medula ( Figures 16D-16E ).
  • HDAC5 Inhibition Suppresses Renal Cyst Formation In Pkd2 " " Mouse Embryos.
  • a prediction based on a pathway where the calcium signal generated by polycytins deactivates HDAC5 is that loss-of-function mutations in HDAC5 should alleviate cyst formation in Pkd2 '/' m ⁇ ce. This possibility was tested by crossing pairs of P/cc/2 +A Hdac ⁇ +/ ⁇ double mutant mice. Pkd2 ⁇ / ⁇ embryos were known to die before or immediately after birth with many large renal cysts (Wu et al., 2000).
  • cyst lining cells exhibited a more normal cuboidal morphology as opposed to the flat morphology of Pkd2 ⁇ ' ⁇ cyst lining cells ( Figures 6J-L).
  • TSA injection did not affect the morphology of renal tubules in P/cc/2 +/+ embryonic kidneys ( Figures 6F and 6J).
  • lmmunoblot analysis showed that MIM and MEF2C expression levels were respectively 10 fold and 3 fold reduced in E18.5 Pkd2 ⁇ ' ⁇ embryonic kidneys compared with those in P/cc/2 +/+ embryonic kidneys ( Figure 6N).
  • Pkd2 ⁇ ' ⁇ embryonic kidneys from TSA-treated mothers showed a 4-fold enhancement of expression of MEF2C and a drastically increased expression of MIM compared with those in Pkd2 ⁇ ' ⁇ kidneys from DMSO-treated mothers ( Figure 6N), suggesting that TSA stimulated the expression of MEF2C target genes.
  • Cardiac hypertrophy in the adult heart is an adaptive mechanism of activation of an embryonic program to improve cardiac pump function in response to an increased workload or mechanical stress (Xu et al., 2006).
  • the similarity between the cardiac hypertrophy pathway and the response to fluid flow in renal epithelial cells raises a question as to whether cystogenesis in ADPKD could in part be an outcome of a lack of the proper response to certain stress or an increase in workload on renal epithelial cells.
  • the nephrons are frequently challenged with fluctuations in fluid shear stress and osmotic pressure, and these changes may be enhanced during certain developmental stages or under certain dietary, hormonal or pathological conditions.
  • Renal hypertrophy is a well known phenomenon that occurs after nephretomy or under other stress conditions (Hostetter, 1995).
  • dDAVP 1 -deamino-8-D- arginine vasopressin
  • MEF2C-based transcriptional activation leads to increased expression of contractile proteins and metabolic enzymes in cardiac tissues. If there is indeed a similar hypertrophic pathway in the kidney, MEF2C may cooperate with other tissue specific transcription factors, such as GATA6, to control the expression of genes involved in strengthening the differentiated state of renal epithelial tubules in response to stress.
  • GATA6 tissue specific transcription factors
  • MIM a founding member of the family of IMD-containing actin binding proteins, as well as another member of this family, BAIAP2L1 (Table 2), were found to be a MEF2C target in epithelial cells.
  • the IMD domain has the ability to induce membrane deformation and actin bundling in cultured cells (Machesky and Johnston, 2007).
  • IMD proteins also possess a COOH- terminal WASP homology-2 (WH2) domain, which binds monomeric actin, and thus may be involved in actin polymerization.
  • WH2 COOH- terminal WASP homology-2
  • the actin cytoskeleton plays important roles in almost every aspect of epithelial cell and tissue organization (Leiser and Molitoris, 1993).
  • Actin structures built through MIM and BAIAP2L1 may serve to strengthen epithelial cell polarity or cell-cell and cell-matrix interactions within epithelial tissues.
  • actin-interacting proteins ⁇ -actinin, tropomyosin, troponin, mDial , etc
  • ⁇ -actinin, tropomyosin, troponin, mDial , etc were found to bind polycystins and some were shown to modulate PC-2 channel activity (Li et al., 2003a; Li et al., 2005a; Li et al., 2003b; Rundle et al., 2004).
  • MIM basal cell carcinoma-enriched gene 4
  • BEG4 basal cell carcinoma-enriched gene 4
  • Shh Sonic hedgehog
  • MEF2C was the only MEF2 member whose expression level was found to increase in response to fluid flow in the microarray analysis
  • MEF2A which functions in parallel with MEF2C in regulating cardiac hypertrophy (Xu et al., 2006), is also expressed in MEK cells (data not shown).
  • the functional redundancy of MEF2C and MEF2A may account for the much milder cystic phenotype in Mef2C knockout kidneys than in polycystin knockout mice.
  • HDAC5 Class Ma HDACs
  • HDAC5 lack intrinsic enzymatic activity and require complex formation with HDAC3, a class I HDAC that is sensitive to TSA, for their transcriptional repression activity
  • HDAC5 Class Ma HDACs
  • MIM MEF2- downstream target genes
  • HDAC5 Inhibitor Suppressed Cyst Formation In cAMP -Induced Cysts In Mouse Embryonic Kidney Organ Cultures
  • Embryonic kidneys were dissected from embryos of C57BL/6 at E15.5 in phosphate buffered saline (PBS), with calcium and magnesium, plus penicillin- streptomycin-glutamine (Invitrogen Corp., Carlsbad, CA).
  • PBS phosphate buffered saline
  • penicillin- streptomycin-glutamine Invitrogen Corp., Carlsbad, CA
  • the dissected kidneys were cultured at 37 0 C in DMEM/F12 media containing 2 mM L-glutamine, 10 mM HEPES (Invitrogen Corp.), 5 mg/ml insulin, 5 mg/ml transferrin, 2.8 mM selenium, 25 ng/ml prostaglandin E1 , 32 pg/ml 2,3,5-thido-L-thyronine (T3) and 250 U/ml penicillin-streptomycin.
  • the kidneys were cultured with or without the HDAC inhibitors in the concentrations shown in Figure 17 for 48 hours. 50 mM 8-bromo- cAMP (Sigma, St. Louis, MO) were then added to the culture medium.
  • the culture was allowed to continue for 5 days.
  • the cultured kidneys were then fixed with 4% paraformaldehyde in PBS for 6 hours, washed with PBS twice for 5 minutes each, and then transferred to 70% ethanol for short-term storage at room temperature or for more extended storage at 4 0 C.
  • the fixed kidney samples were prepared for hematoxylin and eosin staining and lectin staining following histology protocols known in the art.
  • Kidneys were treated with DMSO and 100 ⁇ M cAMP to induce cyst formation or cAMP with various concentrations of TSA or suberoylanilide hydroxamic acid (SAHA) as labeled for the duration of the culture.
  • SAHA suberoylanilide hydroxamic acid
  • FIG 17B E15.5 embryonic kidneys were cultured in vitro and treated with H 2 O and cAMP or valproic acid (VPA) and cAMP for 5 days. Arrows point to cAMP- induced cortical cysts.
  • HDAC5 Expression Is Decreased In Human ADPKD Cyst Lining Cells
  • DMEM/F12 Insulin-Transferrin-Selenium-X.
  • HDAC5 Insulin-Transferrin-Selenium-X.
  • MEF2C Insulin-Transferrin-Selenium-X.
  • MIM Insulin-Transferrin-Selenium-X.
  • the results show that the expression levels of HDAC5, MEF2C and MIM decreased in human ADPKD cyst lining cells compared to normal human kidney epithelial cells ( Figures 19A and B).
  • HDAC5 Inhibitors Improve Kidney Function In A Mouse Model Of PKD
  • PKD ws25/- mjce ( Wu et a/ j 1 998 j were obtained from A
  • PKD WS25/ mice escape the embryonic lethality of the PKD " ⁇ phenotype, but recapitulate the human ADPKD phenotype in that they develop cysts early in life (Wu et al., 1998).
  • SAHA vorinostat
  • vehicle Vehicle
  • PKD1 induces p21 (waf1 ) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2.
  • Calcimimetic inhibits late-stage cyst growth in ADPKD. JAm Soc Nephrol. 20, 1527-1532.
  • Histone-deacetylase inhibitors novel drugs for the treatment of cancer. Nat Rev Drug Discov 1 , 287-299.
  • TRPP2 and TRPV4 form a polymodal sensory channel complex. J Cell Biol.
  • Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice.
  • Polycystin-2 associates with tropomyosin-1 , an actin microfilament component. J MoI Biol 325, 949-962.
  • Alpha-actinin associates with polycystin-2 and regulates its channel activity.
  • Polycystin-2 interacts with troponin I, an angiogenesis inhibitor. Biochemistry 42, 450-457.
  • MIM a multifunctional scaffold protein. J MoI Med 85, 569-576.
  • MEF2 a calcium-dependent regulator of cell division, differentiation and death. Trends Biochem Sci 27, 40-47.
  • PKD2 a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272, 1339-1342.
  • Imaging cellular signals in the heart in vivo Cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proceedings of the National Academy of Sciences of the United States of America 103, 4753-4758.
  • MEF2 activates a genetic program promoting chamber dilation and contractile dysfunction in calcineurin-induced heart failure. Circulation 114, 298-308.
  • the Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development. Development 128, 4623- 4633.
  • HDAC5 and mediates expression of KLF2 and eNOS. Blood.
  • Cyclic AMP activates B-Raf

