WO2014180741A1

WO2014180741A1 - Methods of identifying modulators of osbpl7 and the use of such modulators for treatment of diseases associated with osbpl7

Info

Publication number: WO2014180741A1
Application number: PCT/EP2014/058961
Authority: WO
Inventors: Jeremy Beauchamp; Thomas Hartung; Christian Kemmer; Peter Mohr; Stephan Roever; Matthew Wright
Original assignee: F. Hoffmann-La Roche Ag; Hoffmann-La Roche Inc.
Priority date: 2013-05-06
Filing date: 2014-05-02
Publication date: 2014-11-13

Abstract

The present invention refers to an assay method for determining the ability of compounds to modulate the function and distribution of OSBPL7 protein in cells and its use in drug discovery and screening. The invention also refers to the use of pyridine carboxamides as OSBPL7 modulators that are useful for the treatment of diseases such as diabetes.

Description

Methods of identifying modulators of OSBPL7 and the use of such modulators for

treatment of diseases associated with OSBPL7

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.

Background of the Invention

The 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

intracellulary between the Golgi and cell membrane. 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. No wholly satisfactory HDL-elevating therapies exist. Niacin can significantly increase HDL, but has serious tolerability issues which reduce compliance. Fibrates and the HMG CoA reductase inhibitors raise HDL-cholesterol only modestly (10-12%). As a result, there is a significant unmet medical need for a well tolerated agent which can significantly elevate plasma HDL levels. An assay method for the high throughput screening of compounds that are able to increase ABCAl protein levels or upregulate ABCAl expression is therefore needed.

It has been found that 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. However, 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. 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). SAR studies using a series of various pyridine carboxamides demonstrated that the ability of binding to OSBPL7 correlated with up regulation of ABCAl in cell assays. To date, only a few publications have appeared in the literature regarding OSBPL7. One publication suggested it is enriched in the gastrointestinal tract and in immune cells [1] . A second publication identified GATE28 as an interacting protein. A large meta-analysis of GWAS studies of loci related to human plasma cholesterol levels identified OSBPL7 among 95 genes. Although the specific functions of OSBPL7 are unknown, other OSBP family members have been implicated in sterol sensing [3], membrane lipid and protein trafficking [4], cell signaling [5], and lipid homeostasis [6] .

The identification of 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. Moreover, 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. Moreover, OSBPL7 modulating drugs may be useful as

medicaments for treatment of OSBPL7-related diseases such as diabetes or impaired insulin secretion. Thus, there is a need for novel methods to screen for modulators of OSBPL7 and for new compounds that are able to modulate and OSBL7 with the potential to become new medicaments for the treatment of OSBPL7-related diseases.

Summary of the Invention It is an object of the present invention to provide a method for determining the ability of a test compound bind to Oxysterol Binding Associated Protein-Like 7 (OSBPL7), comprising 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, and d) determining the amount of immunoprecipitated OSBPL7 protein in each aliquot by

immunohistochemistry and the amount of bound radioligand in each aliquot by measuring the radioactive decay, wherein a reduction in radioligand binding indicates that the test compound modulates the radioligand binding to OSBPL7.

In one embodiment, 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 immunohistochemistry and the amount of bound radioligand in each aliquot by measuring the radioactive decay, wherein a reduction in radioligand binding indicates that the test compound modulates the radioligand binding to OSBPL7.

In one aspect, 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.

In another aspect, 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.

In one aspect, 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.

In another aspect, 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.

In a further aspect, 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

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 tagged 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 intracellular protein

translocation in response to binding of a test compound. The accumulation of tagged OSBP7 thus also indicates the pharmacological activity of a test compound.

In another aspect, 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, 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, V618I, V618A, K636A, I641A, I641V, R649A, I651A and I651V.

In one aspect, the invention is concerned with the use of an OSBPL7 protein variant as defined above in the methods as described herein before.

In another aspect, the invention also relates to a compound of the formula I

wherein one of A 1 and A2 is N and the other one of A 1 and A2 is CH;

R¹ is selected from the group consisting of Ci-7-alkyl,

Ci-7-cycloalkyl,

C3_7-cycloalkyl-C₁_7-alkyl,

C_1-7-alkoxy-C_1-7-alkyl,

halogen-Ci-7-alkyl,

lower heterocyclyl-Ci-7-alkyl wherein the heterocyclyl group is unsubstituted or substituted by oxo, and

lower heteroaryl-Ci-7-alkyl wherein the heteroaryl group is unsubstituted or mono- or di- substituted by lower alkyl;

R² and R⁶ independently from each other are hydrogen or halogen;

R 3 and R 5 independently from each other are selected from the group consisting of

hydrogen, Ci-7-alkyl, C^-alkoxy, halogen, halogen-C^-alkyl, halogen-C^-alkoxy and cyano;

R⁴ is selected from the group consisting of hydrogen, Ci-7-alkyl, Ci-7-alkoxy, halogen,

halogen-Ci-7-alkyl, halogen-C^-alkoxy, amino and cyano;

