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WO2002084301A2 - Test de criblage d'agents modulant l'activite de la proteine abca1 - Google Patents

Test de criblage d'agents modulant l'activite de la proteine abca1 Download PDF

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Publication number
WO2002084301A2
WO2002084301A2 PCT/CA2002/000489 CA0200489W WO02084301A2 WO 2002084301 A2 WO2002084301 A2 WO 2002084301A2 CA 0200489 W CA0200489 W CA 0200489W WO 02084301 A2 WO02084301 A2 WO 02084301A2
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Prior art keywords
abca1
agent
protein
cholesterol
activity
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WO2002084301A3 (fr
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Lin-Hua Zhang
Michael R. Hayden
Scott M. Newman
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University of British Columbia
Xenon Pharmaceuticals Inc
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University of British Columbia
Xenon Genetics Inc
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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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates generally to high throughput screening assays for agents capable of modulating the activity of the protein formed by expression of the ABCA1 gene, which assays employ a number of ABCA1- protein interacting polypeptides.
  • HDL-C high density lipoprotein cholesterol
  • CAD coronary artery disease
  • peripheral vascular disease is a blood lipid abnormality which correlates with a high risk of cardiovascular disease (CVD), in particular coronary artery disease (CAD), but also cerebrovascular disease, coronary restenosis, and peripheral vascular disease.
  • CVD cardiovascular disease
  • CAD coronary artery disease
  • cerebrovascular disease coronary restenosis
  • peripheral vascular disease is a blood lipid abnormality which correlates with a high risk of cardiovascular disease (CVD), in particular coronary artery disease (CAD), but also cerebrovascular disease, coronary restenosis, and peripheral vascular disease.
  • HDL, or 'good cholesterol' levels are influenced by both environmental and genetic factors. Epidemiological studies have consistently demonstrated that plasma
  • HDL-C concentration is inversely related to the incidence of CAD.
  • HDL-C levels are a strong graded and independent cardiovascular risk factor. Protective effects of an elevated HDL-C persist until 80 years of age.
  • a low HDL-C is associated with an increased CAD risk even with normal ( ⁇ 5.2 mmol/l) total plasma cholesterol levels.
  • Coronary disease risk is increased by 2% in men and 3% in women for every 1 mg/dL (0.026 mmol/l) reduction in HDL-C and in the majority of studies this relationship is statistically significant even after adjustment for other lipid and non-lipid risk factors.
  • Decreased HDL-C levels are the most common lipoprotein abnormality seen in patients with premature CAD.
  • HDL-C levels are important predictors of CAD.
  • those with isolated low HDL cholesterol had a 65% increased death rate compared to diabetics with normal HDL cholesterol levels (>0.9 mmol/l).
  • HDL cholesterol level is an important predictor of CAD.
  • Low HDL cholesterol levels thus constitute a major, independent, risk for CAD.
  • Absolute levels of HDL cholesterol may not always predict risk of CAD.
  • CETP cholesterol ester transfer protein
  • individuals display an increased risk of developing CAD, despite increased HDL cholesterol levels.
  • What seems to be important in this case is the functional activity of the reverse cholesterol transport pathway, the process by which intracellular cholesterol is trafficked out of the cell to acceptor proteins such as ApoAI or HDL.
  • Other important genetic determinants of HDL cholesterol levels, and its inverse relation with CAD may reside in the processes leading to HDL formation and intracellular cholesterol trafficking and efflux. To date, this process is poorly understood, however, and clearly not all of the components of this pathway have been identified. Thus, defects preventing proper HDL-mediated cholesterol efflux may be important predictors of CAD.
  • HDL particles are central to the process of reverse cholesterol transport and thus to the maintenance of tissue cholesterol homeostasis.
  • This process has multiple steps which include the binding of HDL to cell surface components, the acquisition of cholesterol by passive absorption, the esterification of this cholesterol by LCAT (lecithin cholesterol acyl transferase) and the subsequent transfer of esterified cholesterol by CETP, to VLDL (very low density lipoprotein) and chylomicron remnants for liver uptake. Each of these steps is known to impact the plasma concentration of HDL.
  • TD Tangier disease
  • HDL-C HDL cholesterol
  • OMIM On-line Mendelian Inheritance in Man
  • OMIM 205400 autosomal recessive trait
  • Tangier Disease patients accumulate cholesterol esters in several tissues, resulting in characteristic features, such as enlarged yellow tonsils, hepatosplenomegaly, peripheral neuropathy, and cholesterol ester deposition in the rectal mucosa.
  • Defective removal of cellular cholesterol and phospholipids by ApoAI as well as a marked deficiency in HDL mediated efflux of intracellular cholesterol has been demonstrated in TD fibroblasts. Even though this is a rare disorder, defining its molecular basis could identify pathways relevant for cholesterol regulation in the general population.
  • the decreased availability of free cholesterol for efflux in the surface membranes of cells in Tangier Disease patients appears to be due to a defect in cellular lipid metabolism or trafficking.
  • ABCA1 transcription, translation, processing, transport to cellular site, etc., etc.
  • these interacting proteins are suitable drug targets/intervention sites for modulating cellular phospholipid efflux and cholesterol efflux, which has been shown to be related to the level of plasma high density lipoprotein cholesterol (HDL-C) (Brooks-Wilson et al. 1999).
  • AIPs may be involved in the other aspects of the ABCA1 protein life- cycle, including such things as helping to achieve the correct folding and ER- retention of ABCA1 protein control cholesterol efflux.
  • AIPs may help ABCA1 protein to fold in a transient conformational structure for its normal function (such as passing the quality control process to reach to plasma membranes where it functions normally); AIPs may regulate ABCA1 protein for appropriate retention time with ER, or release off, or degradation; AIPs may interact with ABCA1 to form a physiological active molecule.
  • proteins that interact with ABCA1 regulate or modulate its activity or expression. Because control of ABCA1 function or stability by effector proteins would mediate cholesterol and/or phospholipid levels, especially through efflux, such ABCA1 -interacting proteins (or AIPs) present therapeutic targets for the screening and development of chemical agents able to raise and sustain HDL levels.
  • ABCA1 -interacting proteins or AIPs
  • Such proteins include those disclosed herein and thus these proteins can serve in regulating cellular cholesterol homeostasis and plasma high-density lipoprotein-cholesterol through binding to ABCA1.
  • the yeast two-hybrid CytoTrap system from Stratagene, Inc. was used to isolate ABCA1 interacting proteins from the human lung cDNA library.
  • the bait is prepared with the human ABCA1 cDNA (SEQ ID NO: 34) coding amino acids 1873-2261 fused to the human SOS protein in a yeast expression vector pSOS.