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Abstract

La présente invention concerne, entre autres, des procédés de traitement ou d'atténuation d'un effet d'une maladie polykystique. Ce procédé comprend l'administration à un patient nécessitant celle-ci d'une quantité d'un modulateur d'une voie d'histone désacétylase (HDAC), qui est suffisante pour traiter ou atténuer un effet d'une maladie polykystique, en particulier une maladie rénale polykystique.
PCT/US2010/041481 2009-07-10 2010-07-09 Procédés pour traiter une maladie rénale polykystique (pkd) ou d’autres maladies de formation de kystes Ceased WO2011006040A2 (fr)

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WO2005065681A1 (fr) * 2003-12-19 2005-07-21 Takeda San Diego, Inc. Derives de n-hydroxy-3-(3-(1h-imidazol-2-yl)-phenyl)-acrylamide et composes associes en tant qu'inhibiteurs d'histone deacetylase pour le traitement du cancer
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WO2014071000A1 (fr) * 2012-10-31 2014-05-08 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Classe d'inhibiteurs de hdac multipliant la population de cellules progénitrices rénales et améliorant le taux de récupération après une insuffisance rénale aigüe
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WO2020037224A1 (fr) * 2018-08-17 2020-02-20 National Health Research Institutes COMPLEXE AHR-ROR-γT UTILISABLE EN TANT QUE BIOMARQUEUR ET CIBLE THÉRAPEUTIQUE POUR LES MALADIES AUTO-IMMUNES ET LES MALADIES ASSOCIÉES À L'IL-17A
CN112771160A (zh) * 2018-08-17 2021-05-07 财团法人卫生研究院 作为用于自体免疫疾病及IL-17相关疾病的生物标记及治疗标靶的AhR-ROR-γt复合体
EP4292591A1 (fr) * 2022-06-14 2023-12-20 Universitätsmedizin Greifswald Composés destinés à être utilisés dans le traitement de maladies rénales
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