R is selected from the group consisting of C_1-7-alkyl,

C3_7-cycloalkyl, said cycloalkyl being unsubstituted or substituted by hydroxy,

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

-(CH₂)_m-, wherein m is selected from 0 or 1, and

-NR 8 -, wherein R 8 is hydrogen or Ci-7-alkyl, and pharmaceutically acceptable salts thereof, for use as modulator of OSBPL7. In one aspect, the invention thus also relates to a compound of the formula I

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,

Ci-7-cycloalkyl,

C3_7-cycloalkyl-C₁_7-alkyl,

C_1-7-alkoxy-C_1-7-alkyl,

halogen-Ci-7-alkyl,

R² and R⁶ independently from each other are hydrogen or halogen;

halogen-Ci-7-alkyl, halogen-C^-alkoxy, amino and cyano;

R is selected from the group consisting of C_1-7-alkyl,

C3_7-cycloalkyl, said cycloalkyl being unsubstituted or substituted by hydroxy,

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

-(CH₂)_m-, wherein m is selected from 0 or 1, and

8 8

-NR -, wherein 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. In a particular aspect, the disease is diabetes.

In a further aspect, the invention relates to a compound of formula I, which is

6-(3,4-dichlorophenyl)-N-[(7R,2R)-2-hydroxycyclohexyl]-5-(2,2,2-trifluoroethoxy)pyridine-2- carboxamide.

Detailed Description of the Invention

It is an object of the present invention to provide a method for determining the ability of compounds to bind to OSBPL7 which fulfills the requirements by having a robust and reliable readout. 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, and d) determining the amount of immunoprecipitated OSBPL7 protein in each aliquot by immunohistochemistry and the amount of bound radioligand in each aliquot by measuring the radioactive decay, wherein a reduction in radioligand binding indicates that the test compound modulates the radioligand binding to OSBPL7. In particular, 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

immunohistochemistry and the amount of bound radioligand in each aliquot by measuring the radioactive decay, wherein a reduction in radioligand binding indicates that the test compound modulates the radioligand binding to OSBPL7. 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. After a certain incubation period that may last from 1 to 30 hours, in particular from 1 to 6 hours, more particularly 3 hours the medium is removed and the cells are washed in order to remove unbound compounds. In particular, they are washed with ice cold phosphate buffered saline (PBS). The cells are then irradiated with UV-light (A = 366 nm, 8W) to covalently cros s-link the ( Ill- labeled radioligand to the OSBPL7 protein or variant thereof. 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

immunoprecipated protein by gel electrophoresis. In particular, the separation is done by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 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.

For the method according to the invention 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. The term "protein" or "polypeptide" as used herein, refers to a polymer of amino acids, and not to a specific length. Thus peptides, oligopeptides and protein fragments are included within the definition of "protein".

The term "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. In particular, 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 2μg DNA of the expression vector encoding OSBPL7 or a variant thereof with 200μΙ_^ 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.

"Test compounds" can be any compounds that are to be assayed for their ability to act as OSBPL7 modulator. In particular, organic compounds (small molecules) from a chemical substance library are to be assayed. Typically, 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.

A "small molecule" is defined herein as an organic molecule having a molecular weight below 800 Daltons. The term "labeled" 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.

In particular, 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. When the specific peptide has affinity to metal ions, 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. 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.

A "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.

As an alternative, proteins can also be labeled with fluorophores. Particular useful for the labeling of proteins are 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

(Molecular Probes, Invitrogen).

More particularly, 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. Through employing robotics and software that allows large amounts of data processing and analysis, HTS allows conducting millions of biochemical, genetic or pharmacological tests in a short timeframe. 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.

In one aspect, 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.

Immunohistochemical detection means using an antibody that binds to OSBPL7 or the variant thereof.

In a further aspect, 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. In another aspect, 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. Furthermore, 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. In particular, 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,

V618I, V618A, K636A, I641A, I641V, R649A, I651A and I651V.

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. In particular 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.

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.

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. Such expression vectors can be prepared by methods known in the art. For example 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. In particular, 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

wherein of A¹ and A² is N and the other one of A¹ and A² is CH; is selected from the group consisting of Ci-7-alkyl,

Ci-7-cycloalkyl,

C₃_7-cycloalkyl-C₁_7-alkyl,

C_1-7-alkoxy-C_1-7-alkyl,

halogen-Ci-7-alkyl,

lower heteroaryl-Ci-7-alkyl wherein the heteroaryl group is unsubstituted or mono- or di- substituted by lower alkyl; 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 .

halogen-Ci-7-alkyl, halogen-C^-alkoxy, amino and cyano;

R is selected from the group consisting of C_1-7-alkyl,

C₃_7-cycloalkyl, said cycloalkyl being unsubstituted or substituted by hydroxy,

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

-(CH₂)_m-, wherein m is selected from 0 or 1, and

8 8

-NR -, wherein R is hydrogen or Ci-7-alkyl, and pharmaceutically acceptable salts thereof.

More particularly, the following new compound is described therein:

6-(3,4-dichlorophenyl)-N-[(7R,2R)-2-hydroxycyclohexyl]-5-(2,2,2- trifluoroethoxy)pyridine-2-carboxamide.

The term "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. Examples of straight-chain and branched 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.

The term "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. Examples of lower alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.-butoxy and tert.-butoxy, in particular methoxy.

