WO2014180741A1 - Procédés d'identification de modulateurs d'osbpl7 et utilisation de ces modulateurs pour traiter les maladies associées à osbpl7 - Google Patents

Procédés d'identification de modulateurs d'osbpl7 et utilisation de ces modulateurs pour traiter les maladies associées à osbpl7 Download PDF

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WO2014180741A1
WO2014180741A1 PCT/EP2014/058961 EP2014058961W WO2014180741A1 WO 2014180741 A1 WO2014180741 A1 WO 2014180741A1 EP 2014058961 W EP2014058961 W EP 2014058961W WO 2014180741 A1 WO2014180741 A1 WO 2014180741A1
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osbpl7
alkyl
protein
halogen
group
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PCT/EP2014/058961
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Jeremy Beauchamp
Thomas Hartung
Christian Kemmer
Peter Mohr
Stephan Roever
Matthew Wright
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Publication of WO2014180741A1 publication Critical patent/WO2014180741A1/fr

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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
    • G01N33/5005—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • C07D213/82—Amides; Imides in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • G01N33/5035—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 on sub-cellular localization

Definitions

  • This invention refers to methods for screening and identifying new pharmacophore as binders and functional agonists/antagonists of Oxysterol Binding Associated Protein -Like 7 (OSBPL7). It also refers to the use of pyridine carboxamides as modulators, more particularly molecular antagonists of Oxysterol Binding Associated Protein -Like 7 (OSBPL7). Furthermore, the invention is related to the treatment of diseases associated with OSBPL7 such as diseases associated with detectable expression of OSBPL7 where it is mainly expressed in the pancreatic islets of Langerhans, the heart, the intestine, and the vasculature.
  • diseases associated with OSBPL7 such as diseases associated with detectable expression of OSBPL7 where it is mainly expressed in the pancreatic islets of Langerhans, the heart, the intestine, and the vasculature.
  • ATP-binding cassette transporter Al (ABCAl) protein mediates the efflux of cell cholesterol and phospholipids to lipid-poor apolipoproteins (ApoA-I and Apo E), to form nascent high-density lipoprotein particles (HDL).
  • ABCAl also mediates the transport of lipids
  • ABCAl is expressed as a membrane protein of 220-kD in many tissues.
  • the amino acid sequence is given in Sequence ID NO: 1.
  • ABCAl functions as a pivotal regulator of lipid efflux from cells to apolipoproteins and is thus involved in lowering the risk of cardiovascular diseases.
  • ABCAl is a desirable therapeutic target. Drugs that increase the levels and function of ABCAl protein levels are expected to raise high density lipoprotein (HDL)-cholesterol, reduce atherosclerosis and reduce cardiovascular disease burden. Tangier patients, who lack functional ABCAl, have almost no HDL-cholesterol and may suffer from premature cardiovascular disease.
  • Apolipoprotein A-I (ApoA-I), the major apolipoprotein of HDL, is a ligand of ABCAl and acceptor of exported cell cholesterol. Its amino acid sequence is given in Sequence ID NO: 2 (SwissProt Accession No. P02647.1).
  • HDL-cholesterol raising agents can be useful as medicaments for the treatment and/or prophylaxis of atherosclerosis, peripheral vascular disease, dyslipidemia, hyperbetalipo- proteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial hypercholesterolemia, cardiovascular disorders, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, angioplastic restenosis, hypertension, and vascular complications of diabetes, obesity or endo toxemia.
  • Niacin can significantly increase HDL, but has serious tolerability issues which reduce compliance.
  • pyridine carboxamides are small molecule inducers of the ATP- binding cassette transporter Al protein.
  • Such pyridine carboxamides are for example described in International Patent Applications Publ. Nos. WO 2011/029827, WO 2012/032018 and WO 2013/037703.
  • WO 2012/031817 describes an assay method for determining the ability of a compound to modulate the level of ABCAl protein in cells by employing apolipoprotein A- l labeled with a fluorophore.
  • apolipoprotein A- l labeled with a fluorophore it remained unclear, why the compounds are able to increase the level of ABCAl protein.
  • a target identification approach employing the compound 5-(3,4-Dichloro-phenyl)-N-(2- hydroxy-cyclohexyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinamide as described in WO 2011/029827 identified oxysterol binding protein like protein 7 (OSBPL7) as a molecular target.
  • OSBPL7 oxysterol binding protein like protein 7
  • the amino acid sequence of human OSBPL7 is given in Sequence ID NO: 3 (SwissProt Accession No. Q9BZF2). This indicates that the ability to increase ABCAl protein is due to interaction with a new target Oxysterol Binding Associated Protein-Like 7 (OSBPL7).
  • OSBPL7 as the functional target for pyridine carboxamides that induce ABCAl demonstrate for the first time that small molecule drugs can be developed against OSBPL7.
  • molecules targeting OSBPL7, leading to ABCAl upregulation would, by extension, be useful as medicaments for the treatment and/or prophylaxis of diseases associated with ABCAl as listed above.
  • OSBPL7 modulating drugs may be useful as
  • OSBPL7 Oxysterol Binding Associated Protein-Like 7
  • a test medium comprising a test compound and a tritium ( H)-labeled radioligand
  • a control medium which is identical to the test medium except that it does not include a test compound
  • the invention provides a method for determining the ability of a test compound to bind to Oxysterol Binding Associated Protein-Like 7 (OSBPL7) in cells overexpressing OSBPL7 or a variant thereof, comprising the steps of: a) incubating a first aliquot of OSBPL7 protein expressing cells with a test medium comprising a test compound and a tritium ( H)-labeled radioligand and incubating a second aliquot of said cells with a control medium which is identical to the test medium except that it does not include a test compound, b) cross-linking the ( H)-labeled radioligand to OSBPL7 by UV irradiation of the cells at 366 nm, c) purifying the OSBPL7 from total cell lysates by immunoprecipitation and separating the immunoprecipitated protein by gel electrophoresis, and d) determining the amount of immunoprecipitated OSBPL7 protein in each aliquot by immunohis
  • the invention relates to methods as defined above, wherein the ( H)-labeled radioligand is 5-(4-azido-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(7R,2R)-2- hydroxycyclohexyl]-2,4-ditritiopyridine-3-carboxamide.
  • the invention relates to the method as defined herein before, wherein the immunoprecipitation is carried out with an antibody that binds OSBPL7 or a variant thereof.
  • the invention further relates to the method as defined above, wherein the OSBPL7 protein expressing cells are produced by transfection with a vector expressing OSBPL7 or a variant thereof.