  • the prey cDNAs from human lung tissue in vector pMyr fused in myristoylation signal sequence, which helps the prey hybrid to attach to plasma membranes driven by the promoter Gal 1 were screened by the pSOS-ABCA1 (AA 1873-2261) bait.
  • ABCA1 (AA 1873-2261) and its partners has been confirmed in yeast two-hybrid assays showing this interaction does not happen between AIPs and the empty bait vector, or the unrelated baits, and other sections of ABCA1 protein (AA 1 -639, 843-1348).
  • SEQ ID NO: 45 The correct full length amino acid sequence for ABCAIis shown herein as SEQ ID NO: 45. It has also been published in Hayden et al (WO 00/55318, Fig. 9A, SEQ ID NO: 1 and WO 01/15676, Fig. 2A, SEQ ID NO: 5 therein.
  • the present invention relates to a method for identifying an agent that modulates ABCA1 biological activity comprising:
  • step (b) determining the activity of an ABCA1 polypeptide in the presence of the agent-contacted AIP of step (a) under conditions promoting said activity, and (c) determining a difference in the activity of said ABCA1 polypeptide of step (b) compared to when said agent-contacted AIP is not present, thereby identifying said agent as a modulator of ABCA1 protein activity.
  • the AIP is a polypeptide encoded by a human gene selected from the group consisting of LIMD1 , KIAA0528, RAD23A, DNAJA2, PDI-ERp58, OAZ1 , LOC51691 , PFDN5, My001 , RP4-744I24, KPNB1 , Bip/GRp78, TPT1, NAP1 L1 , E46, PRB4, Rhoip2, Ubiquinone, AK011474, CGI- 24, and TCP1 , most preferably LIMD1 or KIAA0528.
  • a human gene selected from the group consisting of LIMD1 , KIAA0528, RAD23A, DNAJA2, PDI-ERp58, OAZ1 , LOC51691 , PFDN5, My001 , RP4-744I24, KPNB1 , Bip/GRp78, TPT1, NAP1 L1 , E46, PRB4, Rhoip2, Ubiquinon
  • the contacting may be carried out in vitro or in vivo and the measured activity may be any of reverse cholesterol transport, cholesterol efflux, HDL efflux, and phospholipid efflux, preferably cholesterol efflux or phospholipid efflux.
  • the testing occurs where the agent-contacted AIP is physically bound to said agent, including where there is some type of chemical linkage, which linkage may be covalent, electrostatic or hydrophobic in character.
  • the source of ABCA1 protein activity of step (b) is a cell expressing ABCA1 polypeptide and said comparison is made between said cell contacted with agent-bound AIP and a corresponding cell contacted with AIP free of said agent and wherein any difference in the activities compared in step (c) is at least partly the result of a change in stability of said ABCA1 protein or a change in catabolism of said ABCA1 protein.
  • the present invention relates to a method for identifying an agent that modulates cholesterol or phospholipid levels in an animal, comprising administering to an animal an effective amount of an ABCA1 -protein modulating agent first identified as such using a method of the invention and detecting a change in cholesterol or phospholipid level in said animal thereby identifying an agent said agent as a modulator of cholesterol or phospholipid activity.
  • Plasma cholesterol or phospholipid are especially evaluated, preferably where the result is a decrease in these levels.
  • the animal may be a bird, such as a chicken, or a mammal, including a human being.
  • a transgenic animal may also be utilized, such as one that has been engineered for elevated cholesterol of phospholipid levels.
  • the present invention includes methods of identifying ABCA1 modulating agents by their character as an antagonist of AIPs, such as where they are able to bind AIPs, and wherein the comparison of ABCA1 activity of step (c) is a comparison of lipid transport across a membrane containing ABCA1 polypeptide.
  • ABCA1 may be part of a membrane, such as a liposome, or such membrane may be part of an intact cell.
  • the intact cell is from a mammal, most preferably a human.
  • the intact cell is a recombinant cell, preferably wherein said recombinant cell has been engineered to comprise ABCA1 protein, such as where the recombinant cell does not comprise ABCA1 protein absent said engineering.
  • the engineering is genetic engineering and said cell expresses a gene that encodes ABCA1 protein.
  • Cells useful in the invention need not express a gene that encodes ABCA1 protein but may be physically manipulated to incorporate ABCA1 in their membrane by other than genetic means, such as by physical insertion.
  • the lipid is preferably one of cholesterol, phospholipid, triglyceride and/or HDL-cholesterol.
  • the ABCA1 activity is binding of ATP, or hydrolysis of ATP, by the ABCA1 polypeptide.
  • the present invention also relates to a method for producing a product comprising identifying an agent according to the assay methods of the invention, wherein said product is the data collected with respect to said agent as a result of said process and wherein said data is sufficient to convey the chemical structure and/or properties of said agent.
  • the present invention further relates to methods of treating disorders of lipid metabolism, including elevated cholesterol and phospholipid levels, comprising administering to an animal, especially a human patient, afflicted with such disorder an effective amount of an ABCA1 -modulating agent identified according to the methods disclosed herein.
  • the present invention relates to a process for identifying agents that modulate the activity of ABCA1 protein through their effects on the ability of ABCA1 interacting proteins (AIPs) to bind to and interact with the ABCA1 protein.
  • AIPs ABCA1 interacting proteins
  • Such process permits high throughput screening assays to be performed for the identification of chemical agents, such as small organic compounds, that bind to such AIPs.
  • ABCA1 interacting polypeptides i.e., proteins and polypeptides that interact with the protein produced from the ABCA1 gene.
  • AIPs nucleic acid sequences and amino acid sequences of such AIPs are known to those of skill in the art and such sequences are readily provided by resort to GenBank.
  • GenBank GenBank accession numbers
  • the corresponding sequences along with their respective GenBank accession numbers are tabulated below.
  • the characteristics of such AIPs as individual genes and gene products, and their physiological functions (such as are known) are included.
  • the present invention relates to the use of the gene LIMD1 (SEQ ID NO: 1) and its expression product or polypeptide (SEQ ID NO: 2).
  • This is the Lim-domains-containing-1 gene with GenBank Accession No. NM_014240.
  • the protein is an intracellular signaling structure associated with protein kinases, such as PKA and PKC. It has OMIM Ref.: *604543 and is located on Chromosome 3 at 3p21.3 with a mouse homolog LIMD1 (GenBank# CAB63700). 675 amino acids, having 77% homology (amino acid sequence is SEQ ID NO: 2 for the human).
  • the tissue Expression pattern includes adrenal gland, CNS, kidney, ovary, testis, bladder, breast, colon, head, neck, pancreas, placenta, skin.