The term "lower alkoxyalkyl" or "C₁_7-alkoxy-C₁_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. Examples of lower alkoxyalkyl groups are e.g. -CH₂-0-CH₃, -CH₂-CH₂-0-CH , -CH₂-0-CH₂-CH and the groups specifically exemplified herein. More particularly, lower alkoxyalkyl is methoxyethyl.

The term hydroxy means the group -OH.

The term "cycloalkyl" or "C₃_7-cycloalkyl" denotes a saturated carbocyclic group containing from 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl cycloheptyl.

The term "lower cycloalkylalkyl" or "C₃_7-cycloalkyl-C₁_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. Among the lower cycloalkylalkyl groups of particular interest resides cyclopropylmethyl.

The term "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.

The term "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. Examples of lower halogenalkyl groups are e.g. -CF₃, -CHF₂, -CH₂C1, -CH₂CF₃, -CH(CF₃)₂, -CF₂-CF_3> -CH₂-CH₂-CF₃, -CH(CH₃)-CF₃ and the groups specifically exemplified herein. Of particular interest are the groups trifluoromethyl (-CF ) and 2,2,2- trifluoroethyl (-CH₂CF₃).

The term "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. Among the lower halogenalkoxy groups of particular interest are trifluoromethoxy, difluoromethoxy, fluormethoxy and chloromethoxy, more particularly trifluoromethoxy.

The term amino means the group -NH₂.

The term "cyano" means the group -CN.

The term "azido" means the group -N₃. The term "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.

furanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl, isoxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, imidazolyl, pyrazolyl, triazolyl, oxadiazolyl, oxatriazolyl, tetrazolyl, pentazolyl, or pyrrolyl. The term "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.

The term "lower 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. The term "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,

pyrrolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl,

morpholinyl, thiazolidinyl, isothiazolidinyl, oxiranyl, thiadiazolylidinyl, oxetanyl, dioxolanyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, and thiomorpholinyl. Of particular interest are piperidinyl and tetrahydropyranyl.

The term "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 term "oxo" means that a C-atom of the heterocyclyl or heteroaryl ring may be substituted by =0, thus meaning that the heterocyclyl or heteroaryl ring may contain one or more carbonyl (-CO-) groups.

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.

Description of the Figures

Figure 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. In case 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).

Figure 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.

Figure 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: 20μιη.

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.

Figures 6a to 6d: These figures 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

Insle and co-localizes with insulin.

Figure 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. 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.

Examples

Preparation and Purification of the OS BPL7 protein variants

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. 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. Cloning of said cDNAs into pcDNA3.1-OSBPL7-GFP-10xHis while replacing wildtype OSBPL7 variant was performed using Nhel/Notl. Protein purification was performed from either mammalian cells or bacterial cultures expressing said proteins. In particular, OSBPL7(379-842)-GFP-10xHis was expressed by transfection of 4xl0⁶ HEK293 with a transfection mix containing 10μg expression vector DNA, ΙΟΟΟμΕ OptiMEM® I reduced serum medium (Gibco) and 30μί 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. 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.

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.

A schematic representation of the prepared OSBPL7 variants can be found in 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. Preparation of the Pyridine Carboxamides

List of compounds:

Preparation of Compound A

5-(4-Azido-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(lR,2R)-2-hydroxycyclohexyl]-2,4- ditritiopyridine-3-carboxamide a) 5-(4-Amino-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(7R,2R)-2-hydroxycyclohexyl]- pyridine-3-carboxamide

5-Bromo-6-(cyclopropylmethoxy)-N-[(7R,2R)-2-hydroxycyclohexyl]-3- pyridinecarboxamide (CAS Reg. No. 1018783-91-9, 2.4 g, 6.06 mmol) was dissolved in a mixture of toluene (50 mL) and dimethylformamide (5 mL). To this solution was added [1,1 '- bis(diphenylphosphino)-ferrocene]-dichloropalladium(II) dichloro-methane complex (276 mg), 2-chloro-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-benzeneamine (CAS Reg. No. 721960- 43-6, 3.3 g, 13 mmol) and 2 N sodium carbonate solution (6.5 mL). The whole mixture was heated with stirring at 90 °C for 16 h, cooled to room temperature and extracted with ethyl acetate and water. Ethyl acetate phases were pooled, dried with Na₂S0₄, filtered and solvent was removed in vacuo. The residue was purified by chromatography on silica with heptane / ethyl acetate 1: 1. The title compound was isolated (2.5 g, 92 %) as off-white solid; 416.3 (MH)⁺. b) 5-(4-Amino-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(lR,2R)-2-hydroxycyclohexyl]-2,4- ditritiopyridine-3-carboxamide 5-(4-Amino-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(7R,2R)-2-hydroxycyclohexyl]- pyridine-3-carboxamide (8.5 mg, 0.02 mmol) and [l,3-bis(2,4,6-trimethylphenyl)imidazol-2- ylidene]-dimethylphenylphosphine-(l,5-cyclooctadiene) iridium(I) hexafluorophosphate (2.7 mg, 0.15 eq.) were dissolved in dichloromethane (1 mL) and tritiated with tritium gas for 1.5 h. 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 μΜ 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. c) 5-(4-Azido-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(7R,2R)-2-hydroxycyclohexyl]-2,4- ditritiopyridine-3-carboxamide