  • the invention relates to the method as defined herein before, wherein the ability of a test compound to bind to OSBPL7 in cells is determined.
  • the invention relates to the method as defined above, wherein the OSBPL7 expressing cells are selected from the group consisting of CHO cells, HeLa cells, BHK-21 cells, HEK293 cells, macrophages, particularly THPl macrophages, and pancreatic beta cells, particularly Insle cells.
  • the invention relates to the method as defined herein before, wherein the incubation period in step a) lasts 1 to 30 hours, particularly 3 hours.
  • the invention also relates to a method for determining the ability of a test compound to promote or prevent the redistribution of OSBPL7 to accumulations to specific points in cells that are expressing an OSBPL7 variant that is fused with an affinity or fluorescence protein tag, comprising the steps of: a) incubating a first aliquot of the cells expressing tagged OSBPL7 with a test medium
  • the accumulation of tagged OSBP7 thus also indicates the pharmacological activity of a test compound.
  • the invention relates to an OSBPL7 protein variant selected from the group consisting of a) OSBPL7 fused with an affinity tag and/or a fluorescent protein tag, in particular a protein comprising the amino acid sequence of SEQ ID NO:4 or a protein comprising the amino acid sequence of SEQ ID NO:5, b) N- or C-terminal truncated OSBPL7 fused with an affinity tag and/or a fluorescent protein tag, in particular OSBPL7(379-842) comprising the amino acid sequence of SEQ ID NO:6, c) an OSBPL7 variant fused with an affinity tag and/or a fluorescent protein tag that carries at least one loss of function mutation in the PH- and FFAT domain, in particular the OSBPL7mutPH2 carrying point mutations K56I/K57A/K67A/R68I comprising the amino acid sequence of SEQ ID NO:7 and OSBPL7mutFFAT carrying point mutation FF403/404AA comprising the amino acid sequence of SEQ ID NO:8,
  • the invention is concerned with the use of an OSBPL7 protein variant as defined above in the methods as described herein before.
  • the invention also relates to a compound of the formula I
  • a 1 and A2 wherein one of A 1 and A2 is N and the other one of A 1 and A2 is CH;
  • R 1 is selected from the group consisting of Ci-7-alkyl
  • heterocyclyl-Ci-7-alkyl wherein the heterocyclyl group is unsubstituted or substituted by oxo
  • heteroaryl-Ci-7-alkyl wherein the heteroaryl group is unsubstituted or mono- or di- substituted by lower alkyl
  • R 2 and R 6 independently from each other are hydrogen or halogen
  • R 3 and R 5 independently from each other are selected from the group consisting of
  • R 4 is selected from the group consisting of hydrogen, Ci-7-alkyl, Ci-7-alkoxy, halogen,
  • R is selected from the group consisting of C 1-7 -alkyl
  • heterocyclyl said heterocyclyl having 3 to 7 ring atoms, comprising one, two or three heteroatoms selected from N, O and S and being unsubstituted or substituted by hydroxy or oxo,
  • phenyl wherein phenyl is unsubstituted or substituted by one or two groups selected from the group consisting of C 1-7 -alkyl, hydroxy, Ci-7-alkoxy, cyano, halogen and halogen-Ci-7- alkyl, and
  • heteroaryl wherein heteroaryl is unsubstituted or substituted by one or two groups selected from the group consisting of C 1-7 -alkyl, hydroxy, Ci-7-alkoxy, cyano, halogen and halogen- C 1-7 -alkyl, and
  • G is selected from the group consisting of
  • the invention thus also relates to a compound of the formula I
  • a 1 and A2 wherein one of A 1 and A2 is N and the other one of A 1 and A2 is CH;
  • R is selected from the group consisting of Ci-7-alkyl
  • heterocyclyl-Ci-7-alkyl wherein the heterocyclyl group is unsubstituted or substituted by oxo
  • heteroaryl-Ci-7-alkyl wherein the heteroaryl group is unsubstituted or mono- or di- substituted by lower alkyl
  • R 2 and R 6 independently from each other are hydrogen or halogen
  • R 3 and R 5 independently from each other are selected from the group consisting of
  • R 4 is selected from the group consisting of hydrogen, Ci-7-alkyl, Ci-7-alkoxy, halogen,
  • R is selected from the group consisting of C 1-7 -alkyl
  • heterocyclyl said heterocyclyl having 3 to 7 ring atoms, comprising one, two or three heteroatoms selected from N, O and S and being unsubstituted or substituted by hydroxy or oxo,
  • phenyl wherein phenyl is unsubstituted or substituted by one or two groups selected from the group consisting of C 1-7 -alkyl, hydroxy, Ci-7-alkoxy, cyano, halogen and halogen-Ci-7- alkyl, and heteroaryl, wherein heteroaryl is unsubstituted or substituted by one or two groups selected from the group consisting of C ⁇ -allcyl, hydroxy, Ci-7-alkoxy, cyano, halogen and halogen- Ci-7-alkyl, and
  • G is selected from the group consisting of
  • R is hydrogen or Ci-7-alkyl, and pharmaceutically acceptable salts thereof, for use in the treatment of a disease that is modulated by OSBPL7.
  • the invention also relates to a compound of formula I as defined above for use in the treatment of a disease that is modulated by OSBPL7, wherein the disease is selected from the group consisting of diabetes, impaired insulin secretion, insulin resistance, beta cell protection, and cancer.
  • the disease is diabetes.
  • the invention relates to a compound of formula I, which is
  • This method comprises the steps of: a) incubating a first aliquot selected from the group consisting of cells overexpressing OSBPL7 protein or a variant thereof, lysates of cells overexpressing OSBPL7 protein or a variant thereof, and isolated OSBPL7 protein or a variant thereof, with a test medium comprising a test compound and a tritium ( H)-labeled radioligand and incubating a second aliquot of said cells, lysates or isolated protein or variant thereof with a control medium which is identical to the test medium except that it does not include a test compound, b) cross-linking the ( H)-labeled radioligand to OSBPL7 by UV irradiation, c) purifying the OSBPL7 by immunoprecipitation and separating the immunoprecipitated protein by gel electrophoresis
  • the invention refers to the method for determining the ability of a test compound to bind to Oxysterol Binding Associated Protein-Like 7 (OSBPL7) in cells overexpressing OSBPL7 or a variant thereof comprising the steps of: a) incubating a first aliquot of OSBPL7 protein expressing cells with a test medium comprising a test compound and a tritium ( H)-labeled radioligand and incubating a second aliquot of said cells with a control medium which is identical to the test medium except that it does not include a test compound, b) cross-linking the ( H)-labeled radioligand to OSBPL7 by UV irradiation of the cells at 366 nm, c) purifying the OSBPL7 from total cell lysates by immunoprecipitation and separating the immunoprecipitated protein by gel electrophoresis, and d) determining the amount of immunoprecipitated OSBPL7 protein in each aliquot by: a) in
  • the described method allows for screening of small molecule drugs that modulate the function or distribution of OSBPL7 protein in cells. Briefly, cells overexpressing OSBPL7 or a variant thereof are exposed to a medium comprising a tritium ( H)-labeled radioligand and fixed or increasing concentrations of test substances. As a control medium an identical medium is used that comprises the tritium ( H)-labeled radioligand but not the test substances.