  • LIM domains are cysteine-rich motifs which bind zinc and mediate protein-protein interactions. LIM is named from three zinc-binding domain containing proteins, Lin-11 , lsl-1 and Mec-3 ( ref. Dawid I.B., Toyama R and Taira M, 1995 C.R. Acad. Sci. Paris 318:295-306; Gill GN 1995 Structure 3: 1285-1289).
  • the LIM motifs had been previously identified in many key regulators of development pathways.
  • LIM domains bind protein partners via tyrosine-containing motifs; some have PDZ domain.
  • the LIMD1 protein belongs to the third group of LIM domains which has a heterogeneous collection of LIM domains that are generally localized near the carboxyl terminus and are often associated with additional domains, some of which bind to cytoskeletal component.
  • the role of the protein may be to recruit other transcriptional activators depending on the promoter context and LIM-homeodomain partner. This protein is not known to interact indiscriminantly with other proteins so that its interaction with ABCA1 may be physiologically important as a means of regulation of lipid levels.
  • LIMD1 It has been suggested previously that LIMD1 deserves further study for its possible role as a tumor suppressor. (Kiss et al. Hum. Genet. 105(6): 552- 559 (1999).
  • This AIP Interacts with human ABCA1 protein to enable relationship with protein kinase (i.e. PKA or PKC).
  • protein kinase i.e. PKA or PKC.
  • LIM proteins are known to be involved in protein binding. Actual role suggested by other LIM family members. Proper functioning confers a normal function for ABCA1 in regulating cellular cholesterol efflux.
  • the present invention also utilizes the protein product of gene KIAA0528 (SEQ ID NO: 17 -GenBank # #AB011100). No specific chromosomal location has been assigned, although it has BAG assignment RP11 -359J14, and no natural homologs are known. This protein is not known to interact indiscriminantly with other proteins so that its interaction with ABCA1 may be physiologically important as a means of regulation of lipid levels.
  • This clone of the gene was isolated from human brain library with unknown function (see: Nagase T et al: DNA Res, 5(1):31-39 (1998)). This AIP has not heretofore been associated with any known disease process.
  • this protein contains a C2 domain near the N-terminus (at amino acids 28-121 or 29-113 of the KIAA0528 protein).
  • ABCA1 interacts with the C-terminus of this protein.
  • the N-terminus of KIAA0528 protein is associated with protein kinase C and phospholipids and such C2 domains have properties including a protein kinase C region 2 (CalB); Ca2+-binding motif present in phospholipases, protein kinases C, and synaptotamins (among others). Some do not appear to contain Ca2+-binding sites. Particular C2s appear to bind phospholipids, inositol polyphosphates, and intracellular proteins. Unusual occurrence in perforin.
  • Synaptotagmin and PLC C2s are permuted in sequence with respect to N- and C-terminal beta strands.
  • SMART detects C2 domains using one or both of two profiles.
  • KIAA0528 can play an adaptor role.
  • the present invention also utilizes gene RAD23A (SEQ ID NO: 3), which is also known as Homo sapiens RAD23 (Sacctiaromyces cerevisiae) homolog A; HHR23A protein (SEQ ID NO: 4), with GenBank # XM_009054, and #D21235, OMIM: 600061 and is located at chromosome19p13.2. It has a mouse homolog (Mouse RAD23A), with tissue expression in pre-B cell and heart and sub-cellular localization in the nucleus. This gene contains a "ubiquitin associated domain," which is involved in a proteolytic pathway. The HHR23A protein was also identified in relation to a nucleotide excision repair complex. (EMBO J.
  • HHR23 genes have been discussed, (van der Spek, PJ. Et al. Genomics 23(3):651-658 (1994)), however, later work has not found any HHR23A protein bound to the excision repair complex, suggesting this disease association is incorrect. ( Van der Spek, Nucleic Acids Res. 24(13):2551 -9 (1996)).
  • This AIP may help ABCA1 protein (especially mis-folded/mutated) to go to a proteosome for degradation as it contains the ubiquitin-associated domains which have been found to interact with S ⁇ alpha, a subunit of the human 26S proteosome (See: Hiyama H, et al: J. Biol. Chem. 274:28019-28025 (1999)).
  • the present invention also utilizes gene DNAJA2 (SEQ ID NO: 5), called Homo sapiens HIRA interacting protein 4 (dnaJ-like) (HIRIP4) or Homo sapiens DnaJ (Hsp40) homolog, subfamily A, member 2 (DNAJA2).
  • Other names include DnaJ protein, Cell cycle progression 3 protein (DNJ3), CPR3, and PRO3015 mRNA. It has GenBank # NM_005880 and is located on chromosome 16 at 16q11.1-q11.2. Its nearest homolog is Mouse DNA J protein (DnaJ3) 99 % / 411 aa; Rat DnaJ homolog.
  • Tissue expression includes bone, brain, colon, foreskin, heart, lung, lymph, ovary, pooled, prostate, testis, uterus, adrenal gland, and elsewhere. Its full-length cDNA sequence with 1993 nucleotides is (SEQ ID NO: 5) encoding a polypeptide of 412 amino acids (SEQ ID NO: 6).
  • the protein encoded by this gene shares sequence similarity with Hir1 p and Hir2p, the two corepressors of histone gene transcription characterized in the yeast, Saccharomyces cerevisiae. It was originally called HIRIP4. The structural features of this protein suggest that it may function as part of a multiprotein complex.
  • HIRIPs Several cDNAs encoding interacting proteins, HIRIPs, have been identified.
  • DNAJA2 has sequence identity with a DnaJ protein.
  • Eukaryotic DnaJ proteins have also been found to interact with luciferase during the luciferase folding process and thus have a putative chaperonin role. Frydman et al. 1994. Nature 370:111 -117. This AIP does not appear to be involved with, or related to, any known disease.
  • This AIP together with other molecular chaperones, forms a multiprotein complex for stablizing the newly synthesized ABCA1 protein, or correct folding of ABCA1 protein.
  • the present invention also utilizes gene ERp58 (SEQ ID NO: 7), called human liver endoplasmic reticulum P58, ER-resided p57, Erp57, protein disulfide isomerase isoform; Homo sapiens glucose regulated protein, 58kD (GRP58), human ER-60 protease, or human phospholipase C-alpha (re- designated).
  • GenBank #'s include U42068, XM_007536, D16234, D83485, Z49835, with OMIM: 602046. It is located on chromosome 15, at 15q15 and has rat, mouse, and bovine homologs.
  • Tissue expression is most abundant in liver, placenta, and lung, and at lower levels in all other tissues tested with the amino acid sequence shown as SEQ ID NO: 8.
  • Proteins of this kind i.e., ER-chaperones
  • ER-chaperones have been found to assist the folding and assembly process of newly synthesized protein. They contain ER- retention/retrieval motif and thioredoxin-like domains serving as retention anchors for immature proteins. Excluded as a protease or as a carnitine acyltransferase. It has been suggested that GRP58 functions in combination with calnexin and calreticulin as a molecular chaperone of glycoprotein biosynthesis.