5-(4-Amino-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(7R,2R)-2-hydroxycyclohexyl]- 2,4-ditritiopyridine-3-carboxamide (1.6 mg, 304 mCi, 79.4 Ci/mmol) was dissolved in THF (0.6 mL). Trifluoroacetic acid ( 50 μί) was added and the mixture was cooled to - 10 °C before adding sodium nitrite (1. 0 mg). The reaction mixture was stirred for 30 min at -10 °C and afterwards sodium azide (1.0 mg) was added and stirring was continued for 10 min at -5 °C and for 1 hour at room temperature. Solvent was removed in vacuo and the residue was partitioned with ethyl acetate and 2 N sodium bicarbonate. Organic phases were pooled, dried and solvent was removed. The residue was purified by filtration over silica with dichloromethane/ methanol 95/5 as eluent and subsequent HPLC purification on a Zorbax Eclipse XDB C- 18 column. The product was isolated as a mixture of tritium isomers (104 mCi) with a specific activity of 78.0 Ci/mmol.

Preparation of Compound B

5-(3,4-dichloro-phenyl)-N-((lR,2R)-2-hydroxy-cyclohexyl)-6-(2,2,2-trifluoro-ethoxy)- nicotinamide The compound was prepared from 5-bromo-6-chloro-3-pyridinecarboxylic acid, (1R,2R)-

2-amino-cyclohexanol and 3,4-dichlorophenylboronic acid in accordance with the procedure described in WO 2011/1029827, Example 3. MS 463.079, 465.077 (M+H)⁺. Preparation of Compound C

N'-(6-chloropyridazin-3-yl)-5-(4-cyanophenyl)-N'-methyl-6-(2,2,2-trifluoroethoxy)pyridine-3- carbohydrazide a) 5-(4-Cyano-phenyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinic acid methyl ester In a 50 mL 4-necked flask, methyl 5-bromo-6-(2,2,2-trifluoroethoxy)nicotinate (1 g, 3.18 mmol, Eq: 1.00, CAS Reg. No. 1211589-51-3) and cesium carbonate (3.11 g, 9.55 mmol, Eq: 3) were combined with toluene (25 mL) and water (2.8 mL) to give a colorless solution. The reaction mixture was 3x degassed and purged with argon; then palladium(II) acetate (14.3 mg, 63.7 μιηοΐ, Eq: 0.02), potassium (4-cyanophenyl)trifluoroborate (732 mg, 3.5 mmol, Eq: 1.1, CAS Reg. No. 850623-36-8) and butyldi- l-adamantylphosphine (68.5 mg, 191 μηιοΐ, Eq: 0.06, CAS Reg. No. 321921-71-5) were successively added. The degassing-purging cycle was repeated after each addition. The reaction mixture was then heated to 120 °C for 5 hours. After cooling, the reaction mixture was poured onto 50mL H₂0 and extracted with AcOEt (2x50mL). The organic layers were washed with H₂0/brine, combined, dried over Na₂S0₄ and concentrated in vacuo. Purification by flash chromatography (silica gel, 50 g, 50% to 100 % CH₂C1₂ in heptane) yielded finally 898mg of the title compound as white foam; MS (ESI) 337.2 (M+H)⁺. b) 5-(4-Cyano-phenyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinic acid

In a 25 mL round-bottomed flask, the above prepared 5-(4-cyano-phenyl)-6-(2,2,2- trifluoro-ethoxy)-nicotinic acid methyl ester (0.891 g, 2.65 mmol, Eq: 1.00) was combined with THF (7 mL) and water (3.5 mL) to give a light yellow biphasic system. Lithium hydroxide (127 mg, 5.3 mmol, Eq: 2) was added and the reaction mixture was stirred at 40 °C for 3 hours when TLC indicated the reaction to be complete. Work up: lOmL H₂0 and 7 mL HC1 IN were added, the mixture extracted with AcOEt (2x50mL), the organic layers were combined, washed with brine, dried over Na₂S0₄ and concentrated in vacuo. Trituration with heptane / EtOAc 9: 1 afforded finally 794 mg of the desired title product as white solid; MS (ESI) 321.2 (M-H)^~. c) N'-(6-chloropyridazin-3-yl)-5-(4-cyanophenyl)-N'-methyl-6-(2,2,2-trifluoroethoxy)pyridine-3- carbohydrazide

In a 5mL round-bottomed flask, the above prepared 5-(4-cyano-phenyl)-6-(2,2,2-trifluoro- ethoxy)-nicotinic acid (0.050 g, 155 μιηοΐ, Eq: 1.00) was combined with THF (2 mL) to give a colorless solution. TBTU (0-(benzotriazol- l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, 74.7 mg, 233 μηιοΐ, Eq: 1.5, CAS Reg. No. 125700-67-6) and N,N-diisopropylethylamine (100 mg, 135 μί, 776 μιηοΐ, Eq: 5) were added. The reaction mixture was stirred for lOmin at RT, then 3-chloro-6-(l-methylhydrazinyl)-pyridazine (29.5 mg, 186 μιηοΐ, Eq: 1.2, CAN 76953-33-8) was added and the reaction mixture kept at RT overnight. Pouring into 25 mL 1 M HC1, extraction with EtOAc (2 x 50 mL), washing with 1 M NaOH, drying over Na₂S0₄ and evaporation of all solvents in vacuo, followed by flash chromatography (silica gel, 10 g, 2% to 10 % MeOH in CH₂C1₂) and crystallization from heptane / AcOEt generated eventually 28mg of the title compound as white solid; MS (ESI) 463.1 , 465.3 (M+H)⁺.