  • the medium is removed and the cells are washed in order to remove unbound compounds.
  • they are washed with ice cold phosphate buffered saline (PBS).
  • PBS ice cold phosphate buffered saline
  • the UV irradiation time may vary from 3 min to 30 min, in particular 15 min.
  • the OSBPL7 protein or variant thereof is then isolated from total cell lysates by immunoprecipitation from the soluble protein lysate fraction using an antibody that binds OSBPL7 or the variant thereof and separating the
  • the amount of OSBPL7 or the variant thereof in each aliquot is determined by immunohistochemistry, e.g. by quantitative image-based analysis, i.e. by fluorescence spectroscopy, and in parallel, the amount of the ( H)- labeled radioligand in each aliquot is detected by autoradiography.
  • OSBPL7 protein expressing cells are used.
  • OSBPL7 refers to Oxysterol Binding Associated Protein-Like 7 and may also be used to refer to the gene encoding this protein.
  • OSBPL7 is herein defined as either the full sequence according to SEQ ID No. 1 (OSBPL7_HUMAN, SwissProt Accession No. Q9BZF2) having 842 amino acids or variants thereof such as the ones described below, and also includes OSBPL7 sequences of non-human origin.
  • OSBPL7 protein expressing cells refers to any cell type that naturally expresses OSBPL7 or which has been genetically engineered to express OSBPL7, including both prokaryotic and eukaryotic cell types.
  • the cells are selected from the group consisting of monocytes, macrophages, hepatocytes, endothelial cells, fibroplasts and enterocytes.
  • the OSBPL7 protein expressing cells are selected from the group consisting of Chinese hamster ovary cells (CHO cells), HeLa cells, BHK-21 cells, HEK293 cells, macrophages, particularly THP1 macrophages, and pancreatic beta cells, particularly Insle cells.
  • Cells can for example be transfected by seeding the said cells to cell culture plates. 24h after seeding the cells are transfected by mixing 2 ⁇ g DNA of the expression vector encoding OSBPL7 or a variant thereof with 200 ⁇ ⁇ OptiMEM® I reduced serum medium (Gibco). After addition of 6 ⁇ L Megatran 1.0 transfection reagent (Origene) (ratio DNA:Megatranl.O (w/v) 1:3) the transfection mix is vortexed, incubated for 15min at room temperature, and finally added to the cells. After eight hours the transfection medium is replaced by fresh growth medium and the cells are grown in a time range of 24 to 48 hours for OSBPL7 expression.
  • Megatran 1.0 transfection reagent Origene
  • Test compounds can be any compounds that are to be assayed for their ability to act as OSBPL7 modulator.
  • organic compounds small molecules from a chemical substance library are to be assayed.
  • solutions of the test substances in DMSO are used for testing. More particularly, the test compounds are serially diluted with DMSO and then added in different amounts to the cell medium (Dulbeccos Modified Eagle medium (DMEM)) to provide the test medium in the desired final concentrations.
  • DMEM Dulbeccos Modified Eagle medium
  • a "small molecule” is defined herein as an organic molecule having a molecular weight below 800 Daltons.
  • label when used herein refers to a compound, protein or composition which is conjugated or fused directly or indirectly to a reagent such as a radioisotope or a fluorophore which facilitates detection of the compound, protein or composition.
  • the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound, protein or composition which is detectable.
  • Tritium labeled or tritiated ( H)-labeled radioligands are particularly valuable for studies involving high resolution autoradiography.
  • the physical (nuclear) properties of tritium, the low maximum beta energy (18 keV) of the radiation and the high maximum specific activity (29 Ci/mg atom of hydrogen), are the reasons why tritium is considered the ideal isotope for determining the precise localization of the radioligands.
  • the ( H)-labeled radioligand is 5-(4-azido-3-chlorophenyl)-6- (cyclopropylmethoxy)-N-[(7R,2R)-2-hydroxycyclohexyl]-2,4-ditritiopyridine-3-carboxamide.
  • Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes.
  • Affinity chromatography is often the preferred method for protein purification and can be used to purify proteins from complex mixtures with high yield.
  • Affinity chromatography is based on the ability of proteins to bind non-covalently but specifically to an immobilized ligand for the desired protein, e.g. an antibody for a protein antigen.
  • an immobilized ligand for the desired protein e.g. an antibody for a protein antigen.
  • isolation of the fusion protein can be done using metal affinity chromatography.
  • Affinity tags are chelating peptides ("tags") that specifically bind to immobilized metal ions and are therefore a tool for purifying proteins that contain such a chelating peptide.
  • tags One of the most commonly used tags for affinity purification of proteins is the hexahistidine or 6xHis- tag.
  • Affinity tags as used in the present invention are selected from the group consisting of His- tag, in particular lOxHis-tag, myc-tag and FLAG-tag.
  • the myc-tag and the FLAG-tag are tags that can be fused to the protein if there is no antibody against the studied protein. Adding a myc- tag or a FLAG-tag provides the possibility to recognize the fusion protein with an antibody against the myc epitope or the FLAG epitope, respectively. Such antibodies are commercially available.
  • fluorophore is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re- emit energy at a different (but equally specific) wavelength. However, in a common sense, the term “fluorophore” also means the compound (molecule) that comprises the functional group able to re-emit energy at different wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore.
  • Fluorescence protein tags can be used to provide a visual readout on a protein.
  • Typical fluorescence protein tags that can be fused with OSBPL7 or a variant thereof, are for example selected from the group consisting of green fluorescent protein (GFP), eGFP (a mutation of GFP), red fluorescent (RFP) and yellow fluorescent protein (YFP).