  • mutant peptides have been implicated in the accelerated degradation of mutant peptides.
  • the delta-508 CFTR protein fails to reach its intended cellular location because it is degraded at the ER. See Kopito, RR. 1999. Physiol. Rev. 79 S167. A similar result is found with mutant low density lipoprotein receptors. See Jorgensen, MM., et al. J. Biol. Chem. 275(43):33861 -33868 (2000).
  • This AIP has not heretofore been associated with any known disease process but is responsible for assisting the folding, maturation and ER- association of newly synthesized ABCA1 protein and ABCA1 intracellular transport. Appropriate ER-retention time and correct folding of ABCA1 protein may be involved in regulation of cellular cholesterol efflux.
  • the present invention also utilizes gene OAZ1 (omithine decarboxylase 1) with cDNA sequence in SEQ ID NO: 9). It is also referred to as human ornithine decarboxylase antizyme 1 and has GenBank # XM__009414, U09202, AF059317 with OMIM: 601579. It is located on chromosome 19 at 19p13.3. Homologs include Mouse (84%/226 a.a.); Rat (4%/226 a.a.); D. melongaster (7%/165 a.a.). The protein (SEQ ID NO: 10) is widely expressed.
  • Antizyme (ornithine decarboxylase antizyme) is the sole animal gene product known to be expressed with translational frameshifting (Rom and Kahana, 1994; Matsufuji et al., 1995). Elevation of cellular polyamine levels induces antizyme synthesis by raising frameshift efficiency. Matsufuji et al. (1996) noted that induced antizyme prevents further increase in polyamines by binding to ornithine decarboxylase (ODC; 165640), a key regulatory enzyme in polyamine biosynthesis, which accelerates degradation of ODC by the 26S proteasome. Antizyme also inhibits cellular uptake of polyamines. This AIP has not heretofore been associated with any known disease process. An alternative expression product is available (SEQ ID NO: 39).
  • the association of this AIP with ABCA1 is quite novel.
  • the AIP could be involved in ABCA1 degradation process. In any event, proper functioning confers a normal function for ABCA1 in regulating cellular cholesterol efflux.
  • the present invention also utilizes the gene LOC51691 (SEQ ID NO: 11), called human U6 snRNA-associated Sm-like protein LSm8, with GenBank # XM_004979, AF182294, and located on chromosome 7 at 7q31.1-q31.3.
  • the protein is homologous to S. pombe splicing factor.
  • tissue expression profile includes bone, colon, ear, foreskin, germ cell, kidney, lung, lymph, ovary, parathyroid, placenta, prostate, skin, tonsil, uterus, adrenal gland, bone marrow, brain, and elsewhere.
  • this protein binds to the 3' end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.
  • This class of protein has been identified in establishment of complexes for RNA splicing, ribosomal translation complexes (tRNA binding) and translation checkpoint controls.
  • This AIP has not heretofore been associated with any known disease process.
  • the present invention also utilizes the gene PFDN5 (SEQ ID NO: 13, encoding SEQ ID NO: 14), known as human prefoldin 5.
  • Other names include EIG-1 and c-myc binding protein MM-1.
  • Prefoldin 5 is a heterohexameric chaperone protein which assists in the correct folding of other proteins. It binds specifically to cytosolic chaperonin and transfers target proteins. Prefoldin may function by selectively targeting nascent actin and tubulin chains pending their transfer to cytosolic chaperonin for final folding and/or assembly. It promotes folding in an environment in which there are many competing pathways for nonnative proteins. Prefoldin 5 is associated with c-myc and is thought to repress the transcriptional activity of c-myc. (See Mori et al. J. Biol. Chem. 273(45): 29794-29800 (1998)).
  • This AIP has not heretofore been associated with any known disease process. It may help ABCA1 folding correctly and assume its proper sub-cellular localization for its normal function in regulating cholesterol efflux.
  • the present invention also utilizes the gene My001 (SEQ ID NO: 15), which encodes My001 protein (SEQ ID NO: 16). It has GenBank # AF059317. This AIP has not heretofore been associated with any known disease process.
  • the present invention also utilizes human ABCA1 interacting protein #107, a gene in RP4-744I24 human genomic clone. This is a peptide of 31 amino acid residues in length, with the sequence of SEQ ID MO: 40:
  • the present invention also utilizes gene KPNB1 (SEQ ID NO: 43), called Karyopherin (importin) beta 1 , Importin-beta, NTF97, nuclear protein import factor, or Impnb (SEQ ID NO: 44). It has GenBank # NM_002265 and OMIM 602738. The protein is homologous with rat (98%/875 a.a.), mouse (98%/875 a.a.), fruit fly (59%/ 864a.a.), yeast (33% /869 a.a.) and C. elegans (22%/726/a.a.) and is expressed in the brain.
  • Importin-beta 1 also called the nuclear transport factor p97 (NTF97), b- karyopherin in cytoplasm, together with the karyopherin-alpha which recognizes the nuclear localization signal-bearing proteins, mediates protein docking and translocation across the nuclear envelope. Also described as part of a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope (See: Gorlich, D. et al. 1995. Curr. Biol. 5(4): 383-92). Importin protein is also expressed in fibroblasts (of skin).
  • This AIP has not heretofore been associated with any known disease process. It binds to ABCA1-loop 4 region (extra-cellular loop; amino acids 1371- 1650 of the human ABCA1 , GenBank # AF285167). Importin-beta may mediate newly synthesized ABCA1 protein docking/passing through intracellular membranes along with the trans-Golgi network movement to reach plasma membrane where it acts as a gate-keeper of cholesterol and thus suggests a new mechanism to control cell cholesterol and plasma high-density lipoprotein- cholesterol concentrations. ABCA1 is a key regulatory protein to control cellular lipid (cholesterol and phospholipid) efflux.
  • the present invention also utilizes gene Bip/GRp78 (SEQ ID NO: 19), the protein being called human endoplasmic reticulum lumenal Ca2+ binding protein grp78, with GenBank # AF216292. Suggested functions with ABCA1 have included ER-retention.
  • the present invention also utilizes gene TPT1 (SEQ ID MO: 21), which encodes human tumor protein, tranlationally-controlled 1 (SEQ ID NO: 22), with GenBank # XM 012282, OMIM: *600763, and located on chromosome:13 and mapped to 13q12-q14.
  • TPT1 has been suggested as a candidate gene for allergic asthma.