Preparation of Compound D

6-(4-Chloro-phenyl)-5-(2,2,2-trifluoro-ethoxy)-pyridine-2-carboxylic acid (3-isopropyl-isoxazol- 5 -ylmethyl) - amide

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.

543713-30-0) in accordance with the method described in WO 2012/032018, Example 64. LC- MS (UV peak area/ESI) 100.0%, 454.4 (M+H)⁺.

Preparation of Compound E

6-(3,4-Dichlorophenyl)-N-[(7R,2R)-2-hydroxycyclohexyl]-5-(2,2,2-trifluoroethoxy)pyridine-2- carboxamide a) 6-Chloro-2-(3,4-dichlorophenyl)-3-fluoropyridine

In a 100 mL four-necked flask, 2,6-dichloro-3-fluoropyridine (765 mg, 4.61 mmol, Eq: 1.00, CAS Reg. No. 52208-50-1) and potassium (3,4-dichlorophenyl)trifluoroborate (1.21 g, 4.61 mmol, Eq: 1.00, CAN 850623-68-6) were combined with dioxane (23 mL) and water (13 mL). 2M Na₂C0₃ (6.91 mL, 13.8 mmol, Eq: 3) was added, followed by PdCl₂(DPPF)-CH₂Cl₂ adduct (169 mg, 230 μιηοΐ, Eq: 0.05, CAS Reg. No. 95464-05-4). The reaction mixture was 3x degassed and purged with argon and then heated under stirring overnight to 60°C. The reaction mixture was cooled to ambient temperature, poured into 50 mL H₂0 and extracted with tert. butyl-methyl-ether (2xl00mL). The organic layers were washed with H₂0/brine, combined, dried over Na₂S0₄ and concentrated in vacuo. The crude material was purified twice by flash chromatography (silica gel, 70g, 5% to 20% dichloromethane in heptane) to yield 540mg of the title compound as white semisolid. b) 6-Chloro-2-(3,4-dichlorophenyl)-3-(2,2,2-trifluoroethoxy)pyridine

In a 25 mL pear-shaped flask, the above prepared 6-chloro-2-(3,4-dichlorophenyl)-3- fluoropyridine (530 mg, 1.44 mmol, Eq: 1.00) was combined with DMSO (8 mL). KOH (118 mg, 2.1 mmol, Eq: 1.46) and 2,2,2-trifluoroethanol (21 1 mg, 153 μΐ, 2.11 mmol, Eq: 1.47) were added and the reaction allowed to proceed for 2h at ambient temperature. The mixture was poured into 30mL H₂0 and extracted with tert. butyl-methyl-ether (2x40mL). The organic layers were washed with H₂0/brine, combined, dried over Na₂S0₄ and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 70g, 5% to 20% EtOAc in heptane) to yield 444mg of the title compound as colorless liquid; MS (ESI) 356.3, 358.3, 360.3 (M+H)⁺. c) Methyl 6-(3,4-dichlorophenyl)-5-(2,2,2-trifluoroethoxy)pyridine-2-carboxylate

In a 35 mL autoclave the above prepared 6-chloro-2-(3,4-dichlorophenyl)-3-(2,2,2- trifluoroethoxy)pyridine (437 mg, 1.22 mmol, Eq: 1.00) was dissolved in 5 mL MeOH. Protected from oxygen and moisture by Argon, PdCl₂(DPPF)-CH₂Cl₂ adduct (75.2 mg, 92 μιηοΐ, Eq: 0.083, CAS Reg. No. 95464-05-4) was added, followed by triethylamine (233 mg, 321 μL·, 2.31 mmol, Eq: 2.31). The reactor was then flushed three times with CO, pressurized to 70 bar, and the carbonylation then allowed to proceed for 20 h at 1 10 °C. After cooling and release of the pressure, the crude reaction mixture was concentrated in vacuo. The residue was then redissolved in AcOEt/H₂0 and transferred to a separatory funnel. The aqueous layer was back-extracted with EtOAc, the organic layers were washed with H₂0 and brine, combined, dried over Na₂S0₄ and concentrated in vacuo. Flash chromatography (silica gel, 70g, 10% to 40% EtOAc in heptane) delivered finally 327 mg of the title product as white solid; MS (ESI) 380.4, 382.4 (M+H)⁺. d) 6-(3,4-Dichlorophenyl)-5-(2,2,2-trifluoroethoxy)pyridine-2-carboxylic acid