  • GFP for example is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.
  • proteins can also be labeled with fluorophores.
  • fluorophores selected from the group consisting of fluorescein, a rhodamine, a cyanine dye, a DyLight Fluor dye (Thermo Fisher Scientific), an Alexa Fluor dye (Molecular Probes, Invitrogen), an ATTO dye (ATTO-TEC GmbH) and a BODIPY dye
  • the fluorophore is selected from the group consisting of Fluorescein, Rhodamine B, Rhodamine Red, Texas Red, Tetramethylrhodamine, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Cy3, Cy5, Cy7, ATTO 610, ATTO 633, ATTO 647N, ATTO 655, Dylight Fluor 633, Dylight Fluor 650, Dylight Fluor 680, BODIPY 630/650 and BODIPY 650/665.
  • the labeling (coupling) of proteins with the fluorophore can be carried out according to methods well-known in the art.
  • the manufacturers of the fluorophores e.g. Invitrogen also offer kits for the labeling of proteins.
  • the readout i.e. determining the levels of labeled OSBPL7 in the portions, is performed by quantitative image -based analysis, i.e. by fluorescence spectroscopy. Any fluorometer which is able to detect the fluorophore can be used. More particularly, the readout can be performed with an OPERA QEHS high content screening microplate imaging reader (Perkin Elmer). This imaging reader is specifically suitable for high throughput and high content screening of fluorescently labeled cell samples and the readout is determined from the acquired images with an image analysis software (Acapella, www.perkinelmer.com) .
  • the assay method of the invention may be suitable for measuring the ability of test compounds to modulate the level of ABCA1 protein in cells in a high throughput (HTS) format.
  • HTS high throughput
  • the key labware of HTS are microtiter plates that are small plates containing small wells in multiples of 96, for example 384, 1536 or 3456 wells.
  • the invention also relates to a method, wherein the ability of a test compound to promote or prevent the redistribution of OSBPL7 from diffuse to punctate structures / vesicles / accumulations to specific points within cells is used as the readout.
  • This method is a method for determining the ability of a test compound to promote or prevent the redistribution of OSBPL7 to accumulations to specific points in cells that are expressing an OSBPL7 variant that is fused with an affinity or fluorescence protein tag, comprising the steps of: a) incubating a first aliquot of the cells expressing OSBPL7 with a test medium comprising a test compound and incubating a second aliquot of said cells with a control medium which is identical to the test medium except that it does not include a test compound, and b) analyzing the cellular localization of OSBPL7 by microscopy, fluorescent microscopy or fluorescence assisted cell sorting (FACS) in both aliquots, wherein an accumulation of tagged OSBPL7 in the first aliquot indicates protein translocation in response to binding of the test compound.
  • FACS fluorescence assisted cell sorting
  • the invention relates to the method as defined above, wherein the incubation period in step a) lasts 0 to 30 hours, particularly 8 to 24 hours.
  • the analysis of the cellular OSBPL7 localization can be carried out by microscopy, fluorescent microscopy or fluorescence assisted cell sorting (FACS) in living cells or after fixation, permeabilization and/or immunohistochemical detection of OSBPL7.
  • FACS fluorescence assisted cell sorting
  • the invention relates to the methods as defined above, wherein a test compound is instead substituted by incubation with a mixture of a transfection reagent and a short hairpin RNA (shRNA), small interfering RNA (siRNA) and/or an sh/siRNA pool against a specific gene to measure the effect of knockdown of expression of the corresponding mRNA on glucose stimulated insulin secretion, ABCA1 levels, or OSBPL7 redistribution in cells.
  • shRNA short hairpin RNA
  • siRNA small interfering RNA
  • sh/siRNA pool against a specific gene to measure the effect of knockdown of expression of the corresponding mRNA on glucose stimulated insulin secretion, ABCA1 levels, or OSBPL7 redistribution in cells.
  • the invention is directed to the methods as defined above, wherein the OSBPL7 protein expressing cells are human or animal cells such as Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, macrophages, particularly THP1 macrophages or pancreatic beta cells, particularly Insle cells.
  • the invention relates to the methods as defined above, wherein a OSBPL7 protein or variant thereof is employed that is fused with an affinity tag and/or a fluorescent protein tag.
  • Such OSBPL7 protein or variant thereof may contain various truncations, fusions or mutations, or be endogenously expressed.
  • the invention relates to an OSBPL7 protein variant selected from the group consisting of a) OSBPL7 fused with an affinity tag and/or a fluorescent protein tag, b) N- or C-terminal truncated OSBPL7 fused with an affinity tag and/or a fluorescent protein tag, c) an OSBPL7 variant fused with an affinity tag and/or a fluorescent protein tag that carries at least one loss of function mutation in the PH- and FFAT domain, and d) an OSBPL7 variant fused with an affinity tag and/or a fluorescent protein tag that carries point mutations in the C-terminal oxysterol recognition domain (ORD), in particular at least one mutation selected from the group consisting of R463A, A469V, I482A, I482F, N485A, A540V, A551V, C553A, K554A, M602A, F608A, E614A, V616I, V616A,
  • ORD C-terminal
  • the invention is concerned with the use of an OSBPL7 protein variant as defined above in the methods as described herein before.
  • An affinity tag that can be fused with OSBPL7 or a variant thereof is for example selected from the group consisting of His-tag, in particular lOxHis-tag, myc-tag and FLAG-tag.
  • a fluorescence protein tag that can be fused with OSBPL7 or a variant thereof is for example selected from the group consisting of GFP, eGFP, RFP, YFP, and eYFP.
  • a particular OSBPL7 protein variant according to group a) is for instance human wild-type OSBPL7 carrying a C-terminal GFP and lOxHistidine tag comprising the amino acid sequence of SEQ ID NO:4 (OSBPL7-GFP- lOxHis).
  • Another OSBPL7 protein variant according to group a) is human wild-type OSBPL7 carrying a C-terminal myc and FLAG tag comprising the amino acid sequence of SEQ ID NO:5 (OSBPL7-myc-FLAG).
  • Group b refers to N- or C-terminal truncated OSBPL7 protein variants that are fused with an affinity tag and/or a fluorescent protein tag.
  • protein variants that lack the regulatory pleckstrin homology (PH)-domain and/or FFAT-motif are used, such as
  • OSBPL7 (379-842).
  • a particular N-terminal truncated OSBPL7 protein variant fused with an affinity tag and/or a fluorescent protein tag is OSBPL7(379-842)-GFP-10xHis comprising the amino acid sequence of SEQ ID NO:6.