  • the present invention also utilizes gene NAP1 L1 (SEQ ID NO: 23), encoding Homo sapiens nucleosome assembly protein 1-like 1 (SEQ ID NO: 24), with GenBank # XM_006560, OMIM: 164060, and having a possible role in nucleosome assembly.
  • the transcript was detected in all human tissues and cell lines studied, but levels were somewhat increased in rapidly proliferating cells. This and other observations suggest that the gene product participates in DNA replication and therefore plays an important role in the process of cell proliferation.
  • the present invention also utilizes gene E46 (SEQ ID NO: 25), encoding human-like mouse brain protein E46 (also called E46L) with amino acid sequence of (SEQ ID NO: 26). It has GenBank # NM_013236 and Locus ID: 25814.
  • the present invention also utilizes gene PRB4 (SEQ ID NO: 27), encoding human proline-rich protein BstNI subfamily 4 (Basic Salivary Proline-
  • the Po protein and a glycoprotein (11-1) are encoded in the PRB4 gene. It is noted that concanavalin-A binding proteins are of special biologic interest, since they, like other proline-rich proteins of the saliva, might bind to specific oral bacteria and modulate intraoral disease susceptibility. It has been suggested to function in post-translational cleavage and RNA splicing. (See: Maeda et al, Differential RNA splicing and post-translational cleavages in the human salivary proline-rich protein gene system, J. Biol. Chem. 260 (20), 11123-11130 (1985)).
  • the present invention also utilizes the gene Rhoip2 (SEQ ID NO: 29), encoding human cDNA: FLJ22163 fis, clone HRC00430, similar to Mouse Rhoip2, with GenBank # AK025816.
  • the polypeptide is shown as SEQ ID NO: 30.
  • the mouse Rho interacting protein 2 is involved in small GTPase activity.
  • the present invention also utilizes the gene ubiquinone (SEQ ID NO: 31), encoding SEQ IS NO: 32, which is similar to NADH dehydrogenase (ubiquinone) flavoprotein 2 (24kD), clone MGC:2144. It has GenBank # BC001632.
  • the present invention also utilizes the gene for the human homolog of mouse AK011474 (SEQ ID MO: 33), encoding the human homolog of Mus musculus 10 days embryo cDNA, with 87% similarity. It has GenBank # AK011474. The cDNA for this gene is shown as SEQ ID NO: 34 and the encoded protein is used in the methods of the invention.
  • the cDNA library was prepared and sequenced in Mouse Genome
  • cDNA was cleaved with Xhol and Sstl. Cloning sites, 5' end: Xhol; 3' end: Sstl.
  • SEQ. ID No. 34 is a partial nucleotide sequence of the human homolog. Using this sequence, those skilled in the art of molecular biology can clone and sequence the corresponding full length human gene. It is both the partial sequence and its corresponding full length gene which are useful in the compositions and methods of this invention.
  • the present invention also utilizes the gene for CGI-24 protein (SEQ ID NO: 35 and amino acid sequence in SEQ ID NO: 36), or CGI-24 protein (LOC51610), PTD013 protein, with GenBank # XMJD04309, Locus ID: 51610. Its predicted function is similar to that for rat small androgen-receptor interacting protein.
  • the present invention also utilizes the gene TCP1 or CCT2 (SEQ ID NO: 37) for Chaperonin containing TCP1 , subunit 2(beta) CCT2 (SEQ ID NO: 38), with GenBank # XMJD06861 , OMIM 605139, and located on chromosome 12.
  • the chaperonin containing TCP1 complex also called the TCP1 ring complex, consists of 2 back-to-back rings, each containing 8 unique but homologous subunits (e.g., CCT3).
  • CCT assists the folding of newly translated polypeptide substrates through multiple rounds of ATP-driven release and rebinding of partially folded intermediate forms.
  • Substrates of CCT include the cytoskeletal proteins actin and tubulin, as well as alpha- transducin.
  • Cyclin E CCNE1
  • CCNE1 degradation is regulated by ubiquitination and proteasomal action, which occur upon autophosphorylation and activation of the CCNE1-CDK2 complex.
  • Won et al. (1998) obtained a cDNA encoding CCT2, which they called CCT- beta. Mutational analysis indicated that CCT is essential for CCNE1 maturation and accumulation.
  • the present invention relates to a method for identifying an agent that modulates ABCA1 biological activity comprising: (a) contacting an agent with an ABCA1 -interacting protein (AIP) under conditions promoting said contacting,
  • step (b) determining the activity of an ABCA1 polypeptide in the presence of the agent-contacted AIP of step (a) under conditions promoting said activity
  • AIP is a polypeptide encoded by a human gene selected from the group consisting of LIMD1 , RAD23A, DNAJA2, PDI-ERp58, OAZ1 , LOC51691 , PFDN5, My001 , KIAA0528, RP4-744I24, KPNB1 , and those AIPs set out in Table 2 (Bip/GRp78, TPT1 , NAP1 L1 , E46, PRB4, Rhoip2, Ubiquinone, AK011474, CGI-24, and TCP1).
  • the process of the invention can be carried out either in vitro or in vivo, and using cells in culture, including wild type or mutant or recombinant cells, as well as in animal models, including birds, such as chickens, or mammals, including rodents, primates and humans, any one of which may provide the source of ABCA1 protein whose biological or chemical activity is to be measured.
  • any of the AIPs listed herein may be utilized in the processes of the invention.
  • the methods of the invention utilize the Lim and KIA0528 genes and their expression products.
  • interferons especially interferon- ⁇ (IFN- ⁇ )
  • IFITM2 interferon-induced transmembrane protein 2
  • PKC protein kinase C
  • interferons are known to affect cell proliferation, differentiation and cell-cell interactions, as well as altering plasma membrane processes and protein composition. They modulate as many as 100 different cellular proteins. For example, they increase IFITMs 1 , 2 and 3 and also decrease ABCA1.
  • IFN- ⁇ has been shown to decrease cholesterol efflux in macrophages by down-regulating ABCA1 mRNA (by 2 to 3 fold) and by decreasing ApoAI - mediated cholesterol and phospholipid efflux (by 5 to 10 fold).
  • ABCA1 mRNA does not account for the larger overall decrease in ABCA1 function.
  • over- expression of IFITM2 in cells serves to decrease efflux while decreased expression if IFITM2, such as by anti-sense knockdown, would increase efflux.
  • IFITM2 antagonists are expected to increase ABCA1 function.
  • ABCA1 protein for AIP binding Among the target sites on ABCA1 protein for AIP binding are the C- terminal domain and the first intracellular loop.