In a 25 mL pear-shaped flask, the above prepared methyl 6-(3,4-dichlorophenyl)-5-(2,2,2- trifluoroethoxy)picolinate (326 mg, 858 μιηοΐ, Eq: 1.00) was combined with tetrahydrofuran (5 mL) to give a colorless solution. Water (2.5 ml) was added, followed by LiOH (41.1 mg, 1.72 mmol, Eq: 2) and the reaction mixture was stirred for 2hr at 40 °C. The reaction mixture was poured into 5mL sat. NH₄C1 sol. and 3mL IN KHS0₄ sol. and extracted with EtOAc (2 x 30 mL).The organic layers were combined, dried over Na₂S0₄ and concentrated in vacuo to leave 322 mg of the title acid as white foam; MS (ESI) 366.4, 368.4 (M+H)⁺. e) 6-(3,4-Dichlorophenyl)-N-[(lR,2R)-2-hydroxycyclohexyl]-5-(2,2,2-trifluoroethoxy)pyridine- 2-carboxamide

In a 25 mL pear-shaped flask, the above synthesized 6-(3,4-dichlorophenyl)-5-(2,2,2- trifluoroethoxy)-picolinic acid (322 mg, 879 μιηοΐ, Eq: 1.00) was dissolved in DMF (12 mL). TBTU (0-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, 424 mg, 1.32 mmol, Eq: 1.5, CAS Reg. No. 125700-67-6) and N,N-diisopropylethylamine (568 mg, 768 μΐ, 4.4 mmol, Eq: 5) were added and the reaction mixture stirred for lOmin at ambient temperature before (lR,2R)-2-aminocyclohexanol hydrochloride (160 mg, 1.06 mmol, Eq: 1.2, CAS Reg. No. 13374-31-7) was added without additional solvent. The reaction was then allowed to proceed for 3 h at room temperature. The crude mixture was diluted with H₂0 and extracted with CH₂C1₂. The organic layers were combined, dried over Na₂S0₄ and concentrated in vacuo. Purification by flash chromatography (basic alumina, 50g, 10% to 80% EtOAc in heptane) and precipitation of the crude product from EtOAc and heptane afforded the title amide as white solid; high resolution MS (ESI) 463.0798, 465.0769 (M+H)⁺; expected: 463.0798, 465.0768.

Preparation of Compound F

6-(3,4-Dichlorophenyl)-N-[(7R,2R)-2-hydroxycyclohexyl]-5-(2,2,2-trifluoroethoxy)pyridine-2- carboxamide

The title compound was synthesized in analogy to the procedure described in WO

2012/049190, Example 3, using 5-(4-chloro-phenyl)-6-(2,2,2-trifluoro-ethoxy)-pyridin-3- ylamine and nicotinic acid as starting materials, MS (LC/MS): 408.0 (M+H).

Assay format 1: Cell-based OSBPL7 ligand binding assay

120.000 baby hamster kidney cells (BHK21) were seeded in 12well plates. After 16h cells were transfected by mixing 2μg expression vector, which encodes the c-terminal FLAG-tagged OSBPL7 variant, with 200μί OptiMEM® I reduced serum medium. After addition of 6 iL Megatran 1.0 transfection reagent (ratio DNA:Megatranl.O (w/v) 1:3) the transfection mix was vortexed, incubated for 15min at room temperature, and finally added to the cells. 24h after transfection the medium was replaced by fresh Dulbeccos Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) Penicillin/Streptomycin mix (Gibco) and containing ΙμΜ tritium labeled compound A and either ΟμΜ, 10μΜ or 50μΜ of test compounds. After incubation for 3h at 37°C, 5% C0₂, the medium was removed and unbound compounds were removed by repeated washing with 2mL ice cold Dulbeccos

Phosphate Buffered Saline (DPBS) (Gibco). Cells were covered with 2mL ice cold DPBS, placed on ice and irradiated for 15min with UV-light ( =366nM, 8W) to covalently cross-link the bound compound A to the protein. 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 100μg of the soluble protein lysate fraction by overnight incubation with EZview Red anti-flag M2 affinity gel (Sigma) at 4°C. Unbound proteins were removed by repeated washing with immunoprecipitation buffer (50mM Hepes (pH 7.4), 150mM NaCl, l%(v/v) Triton X100, 5mM EDTA). The immunoprecipitated proteins were eluted in 50μί NuPAGE LDS Sample Buffer (Invitrogen) containing 10% (v/v) NuPAGE Sample Reducing Agent (Invitrogen). For OSBPL7 quantification 10μg of eluted proteins were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS- PAGE) using NuPAGE 4-12% Bis-Tris gels (Life Technologies). 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). In parallel, binding of tritiated compound A was analyzed by separation of 50μg eluted proteins by SDS-PAGE as described above. The NuPAGE 4-12% Bis-Tris gels containing the separated proteins were fixed for 30min at room temperature in fixation solution (25% (v/v) isopropanol, 65% (v/v) water, and 10% (v/v) acetic acid). After removal of the fixation solution the gel was incubated for 15-30min in amplifier solution (Amersham) with gentle agitation, followed by lh incubation with gel drying solution (Invitrogen). The gel was vacuum dried at 60-80°C for 2h and finally exposed to Amersham Hyperfilm MP (GE Healthcare Life Sciences) for 3weeks (see also Figure 1). 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 ΟμΜ, 10μΜ or 50μΜ 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

70.000 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), 50μΜ beta mercaptoethanol (Sigma), and l%(v/v) Penicillin/Streptomycin mix (Gibco)). For overexpression experiments the medium was replaced by fresh growth medium containing Lipofectamine 2000 transfection reagent and either lOOng/well DNA of the expression plasmid encoding OSBPL7-GFP or the empty expression vector. 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 200μί 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. 200μί KRBH-buffer containing 2.0mM glucose and 10μΜ 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 10μΜ 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).