  • Group c relates to OSBPL7 protein variants fused with an affinity tag and/or a fluorescent protein tag that carry any loss of function mutation in the PH- and FFAT domain, such as OSBPL7mutPH2 carrying 4 point mutations K56I/K57A/K67A/R68I comprising the SEQ ID NO:7 which disrupts the function of the PH-domain, and OSBPL7mutFFAT carrying 2 point mutations FF403/404AA comprising the amino acid sequence of SEQ ID NO: 8 which disrupts the function of the FFAT-domain.
  • OSBPL7mutPH2 carrying 4 point mutations K56I/K57A/K67A/R68I comprising the SEQ ID NO:7 which disrupts the function of the PH-domain
  • OSBPL7mutFFAT carrying 2 point mutations FF403/404AA comprising the amino acid sequence of SEQ ID NO: 8 which disrupts the function of the FFAT-domain.
  • Group d) comprises OSBPL7 protein variants fused with an affinity tag and/or a fluorescent protein tag that carry point mutations in the C-terminal oxysterol recognition domain (ORD), in particular at least one mutation selected from the group consisting of R463A, A469V, I482A, I482F, N485A, A540V, A551V, C553A, K554A, M602A, F608A, E614A, V616I, V616A, V618I, V618A, K636A, I641A, I641V, R649A, I651A and I651V.
  • ORD C-terminal oxysterol recognition domain
  • the invention is also concerned with the use of such OSBPL7 protein variants as defined above in the methods as described herein before.
  • the OSBPL7 variants with its various mutations and truncations are especially useful for structural and functional studies in the identification and optimization of medicaments.
  • the invention also relates to expression vectors encoding the OSBPL7 protein variants.
  • expression vectors can be prepared by methods known in the art.
  • expression vectors can be prepared by cloning a piece of DNA that encodes a specific protein, such as a complementary DNA (cDNA), under control of a promoter which confers expression in the target cell line.
  • cDNAs can be synthesized from messenger RNA templates by a combined reaction of a reverse transcriptase and DNA polymerase, or by artificial de novo synthesis.
  • mammalian OSBPL7 expression vectors were produced by cloning various cDNAs encoding said human OSBPL7 variant under control of a human cytomegalovirus promoter that confers constitutive expression in mammalian cells.
  • the present invention thus provides assay method to screen for compounds that bind to OSBPL7 and alter its function.
  • the method is especially useful in drug discovery and screening.
  • the method is also useful for discovery of new pathways and targets leading to modulation of OSBPL7, particularly after transfection of cells with siRNAs of overexpressing plasmids.
  • the invention also relates to modulators of OSBPL7, in particular antagonists of OSBPL7, of the formula I
  • a 1 and A 2 is N and the other one of A 1 and A 2 is CH; is selected from the group consisting of Ci-7-alkyl,
  • heterocyclyl-Ci-7-alkyl wherein the heterocyclyl group is unsubstituted or substituted by oxo
  • R and R b independently from each other are hydrogen or halogen
  • R and R independently from each other are selected from the group consisting of
  • R is selected from the group consisting of hydrogen, Ci-7-alkyl, C .
  • R is selected from the group consisting of C 1-7 -alkyl
  • heterocyclyl said heterocyclyl having 3 to 7 ring atoms, comprising one, two or three heteroatoms selected from N, O and S and being unsubstituted or substituted by hydroxy or oxo,
  • phenyl wherein phenyl is unsubstituted or substituted by one or two groups selected from the group consisting of C 1-7 -alkyl, hydroxy, Ci-7-alkoxy, cyano, halogen and halogen-Ci-7- alkyl, and
  • heteroaryl wherein heteroaryl is unsubstituted or substituted by one or two groups selected from the group consisting of C 1-7 -alkyl, hydroxy, Ci-7-alkoxy, cyano, halogen and halogen- C 1-7 -alkyl, and is selected from the group consisting of
  • R is hydrogen or Ci-7-alkyl, and pharmaceutically acceptable salts thereof.
  • lower alkyl or "Ci-7-alkyl”, alone or in combination, signifies a straight-chain or branched-chain alkyl group with 1 to 7 carbon atoms, in particular a straight or branched- chain alkyl group with 1 to 6 carbon atoms and more particularly a straight or branched-chain alkyl group with 1 to 4 carbon atoms.
  • Ci_7 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, the isomeric hexyls and the isomeric heptyls, in particular methyl, ethyl, propyl, isopropyl and tert-butyl.
  • lower alkoxy or "Ci-7-alkoxy” refers to the group R'-O-, wherein R' is lower alkyl and the term “lower alkyl” has the previously given significance.
  • lower alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.-butoxy and tert.-butoxy, in particular methoxy.
  • lower alkoxyalkyl or "C 1 _7-alkoxy-C 1 _7-alkyl” refers to a lower alkyl group as defined above which is mono- or multiply substituted with a lower alkoxy group as defined above.
  • lower alkoxyalkyl groups are e.g. -CH 2 -0-CH 3 , -CH 2 -CH 2 -0-CH , -CH 2 -0-CH 2 -CH and the groups specifically exemplified herein. More particularly, lower alkoxyalkyl is methoxyethyl.
  • hydroxy means the group -OH.
  • cycloalkyl or "C 3 _7-cycloalkyl” denotes a saturated carbocyclic group containing from 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl cycloheptyl.
  • lower cycloalkylalkyl or "C 3 _7-cycloalkyl-C 1 _7-alkyl” refers to lower alkyl groups as defined above wherein at least one of the hydrogen atoms of the lower alkyl group is replaced by a cycloalkyl group.
  • cyclopropylmethyl is particularly preferred.
  • halogen refers to fluoro, chloro, bromo and iodo, with fluoro, chloro and bromo being of particular interest. More particularly, halogen refers to fluoro and chloro.
  • lower halogenalkyl or “halogen-Ci-7-alkyl” refers to lower alkyl groups which are mono- or multiply substituted with halogen, particularly with fluoro or chloro, most particularly with fluoro.
  • lower halogenalkyl groups are e.g. -CF 3 , -CHF 2 , -CH 2 C1, -CH 2 CF 3 , -CH(CF 3 ) 2 , -CF 2 -CF 3> -CH 2 -CH 2 -CF 3 , -CH(CH 3 )-CF 3 and the groups specifically exemplified herein.