  • AIPs disclosed herein have been found to bind to the C- terminal domain of ABCAL These include Lim-domain-containing protein 1 (SEQ ID NO: 2), KIAA0528 protein (SEQ ID MO: 18), OAZI (ornithine decarboxylase antizyme (SEQ ID NO: 10)), My001 protein (SEQ ID NO: 16), U6 Sn RNA-associated Sm-like protein (CGI-24 protein (SEQ ID NO: 12)), E46 protein (SEQ ID NO: 26) and FLJ22163fis hypothetical protein (SEQ ID NO: 30).
  • Lim-domain-containing protein 1 SEQ ID NO: 2
  • KIAA0528 protein SEQ ID MO: 18
  • OAZI ornithine decarboxylase antizyme
  • My001 protein SEQ ID NO: 16
  • the ABCA1 protein activity to be measured is selected from the group consisting of reverse cholesterol transport, cholesterol efflux, HDL efflux, and phospholipid efflux.
  • ABCA1 protein activity may also be measured by ion flux, IL-1beta secretion, and induction of apoptosis of cells.
  • Such functions of ABCA1 are readily measured using methods known to those in the art. For example, any of the methods of Hayden et al (WO 00/55318 (21 September 2000) and Hayden et al WO 01/15676 (8 March 2001), the disclosures of which are hereby incorporated by reference in their entirety.
  • Modulators of ABCA1 protein activity are useful as therapeutic agents.
  • modulators of ABCA1 protein activity can be used to treat cardiovascular disease, conditions relating to reverse cholesterol transport or HDL formation, phospholipid or cholesterol efflux, and/or cholesterol transport from the gut to the blood.
  • Other roles of ABCA1 may be identified in the future, and these roles may be implicated in disease processes.
  • Modulators of ABCA1 protein activity identified according to this invention are also useful for treating such diseases.
  • the present invention also relates to a method for reducing elevated plasma phospholipid and/or cholesterol levels in a patient comprising administering to a patient afflicted with elevated plasma phospholipid and/or cholesterol levels an effective amount of an agent first identified as an agent that reduces plasma phospholipid and/or cholesterol levels using the assay methods disclosed herein.
  • the present invention also relates to a method for preventing elevated plasma phospholipid and/or cholesterol levels in a patient comprising administering to a patient at risk of developing elevated plasma phospholipid and/or cholesterol levels an effective amount of an agent first identified as an agent that reduces plasma phospholipid and/or cholesterol levels using the assay methods disclosed herein.
  • Modulators of the AIPs of this invention are useful as therapeutic agents. In particular, besides their impact on ABCA1 protein activity set out above, such modulators may be implicated in other diseases and conditions not previously associated with AIP activity. Conversely, compounds that interact with ABCA1 protein may have an impact on key functions of an AIP of the invention, and thus have therapeutic impact by this manner.
  • the source of ABCA1 protein activity of step (c), recited above is a cell expressing ABCA1 polypeptide and said comparison is made between said cell contacted with agent-bound AIP and a corresponding cell contacted with AIP free of said agent.
  • the source of the activity, or changes in it may also be monitored.
  • the present invention also contemplates situations wherein any difference in the activities of the ABCA1 protein, regardless of the source of the protein or how it is being assayed, is at least partly the result of a change in stability or regulated catabolism of the ABCA1 protein.
  • ABCA1 protein is involved in a number of processes related to lipid metabolism, the effect of agents, such as small organic molecules, on various aspects of lipid metabolism can be assayed by determining their ability to affect ABCA1 activity by their interaction with AIPs, especially those disclosed herein, thus modulating the activity of ABCA1 protein.
  • the present invention also relates to a process for identifying an agent useful for modulating cholesterol levels, said method comprising: (a) contacting a chemical agent with an ABCA1 interacting polypeptide
  • such animal would include birds, especially chickens, and mammals, including rodents and primates, such as humans.
  • the AIPs disclosed herein are derived from human genes, sufficient sequence homology occurs between the genes encoding these proteins and genes of related animals, such as mouse, that similar AIPs, with potentially similar abilities to regulate the activity, stability, catabolism, etc., of the corresponding ABCA1 proteins may be readily inferred.
  • the animal used for such assays for agents, especially small organic chemicals, able to modulate ABCA1 protein activity via their ability to interact and affect the function of AIPs that themselves interact with ABCA1 protein may include birds, especially chickens, and mammals, especially rodents and primates, such as humans.
  • the comparison of ABCA1 activity of step (c), as recited above is a comparison of lipid transport across a lipid membrane containing ABCA1 polypeptide.
  • said membrane is part of a liposome.
  • said ABCA1 protein is present in a cell-free system.
  • said ABCA1 activity is present as part of a cell, such as a wild type, mutant or recombinant cell, for example, a cell in culture, said cell may be derived from a mammal, especially from a rodent or primate, most especially from a human.
  • such lipid may be cholesterol, phospholipid, a triglyceride or HDL-cholesterol.
  • the ABCA1 activity being monitored may include the binding or hydrolysis of ATP by the ABCA1 polypeptide or the binding of cholesterol to ABCA1 protein.
  • the present invention relates to a method for identifying an agent that modulates cholesterol or phospholipid levels in an animal, comprising administering to an animal an effective amount of an ABCA1 -protein modulating agent first identified as such using a method of the invention and detecting a change in cholesterol or phospholipid level in said animal thereby identifying an agent said agent as a modulator of cholesterol or phospholipid activity.
  • Plasma cholesterol or phospholipid are especially evaluated, preferably where the result is a decrease in these levels.
  • the animal may be a bird, such as a chicken, or a mammal, including a human being.
  • a transgenic animal may also be utilized, such as one that has been engineered for elevated cholesterol of phospholipid levels.
  • the present invention includes methods of identifying ABCA1 modulating agents by their character as an antagonist of AIPs, such as where they are able to bind AIPs, and wherein the comparison of ABCA1 activity of step (c) is a comparison of lipid transport across a membrane containing ABCA1 polypeptide.
  • ABCA1 may be part of a membrane, such as a liposome, or such membrane may be part of an intact cell.
  • the intact cell is from a mammal, most preferably a human.
  • the intact cell is a recombinant cell, preferably wherein said recombinant cell has been engineered to comprise ABCA1 protein, such as where the recombinant cell does not comprise ABCA1 protein absent said engineering.
  • the engineering is genetic engineering and said cell expresses a gene that encodes ABCA1 protein.
  • Cells useful in the invention need not express a gene that encodes ABCA1 protein but may be physically manipulated to incorporate ABCA1 in their membrane by other than genetic means, such as by physical insertion of the protein into the membrane of the ceil or liposome.
  • the lipid is preferably one of cholesterol, phospholipid, triglyceride and/or HDL-cholesterol.
  • the ABCA1 protein can thus be used in screening assays for identification of compounds which modulate its activity because of their ability to modulate the interaction of AIPs with ABCA1 , thereby identifying potential drugs to regulate lipid levels, such as cholesterol and triglyceride levels.