Assay format 4: OSBPL7 -modulator binding leads to intracellular OSBPL7

translocation and protein accumulation 300.000 HeLa cells were seeded in 6 well plates and transfected with 2μg OSBPL7-GFP- lOxHis encoding expression vectors using Megatran 1.0 transfection reagent (Origene) (ratio DNA:Megatranl.O (w/v) 1:3). After 8h, cells were trypsinized and seeded on a 4well Nunc LabTekll chambered coverglass (80.000 cells per well) and grown for 16h for attachment. Cells were treated for 8h with 50μΜ of the indicated compounds, the nuclei were stained for 30min with Hoechst 33342 dye (Invitrogen) and the cellular localization of OSBPL7-GFP was analyzed by fluorescence microscopy using an Axio Observer.Zl Inverse Microscope (Zeiss) (see Figure 4). The arrows in the image in the middle indicate intracellular accumulation of OSBPL7 in response to OSBPL7 modulation by compound E. Scale bar: 20μιη

Modeling of the oxy sterol binding domain identified amino acids essential for ligand binding

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

Based on the Hidden Markov Model of the OSBPL7 binding pocket (Figure 5) a sequential mutational analysis of amino acids lining the binding pocket was performed to test the importance of these residues for ligand / compound A binding. 120.000 BHK21 cells were seeded in 12well plates in DMEM (Gibco) containing 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) Penicillin/Streptomycin mix (Gibco). After 16h cells were transfected with Megatran 1.0 and 2μg expression vector DNA encoding FLAG-tagged variants of either full-length human OSBPL7 carrying the indicated point mutation or native OSBPL7. After protein expression for 24h the medium was exchanged by fresh medium containing ΙμΜ compound A and cells were incubated for 3h. The medium was removed and unbound compound A was removed by repeated washing with 2mL ice cold phosphate buffered saline (PBS). Cells were covered with 2mL ice cold PBS, placed on ice and irradiated for 15min with UV-light ( =366nM, 8W) to covalently cross-link the bound compound A to OSBPL7 variants. The PBS was removed, and cells were lysed with 200uL RIPA buffer followed by ultrasonication. OSBPL7 was

immunoprecipitated from 100μg of the soluble protein lysate fraction using anti-FLAG antibody. The immunoprecipitated proteins were separated by SDS-PAGE and the amount of OSBPL7 protein was estimated by immunohistochemistry. In parallel, compound A binding was detected by autoradiography (Figure 6). Expression of OSBPL7 in human islets and rat pancreatic beta cell line Insle and co- localization with insulin.

Sections, 5 μιη 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 μg/ml for 1 hour: mouse anti-human OSBPL7 (Roche clone, 2/13); guinea pig anti-swine insulin and rabbit anti-glucagon (Dako). The tissue staining was visualized using the secondary antibody detection: Alexa Fluor® 488 donkey anti-mouse IgG; Alexa Fluor® 555 goat anti-guinea pig IgG; and Alexa Fluor® 350 donkey anti-rabbit IgG or DAPI staining (Invitrogen). Insle cells (200.000) were grown for 48h on LabTEK chamber slides. Cells were washed with PBS and fixed and permeabilized using the Image-IT fix -perm kit

(Molecular Probes) according to the manuals. 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).

(Figure 7) In vivo model/assay for OSBPL7 compound activity in diabetic ApoE knockout mice

ApoE knockout mice were fed a western-type diet and treated with lOOmg/kg compound F, or vehicle as control. After a 7 day treatment period the murine islets were explanted and the insulin secretion in response to 2.8mM, 8.3mM glucose, or 2.8mM glucose in the presence of 20mM potassium chloride (KCl) stimulus was evaluated (n=3 (i.e. 3 x 10 islets / condition). 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)

Schematic representation of 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. Literature References:

1. Lehto M, Tienari J, Lehtonen S, Lehtonen E, Olkkonen VM: Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and

intracellular localization of ORP3, ORP6, and ORP7. Cell Tissue Res 2004, 315(l):39-57.

2. Suchanek M, Hynynen R, Wohlfahrt G, Lehto M, Johansson M, Saarinen H,

Radzikowska A, Thiele C, Olkkonen VM: The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket.

Biochem J 2007, 405(3):473-480.

3. Im YJ, Raychaudhuri S, Prinz WA, Hurley JH: Structural mechanism for sterol sensing and transport by OSBP-related proteins. Nature 2005, 437(7055): 154- 158. 4. Du X, Kumar J, Ferguson C, Schulz TA, Ong YS, Hong W, Prinz WA, Parton RG, Brown AJ, Yang H: A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. / Cell Biol 2011, 192(1): 121-135.