  • lower halogenalkoxy or "halogen-Ci-7-alkoxy” refers to lower alkoxy groups as defined above wherein at least one of the hydrogen atoms of the lower alkoxy group is replaced by a halogen atom, particularly fluoro or chloro, most particularly fluoro.
  • halogen atom particularly fluoro or chloro, most particularly fluoro.
  • the lower halogenalkoxy groups of particular interest are trifluoromethoxy, difluoromethoxy, fluormethoxy and chloromethoxy, more particularly trifluoromethoxy.
  • amino means the group -NH 2 .
  • cyano means the group -CN.
  • azido means the group -N 3 .
  • heteroaryl refers to an aromatic 5- or 6-membered ring which can comprise one, two or three atoms selected from N, O and S. Examples of heteroaryl groups are e.g.
  • heteroaryl also includes bicyclic groups comprising two 5- or 6-membered rings, in which one or both rings are aromatic and can contain one, two or three atoms selected from nitrogen, oxygen or sulphur, such as quinolinyl, isoquinolinyl, cinnolinyl, pyrazolo[l,5-a]pyridyl, imidazo[l,2-a]pyridyl, quinoxalinyl, benzothiazolyl, benzotriazolyl, indolyl, indazolyl, and 3,4-dihydro-2H-pyrido[3,2-b][l,4]oxazinyl.
  • Heteroaryl groups of particular interest are of isoxazolyl, pyrazolyl, oxadiazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl. More particularly, heteroaryl is pyridyl or pyridazinyl.
  • heteroarylalkyl or “heteroaryl-Ci-7-alkyl” refers to lower alkyl groups as defined above wherein at least one of the hydrogen atoms of the lower alkyl group is replaced by a heteroaryl group as defined above.
  • heterocyclyl refers to a saturated or partly unsaturated 3-, 4-, 5-, 6- or 7- membered ring which can comprise one, two or three heteroatoms selected from N, O and S. Examples of heterocyclyl rings include piperidinyl, piperazinyl, azetidinyl, azepinyl,
  • morpholinyl thiazolidinyl, isothiazolidinyl, oxiranyl, thiadiazolylidinyl, oxetanyl, dioxolanyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, and thiomorpholinyl.
  • piperidinyl and tetrahydropyranyl are piperidinyl and tetrahydropyranyl.
  • lower heterocyclylalkyl or “heterocyclyl-Ci-7-alkyl” refers to lower alkyl groups as defined above wherein at least one of the hydrogen atoms of the lower alkyl group is replaced by a heterocyclyl group as defined above.
  • the invention further relates to the compounds of formula I for use in the treatment of a disease that is modulated by OSBPL7.
  • a disease that is modulated by OSBPL7 includes diabetes, impaired insulin secretion, insulin resistance, beta cell protection, and cancer.
  • FIG. 1 This figure shows a scheme how the cell-based OSBPL7 ligand binding assay works.
  • Cells overexpressing OSBPL7 or a variant thereof are combined with tritiated Compound A and test compound.
  • Compound A is linked to OSBPL7 or the protein variant thereof by irradiation with UV- light.
  • the test compound competes for the binding of the radioligand, a decrease of the signal is seen in the autoradiography (left side), whereas no or less decrease in signal is seen when the test compound is a non-binder that does not show competition with the radioligand (right side).
  • FIG. 2 This figure shows that OSBPL7 is required for the function of pancreatic beta cells to secrete insulin in response to glucose stimulation.
  • OSBPL7 was manipulated in this case by overexpression heterologous (transfection) or knockdown of endogenous (siRNA) protein.
  • FIG 3 This figure demonstrates that small molecules that bind OSBPL7 (compound D, E) act as functional antagonists in that they decrease glucose-stimulated insulin secretion in pancreatic beta cell line Insle. In contrast, compound C that does not bind OSBPL7 was inactive.
  • Figure 4 This figure shows another functional readout of OSBPL7 small molecule binding. In the presence of compound E, OSBPL7 translocates intracellularly from a rather broad distribution to specific intracellular points. Compound C, a compound that did not bind OSBPL7 did not cause this redistribution. Thus, this is another functional readout for small molecule binding to OSBPL7. Scale bar: 20 ⁇ .
  • Figure 5 This figure illustrates the ligand binding pocket of OSBPL7 as modeled by a sequence alignment with other OSBP family members and the yeast Keslp protein. The model depicts amino acids that are involved in ligand compound binding. Point mutation of these residues abrogated compound binding as demonstrated for mutation K636A, which abolishes binding of compound A.
  • FIGS. 6a to 6d show the effect of mutations predicted to line the oxysterol- binding pocket of OSBPL7 on compound binding. Several mutations abrogated compound binding indicating that the compounds interact functionally in the binding pocket and that mutagenesis may be used to generate a structure-activity relationship.
  • Figure 7 This figure shows that OSBPL7 is expressed in human islets and rat beta cell line
  • FIG 8 This figure shows that treatment of ApoE knockout mice with an OSBPL7 binding compound preserved the ability of the pancreatic islets to secrete insulin in response to glucose. This is an in vivo model/assay for OSBPL7 compound activity.
  • Figure 9 Schematic representation of OSBPL7 variants. The position of the individual protein domains are annotated according to the amino acid sequence of human full-length OSBPL7. Asterisks indicate point mutations in the PH-domain.
  • PH-domain Pleckstrin homology domain
  • FFAT Two phenylalanines in an acidic tract motif (EFFDAxE);
  • ORD oxysterol recognition domain
  • eGFP enhanced green fluorescence protein
  • myc protein tag derived from the c-myc gene
  • lOxHis affinity tag of 10-mer histidine residues.
  • All mammalian protein variants were produced from expression vectors carrying the said cDNAs under control of a human cytomegalovirus promoter.
  • the expression vector encoding OSBPL7-myc-FLAG variant is a TrueORF clone (Origene, RC209112).
  • the expression vector of OSBPL7-GFP-10xHis was prepared by cloning the cDNA encoding human OSBPL7 using restriction endonucleases Nhel/Notl. The resulting expression cassette encodes for full-length OSBPL7 c-terminally tagged with GFP and a decamer of histidines (lOxHis) under control of a human cytomegalovirus derived promoter (P CMV )-
  • the expression vector of OSBPL7(379-842)-GFP-10xHis was produced by polymerase chain reaction (PCR) based amplification of a cDNA encoding OSBPL7(379-842)-GFP-10xHis using oligonucleotides oCK256 (3 ' -GGAATTCCACCATGAAGGGGCGCGAGCTCAC-5 ' , SEQ ID NO:9) and oCK254 (5'- GCTCTAGACTGGCAACTAGAAGGCACAG -3', SEQ ID NO: 10) and full-length OSBPL7-GFP-10xHis as a template.