  • Useful ABCA1 proteins include wild-type and mutant ABCA1 proteins or protein fragments, in a recombinant form or endogenously expressed.
  • Drug screens to identify compounds acting on AIPs that interact with the ABCA1 protein may employ any functional feature of the protein, such as where the phosphorylation state or other post-translational modification is monitored as a measure of ABCA1 biological activity following interaction with an AIP, such as an AIP disclosed herein.
  • ABCA1 has ATP binding sites, and thus assays may wholly or in part test the ability of ABCA1 to bind ATP or to exhibit ATPase activity in the presence and absence of AIP and where said AIP has interacted with a potential modulating agent.
  • the modulating agents identified by the processes of the present invention are those that have their primary effect on AIPs, such as where they increase or enhance the ability of said AIP to interact with, or bind to, or otherwise affect the activity of ABCA1.
  • ABCA1 is thought to be able to form a channel-like structure, which may be affected by interaction with one or more AIPs.
  • Drug screening assays could therefore be based upon assaying for the ability of the ABCA1 protein to form a channel, or upon the ability to transport cholesterol or another molecule, or based upon the ability of other proteins bound by or regulated by ABCA1 to form a channel, in the presence and absence of a drug-bound AIP molecule.
  • phospholipid or lipid transport such as HDL-cholesterol efflux, can also be used as a measure of ABCA1 biological activity as affected by AIPs before and after the latter have bound a potential drug.
  • ABCA1 also transports anions and functional assays can therefore be based upon this property, and would employ drug screening technology such as (but not limited to) the ability of various dyes to change color in response to changes in specific ion concentrations in such assays can be performed in vesicles such as liposomes, or adapted to use whole cells.
  • the drug is screened by its ability to bind to or interact with one or more AIPs, such as those disclosed herein, which drug-bound AIP then interacts with, and alters the activity of, ABCA1 protein.
  • AIPs such as those disclosed herein
  • the ability of ABCA1 protein to interact with AIPs, such as those disclosed herein, are readily determined by a variety of methods known in the art, including, for example, radioimmunoprecipitation, co-immunoprecipitation, co-purification, and yeast two-hybrid screening. Methods for determining such interaction are disclosed herein. Such interactions can be further assayed by means including but not limited to fluorescence polarization or scintillation proximity methods.
  • Such protein-protein interactions are also anticipated based upon functions of the ABCA1 protein deduced upon X-ray crystallography of the protein and comparison of its 3-D structure to that of proteins with known functions.
  • expression of mammalian (e.g., human) ABCA1 in yeast or C. elegans allows for screening of candidate compounds interacting with AIPs and modulating ABCA1 isoforms in wild-type and mutant backgrounds, as well as screens for mutations that enhance or suppress ABCA1 -AIP interactions.
  • Modifier screens can also be performed in ABCA1 transgenic or knock-out mice by administering to said mice one or more drug candidates that bind to and/or interact with one or more AIPs.
  • Human and rodent ABCA1 protein can be used as an antigen to raise antibodies, including monoclonal antibodies, which antibodies are useful in probing the nature of the interaction between a given AIP and ABCA1 , including different isoforms thereof.
  • Monitoring the influence of agents (e.g., drugs, compounds, etc.) on the biological activity of ABCA1 in the presence of one or more AIPs serves to identify compounds capable of modulating ABCA1 -AIP interaction and thus regulating ABCA1 biological activity (and the physiological effects attendant to such activity).
  • agents e.g., drugs, compounds, etc.
  • the effectiveness of an agent determined by a screening assay as described herein to increase the effects of AIPs on ABCA1 biological activity can be monitored in clinical trials of subjects exhibiting altered ABCA1 gene expression, protein levels, or biological activity.
  • the effectiveness of an agent determined by a screening assay to modulate AIP interaction with ABCA1 proteins and thus affect ABCA1 biological activity can be monitored in clinical trials of subjects exhibiting decreased altered gene expression, protein levels, or biological activity.
  • the activity of ABCA1 and other proteins implicated in, for example, cardiovascular disease can be used to ascertain the effectiveness of a particular AlP-interacting drug.
  • the ABCAl gene or a fragment thereof can be used as a tool to express the protein in an appropriate cell in vitro or in vivo (gene therapy), or can be cloned into expression vectors which can be used to produce large enough amounts of ABCA1 protein to use in in vitro assays for drug screening.
  • Expression systems which may be employed include baculovirus, herpes virus, adenovirus, adeno-associated virus, bacterial systems, and eucaryotic systems such as CHO cells. Naked DNA and DNA-liposome complexes can also be used. Such cells may likewise express the AIP as a target for drugs to be screened. Drugs affected the activity of ABCA1 activity directly are not contemplated by the invention disclosed herein. Thus, the ready determination of interaction between candidate agents and AIPs provides a method for high throughput screening of drug candidates for their potential effect on ABCA1 and lipid metabolism.
  • ABCA1 activity may be affected by AIPs through binding to such intracellular interacting proteins, especially where the AIP enhances or inhibits ABCA1 protein activity; such as the latter's interaction with HDL particles or constituents, or interaction with other proteins that facilitate interaction with HDL or its constituents, or the ability of ABCA1 to affect HDL-cholesterol efflux.
  • assays may be based upon the molecular dynamics of macromolecules, metabolites and ions by means of fluorescent-protein biosensors.
  • the effect of candidate modulators on expression or activity may be measured at the level of ABCA1 protein production using the same general approach in combination with standard
  • immunological detection techniques such as Western blotting or immunoprecipitation with an ABCA1 -specif ic antibody.
  • an AIP may bind to and stabilize ABCA1 protein as it is being formed and any drug that binds to said AIP may thereby increase or decrease its ability to stabilize ABCA1 protein, thus leading to an increase or decrease in stability of the nascent ABCA1 protein leading to increase or decrease of ABCA1 activity and thereby modulating lipid metabolism.
  • useful cholesterol-regulating or anti-CVD therapeutic modulators are identified as those which produce any change in ABCA1 polypeptide production because of their direct effects on AIP activity.
  • Such AIPs may also operate by interferring with ABCA1 catabolism and so this aspect of ABCA1 activity would be modulated by agents interacting and modulating AIPs involved in such metabolism. Methods of measuring such stabilities or degradations are well known to those skilled in the art.
  • Agonists, antagonists, or mimetics found to be effective at modulating the level of cellular ABCA1 expression or activity may be confirmed as useful in animal models (for example, mice, pigs, rabbits, or chickens).
  • the compound may ameliorate the low HDL levels of mouse or chicken hypoalphalipoproteinemias through their effects on AIPs, especially those AIPs disclosed herein.
  • Such drugs are expected to have significant metabolic regulating activity.