5. Wang PY, Weng J, Anderson RG: OSBP is a cholesterol-regulated scaffolding protein in control of ERK 1/2 activation. Science 2005, 307(5714): 1472-1476. 6. Beh CT, Rine J: A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution. / Cell Sci 2004, 117(Pt 14):2983-2996. 7. Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, Iwata S: GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 2008, 3(5):784-798.

8. Goto A, Liu X, Robinson CA, Ridgway ND: Multisite phosphorylation of oxysterol- binding protein regulates sterol binding and activation of sphingomyelin synthesis. Mol Biol Cell 2012, 23(18):3624-3635.

9. Banerji S, Ngo M, Lane CF, Robinson CA, Minogue S, Ridgway ND: Oxysterol binding protein-dependent activation of sphingomyelin synthesis in the golgi apparatus requires phosphatidylinositol 4-kinase Ilalpha. Mol Biol Cell 2010, 21(23):4141-4150. 10. Takahashi T, Suzuki T: Role of sulfatide in normal and pathological cells and tissues.

J Lipid Res 2012, 53(8): 1437-1450.

11. Osterbye T, Jorgensen KH, Fredman P, Tranum- Jensen J, Kaas A, Brange J,

Whittingham JL, Buschard K: Sulfatide promotes the folding of proinsulin, preserves insulin crystals, and mediates its monomerization. Glycobiology 2001, ll(6):473-479. 12. Westerlund B, Slotte JP: How the molecular features of glycosphingolipids affect domain formation in fluid membranes. Biochim Biophys Acta 2009, 1788(1): 194-201.

13. Lehto M, Laitinen S, Chinetti G, Johansson M, Ehnholm C, Staels B, Ikonen E,

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

intracellular localization of ORP3, ORP6, and ORP7. Cell Tissue Res 2004, 315(l):39-57. Epub 2003 Oct 2031.

Claims

1. A method for determining the ability of a test compound to bind to Oxysterol Binding Associated Protein-Like 7 (OSBPL7), comprising 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, and d) determining the amount of immunoprecipitated OSBPL7 protein in each aliquot by

2. The method for determining the ability of a test compound to bind to OSBPL7, wherein the binding in cells overexpressing OSBPL7 or a variant thereof is tested, 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, 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

3. The method according to claims 1 or 2, wherein the ( H)-labeled radioligand is 5-(4- azido-3-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(7R,2R)-2-hydroxycyclohexyl]-2,4- ditritiopyridine-3-carboxamide.

4. The method according to any one of claims 1 to 3, wherein the immunoprecipitation carried out with an antibody that binds OSBPL7 or a variant thereof.

5. The method according to any one of claims 1 to 4, wherein the OSBPL7 protein expressing cells are produced by transfection with a vector expressing OSBPL7 or a variant thereof.

6. The method according to any one of claims 1 to 5, wherein the ability of a test compound to bind to OSBPL7 is determined.

7. The method according to any one of claims 1 to 6, 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.

8. The method according to any one of claims 1 to 7, wherein the incubation period in step a) lasts 1 to 30 hours, particularly 3 hours.

9 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 OSBPL7with a test medium

translocation in response to binding of a test compound.

10. 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, 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, V618I, V618A, K636A, I641A, I641V, R649A, I651A and I651V.

11. Use of an OSBPL7 protein variant according to claim 10 in a method as claimed in any one of claim 1 to 9.

12. A compound of the formula I

wherein one of A 1 and A2 is N and the other one of A 1 and A2 is CH; R¹ is selected from the group consisting of Ci-7-alkyl,

Ci-7-cycloalkyl,

C₃_7-cycloalkyl-C₁_7-alkyl,

C_1-7-alkoxy-C_1-7-alkyl,

halogen-Ci-7-alkyl,

R² and R⁶ independently from each other are hydrogen or halogen;

halogen-Ci-7-alkyl, halogen-C^-alkoxy, amino and cyano;

R is selected from the group consisting of C_1-7-alkyl,

G is selected from the group consisting of

-(CH₂)_m-, wherein m is selected from 0 or 1, and

-NR 8 -, wherein R 8 is hydrogen or Ci-7-alkyl, and pharmaceutically acceptable salts thereof,

for use as a modulator of OSBPL7.

13. A compound of the formula I

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,

Ci-7-cycloalkyl,

C3_7-cycloalkyl-C₁_7-alkyl,

C_1-7-alkoxy-C_1-7-alkyl,

halogen-Ci-7-alkyl,

R² and R⁶ independently from each other are hydrogen or halogen;

halogen-Ci-7-alkyl, halogen-C^-alkoxy, amino and cyano;

R is selected from the group consisting of C_1-7-alkyl,

C3_7-cycloalkyl, said cycloalkyl being unsubstituted or substituted by hydroxy,

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- C_1-7-alkyl, and

G is selected from the group consisting of

-(CH₂)_m-, wherein m is selected from 0 or 1, and

8 8

-NR -, wherein 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.

14. A compound of the formula I for use in the treatment of a disease according to claim 13, wherein the disease is diabetes.

15. A compound of formula I, which is