  • PCR polymerase chain reaction
  • the obtained PCR product was introduced by restriction enzyme digestion and subsequent ligation into pcDNA3.1-OSBPL7- GFP-lOxHis using EcoRI/Xbal (New England Biolabs Inc.) restriction endonucleases and T4- DNA Ligase (New England Biolabs Inc.), respectively.
  • Expression vectors for OSBPL7mutPH2- GFP-lOxHis and OSBPL7mutFFAT-GFP-10xHis were produced by de novo synthesis of the cDNAs (GenScript) carrying point mutations K56I/K57A/K67A/R68I and FF403/404AA, respectively.
  • OSBPL7(379-842)-GFP-10xHis was expressed by transfection of 4xl0 6 HEK293 with a transfection mix containing 10 ⁇ g expression vector DNA, ⁇ OptiMEM® I reduced serum medium (Gibco) and 30 ⁇ Megatranl.O (Origene). Six hours after transfection the medium was replaced by fresh DMEM containing 10% (v/v) FBS and the cells were grown for 48h for protein expression.
  • the cells were washed with DPBS and lysed in lmL FSEC buffer (50mM Tris pH8.2, 150mM NaCl, lOmM Tris(2- carboxyethyl)phosphine hydrochloride (TCEP) (Sigma), 12mM pentaethylene glycol monooctyl ether (C8E5) (Sigma), 5% (v/v) glycerol (Invitrogen), complete protease inhibitor cocktail (Roche)) for 15min on ice. The lysate was centrifuged (18.000xg) for 15min at 4°C to remove insoluble cell debris and the soluble protein fraction was collected.
  • TCEP Tris pH8.2, 150mM NaCl, lOmM Tris(2- carboxyethyl)phosphine hydrochloride
  • C8E5 12mM pentaethylene glycol monooctyl ether
  • C8E5 5%
  • glycerol In
  • Proteins were loaded onto a Nickel affinity (Ni-NTA) column, non-specific proteins were removed by washing and the proteins were eluted with elution buffer containing imidazole (50mM Tris pH8.2, 150mM NaCl, 5mM TCEP, 12mM C8E5, 5% (v/v) glycerol, 300mM imidazole). If needed the imidazole was removed by performing an additional size exclusion chromatograph using Superdex200 column material.
  • imidazole 50mM Tris pH8.2, 150mM NaCl, 5mM TCEP, 12mM C8E5, 5% (v/v) glycerol, 300mM imidazole.
  • Sequence ID NO:4 OSBPL7-GFP-10xHis, that is human wildtype OSBPL7 carrying a c- terminal GFP and lOxHistidine tag
  • Sequence ID NO: 5 OSBPL7-myc-FLAG, that is human wildtype OSBPL7 carrying a c-terminal myc and FLAG tag. This variant was the basis for the mutational analysis of the oxysterol recognition domain. Sequence ID NO:6: OSBPL7(379-842)-GFP-10xHis. This variant is an N-terminal truncated OSBPL7 that lacks the first 378 amino acids. This variant lacks the PH-domain, but still contains the FFAT motif and oxysterol binding domain
  • Sequence ID NO:7 OSBPL7mutPH2-GFP-10xHis, this variant carries 4 point mutations (K56I/K57A/K67A/R68I) in the PH-domain which disrupt the function of this domain.
  • Sequence ID NO: 8 OSBPL7mutFFAT-GFP-10xHis, this variant carries 2 point mutations (FF403/404AA) which disrupt the FFAT-domain function.
  • PH-domain Pleckstrin homology domain
  • FFAT Two phenylalanines in an acidic tract motif (EFFDAxE)
  • ORD oxysterol recognition domain
  • eGFP enhanced green fluorescence protein
  • myc protein tag derived from the c-myc gene
  • lOxHis affinity tag of 10- mer histidine residues.
  • Dichloromethane was removed in vacuo and the residue was digested with ethanol/water (1.6 mL, 3x) and toluene (1.5 mL, 2x). The residue was dissolved in ethanol (100 mL) and one half was purified on LichroPrepSi60 25-40 ⁇ with toluene/acetone 3: 1. The product (1.6 mg) was isolated as a mixture of tritium isomers with a specific activity of 79.4 Ci/mmol.
  • the title compound was synthesized from 6-(4-chloro-phenyl)-5-(2,2,2-trifluoro-ethoxy)-2- pyridine carboxylic acid and 3-(l-methylethyl)-5-isoxazolemethanamine (CAS Reg. No.
  • Assay format 1 Cell-based OSBPL7 ligand binding assay
  • DMEM Dulbeccos Modified Eagle Medium
  • fetal bovine serum 10% (v/v) fetal bovine serum and 1% (v/v) Penicillin/Streptomycin mix (Gibco) and containing ⁇ tritium labeled compound A and either ⁇ , 10 ⁇ or 50 ⁇ of test compounds.
  • DMEM Dulbeccos Modified Eagle Medium
  • Penicillin/Streptomycin mix Gibco
  • DPBS Phosphate Buffered Saline
  • the DPBS was removed, and cells were lysed with 200uL Pierce RIPA buffer (Thermo Scientific) containing protease inhibitor cocktail (Roche) followed by ultrasonication.
  • OSBPL7 was immunoprecipitated from 100 ⁇ g of the soluble protein lysate fraction by overnight incubation with EZview Red anti-flag M2 affinity gel (Sigma) at 4°C.
  • the separated proteins were transferred to nictrocellulose membranes using the iBlot system and iBlot Gel Transfer Stacks (Life Technologies). Non-specific membrane binding sites were blocked by incubating the nitrocellulose membrane for lh in Tris buffered saline (Sigma) containing 0.05% (v/v) Tween20 (TBS-T) and 5% (w/v) nonfat dry milk (Biorad). OSBPL7 was detected with anti-FLAG mouse monoclonal Ig-HRP antibody (Sigma A8592)in TBS-T. To remove non-bound antibodies the membranes were washed for 6 times 5min with TBST-T followed by signal development using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).
  • Quantification of protein and autoradiography signals was performed using Quantity One software (Biorad).
  • the amount of bound compound A was normalized to the quantity of expressed OSBPL7 and compared between the conditions with either ⁇ , 10 ⁇ or 50 ⁇ test compound.
  • a reduction in compound A binding with increasing test compound concentrations indicates an efficient competition with the radioligand and therefore binding of the test compound to OSBPL7.
  • Assay format 2 OSBPL7 modulation affects glucose stimulated insulin secretion in beta cell line Insle
  • Insle cells were seeded per well of a 96-well plate and grown for 24h in growth medium (RPMI 1640 medium with GlutaMAX (Gibco) containing 10% (v/v) fetal bovine serum, lOmM HEPES (Gibco), ImM pyruvate (Gibco), 50 ⁇ beta mercaptoethanol (Sigma), and l%(v/v) Penicillin/Streptomycin mix (Gibco)).
  • growth medium RPMI 1640 medium with GlutaMAX (Gibco) containing 10% (v/v) fetal bovine serum, lOmM HEPES (Gibco), ImM pyruvate (Gibco), 50 ⁇ beta mercaptoethanol (Sigma), and l%(v/v) Penicillin/Streptomycin mix (Gibco)
  • the medium was replaced by fresh growth medium containing Lipofectamine 2000 transfection reagent and either lOOng/well DNA of the expression
  • Knockdown of endogenous OSBPL7 was achieved by replacing the medium with fresh growth medium containing Silencer Select siRNA (Ambion, s 155219) targeting rat OSBPL7, or non-coding siRNAs were used as control. After 24h transfection the medium was replaced by fresh growth medium and cells grown for additional 16h. The cells were washed with Krebs- Ringer buffer with Hepes (KRBH) and glucose starvation was performed for 2h in 200 ⁇ KRBH-buffer containing 2.0mM glucose. The KRBH buffer was replaced by fresh KRBH- buffer containing increasing concentrations of glucose and glucose stimulated insulin secretion (GSIS) was performed for lh. The secreted insulin was quantified using the Mercodia High Range Rat Insulin ELISA kit (see also Figure 2).
  • Assay format 3 Small molecule OSBPL7 antagonists inhibit glucose stimulated insulin secretion in beta cell line Insle 70.000 Insle cells were seeded per well of a 96-well plate and grown for 48h in growth medium. The medium was replaced by fresh growth medium and cells grown for additional 16h. The cells were washed with KRBH-buffer. 200 ⁇ KRBH-buffer containing 2.0mM glucose and 10 ⁇ of indicated OSBPL7-modulators were added per well and glucose starvation was performed for 2h. The KRBH buffer was replaced by fresh KRBH-buffer containing increasing concentrations of glucose and 10 ⁇ of indicated OSBPL7 -modulators and glucose stimulated insulin secretion (GSIS) was performed for lh. The secreted insulin was quantified using the Mercodia High Range Rat Insulin ELISA kit (see also Figure 3).
  • the human OSBPL7 sequence was aligned to a Hidden Markov Model constructed from the oxysterol binding protein family and including the yeast Keslp protein.
  • the four amino acid side chains depicted are clustered in the hydrophobic wall of the ligand binding pocket formed by a beta sheet and showed high sequence conservation among OSBPs.
  • Lysine 636 lies at the end of the binding pocket close to the expected position of the sterol -OH group. Mutation of lysine 636 at the base of the sterol binding pocket abolished interaction of the protein with small molecule OSBPL7 modulator compound A, indicating a direct interaction of OSBPL7 modulators with the sterol binding domain (see Figure 5). Mutational analysis of the OSBPL7 binding pocket proofs importance of key interaction residues for compound A binding
  • Sections, 5 ⁇ thick, from commercial paraffin blocks of human adult normal pancreas (Asterand) were deparaffinized, rehydrated, and then microwaved with citrate buffer (Thermo Scientific), followed by incubated with the first antibodies as indicated.
  • the following primary antibodies were used at 1 ⁇ g/ml for 1 hour: mouse anti-human OSBPL7 (Roche clone, 2/13); guinea pig anti-swine insulin and rabbit anti-glucagon (Dako).
  • OSBPL7 was detected with rabbit-anti OSBPL7 antibody (Sigma) and secondary donkey anti-rabbit antibody labeled with Alexa Fluorophore 488 (Invitrogen). Insulin was detected with guinea pig-anti insulin antibody (Dako) and secondary goat anti-guinea pig antibody labeled with Alexa Fluorophore 555 (Invitrogen).
  • Compound F treatment significantly improved islet function as indicated by improvements in glucose and KCl- stimulated insulin secretion ex vivo. This is an in vivo model/assay for OSBPL7 compound activity. ( Figure 8)
  • OSBPL7 protein variants (see Figure 9). The position of the individual protein domains are annotated according to the amino acid sequence of human full- length OSBPL7. Asterisks indicate point mutations in the PH-domain. Abbreviations: PH- domain, Pleckstrin homology domain; FFAT, Two phenylalanines in an acidic tract motif (EFFDAxE); ORD, oxysterol recognition domain; eGFP, enhanced green fluorescence protein; myc, protein tag derived from the c-myc gene; lOxHis, affinity tag of 10-mer histidine residues.
  • Lehto M Tienari J, Lehtonen S, Lehtonen E, Olkkonen VM: Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and
  • Radzikowska A, Thiele C, Olkkonen VM The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket.
  • Olkkonen VM The OSBP-related protein family in humans. J Lipid Res 2001, 42(8): 1203- 1213. 14. Lehto M, Tienari J, Lehtonen S, Lehtonen E, Olkkonen VM: Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and

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Abstract

L'invention concerne un procédé de dosage pour déterminer la capacité de composés à moduler la fonction et la distribution d'une protéine OSBPL7 dans des cellules, et son utilisation dans la découverte et le criblage de médicaments. L'invention concerne également l'utilisation de carboxamides de pyridine en tant que modulateurs d'OSBPL7 qui sont utilisés pour traiter des maladies telles que le diabète.
PCT/EP2014/058961 2013-05-06 2014-05-02 Procédés d'identification de modulateurs d'osbpl7 et utilisation de ces modulateurs pour traiter les maladies associées à osbpl7 WO2014180741A1 (fr)

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JP7478133B2 (ja) 2018-07-26 2024-05-02 エフ． ホフマン－ラ ロシュ アーゲー 腎障害の処置で使用するための化合物
WO2023039063A1 (fr) * 2021-09-08 2023-03-16 River 3 Renal Corp. Forme solide de 5-(3,4-dichlorophényl)-n-((1r,2r)-2-hydroxycyclohexyl)-6-(2,2,2-trifluoroéthoxy)nicotinamide desitnée à être utilisée dans le traitement de maladies rénales
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