  • other ABC transporters can also be affected by AIPs and thus the drugs identified by the assays disclosed herein may have metabolic regulating activity on ABCA1 -related proteins as well though their affects on AIPs.
  • the replacement of ABCA1 with another ABC transporter is possible because it is likely that ABC transporter family members, such as ABC2, ABCR, or ABC8 will have a similar mechanism of regulation.
  • ABCA1 polypeptide (purified or unpurified) is used in an assay to determine its ability to bind an AIP (or even to other proteins) and the effect of a candidate compound on that binding is then determined.
  • ABCA1 protein (or a polypeptide fragment thereof or an epitope-tagged form or fragment thereof) is harvested from a suitable source (e.g., from a prokaryotic expression system, eukaryotic cells, a cell-free system, or by immunoprecipitation from ABCA1 -expressing cells).
  • the ABCA1 polypeptide is then bound to a suitable support (e.g., nitrocellulose or an antibody or a metal agarose column in the case of, for example, a his-tagged form of ABCA1).
  • Binding to the support is preferably done under conditions that allow AIPs to associate with ABCA1 polypeptide. Such conditions may include use of buffers that minimize interference with protein-protein interactions.
  • the binding step can be done in the presence of AIPs alone and, for example, AIPs previously treated with a candidate drug to form an AlP-drug complex.
  • the foregoing assay can be performed using a purified or semipurified protein or other molecule that is known to interact with ABCA1. This assay may include the following steps.
  • the ABCA1 protein can also be tested for its effects on membrane permeability in the presence of an AIP.
  • an AIP that binds to ABCA1 protein and increases such activity would likely be affected by a drug that binds the AIP, especially if such binds greatly interferes with ABCA1 binding.
  • ABCA1 might affect the permeability of membranes to ions.
  • Other related membrane proteins most notably the cystic fibrosis transmembrane conductance regulator and the sulfonylurea receptor, are associated with and regulate ion channels. Thus, such activities would be affected by proteins, such as the AIPs disclosed herein, that bind to and modulate the activity of ABC proteins.
  • ABCA1 or a fragment of ABCA1 is incorporated into a synthetic vesicle, or, alternatively, is expressed in a cell and vesicles, or other cell sub-structures, or an artificial structure, such as a liposome, containing ABCA1 and isolated.
  • the ABCA1 -containing vesicles, cells, or liposomes are loaded with a reporter molecule (such as a fluorescent ion indicator whose fluorescent properties change when it binds a particular ion) that can detect ions (to observe outward movement), or alternatively, the external medium is loaded with such a molecule (to observe inward movement).
  • a reporter molecule such as a fluorescent ion indicator whose fluorescent properties change when it binds a particular ion
  • the external medium is loaded with such a molecule (to observe inward movement).
  • a molecule which exhibits differential properties when it is inside the vesicle compared to when it is outside the vesicle is preferred. For example, a molecule that has quenching properties when it is at high concentration but not when it is at a low concentration would be suitable.
  • ABCA1 activity is also a function of stability of the protein, which stability may be affected by binding of one or more of the AIPs disclosed herein.
  • Methods are available for measuring ABCA1 stability. For example, a cell-based or cell-free system can be used to screen for compounds based on their effect on the half-life of ABCA1 protein.
  • the assay may employ labeled protein. Protein can be quantitated, for example, by fluorescent antibody-based methods.
  • Some AIPs may be involved in ABCA1 phosphorylation and so a screen for drugs interacting with these AIPs presents a means for screening for drugs that can affect ABCA1 activity through this avenue.
  • the effect of a compound on ABCA1 phosphorylation can be assayed by methods that quantitate phosphates on proteins or that assess the phosphorylation state of a specific residue of ABCA1 protein. Such methods include but are not limited to 32 P labelling and immunoprecipitation, detection with antiphosphoamino acid antibodies (e.g., antiphosphoserine antibodies), phosphoamino acid analysis on 2-dimensional TLC plates, and protease digestion fingerprinting of proteins followed by detection of 32 P-labeled fragments.
  • Such assays would be carried out with and without AIP present and then the effects of a candidate drug on the AIP would be determined.
  • Drugs that increase or decrease the ability of an interacting AIP to modulate phosphorylation of ABCA1 thereby serves to identify another potential regulator of lipid metabolism.
  • ABCA1 is based on any method capable of quantitating that particular modification. For example, effects of compounds on glycosylation may be assayed by treating ABCA1 with glycosylase and quantitating the amount and nature of carbohydrate released.
  • ABCA1 The ability of ABCA1 to bind ATP provides another assay to screen for compounds that affect ABCA1.
  • ATP binding can be quantitated using ABCA1 protein at an appropriate level of purity and reconsitituted it in a lipid vesicle, the latter then being exposed to a labeled but non-hydrolyzable ATP analog (such as gamma 35 S-ATP) in the presence or absence of AIPs.
  • azido-ATP analogs can be used to allow covalent attachment of the azido-ATP to protein (by means of UN. light), and permit easier quantitation of the amount of ATP bound to the protein.
  • the amount of ATP analog associated with ABCA1 is then measured.
  • ATPase activity of ABCA1 can also be assayed for the effect of compounds on AIPs that modulate this activity of ABCAL This is preferably performed in a cell-free assay so as to separate ABCA1 from the many other ATPases in the cell.
  • Such an ATPase assay may be performed in the presence or absence of membranes, and with or without integration of ABCA1 protein into a membrane, so long as the ABCA1 is able to interact with at least one AIP.
  • the ATP hydrolysis products produced or the ATP hydrolyzed may be measured within or outside of the vesicles, or both.
  • Such an assay may be based on disappearance of ATP or appearance of ATP hydrolysis products.
  • a transport-based assay can be performed in vivo or in vitro.
  • the assay may be based on any part of the reverse cholesterol transport process that is readily re-created in culture, such as cholesterol or phospholipid efflux.
  • the assay may be based on net cholesterol transport in a whole organism, as assessed by means of a labeled substance (such as cholesterol).
  • AIPs that affect ABCA1 activity in such an assay are likewise screening targets for drugs that interact with such proteins.
  • fluorescent lipids are used to measure ABCA1 -catalyzed lipid efflux.
  • a fluorescent precursor, C6-NBD-phosphatidic acid can be used. This lipid is taken up by cells and , dephosphorylated by phosphatidic acid phosphohydrolase.
  • the product, NBD-diglyceride is then a precursor for synthesis of glycerophospholipids like phosphatidylcholine.
  • the efflux of NBD-phosphatidylcholine can be monitored by detecting fluorescence resonance energy transfer (FRET) of the NBD to a suitable acceptor in the cell culture medium.
  • FRET fluorescence resonance energy transfer
  • This acceptor can be rhodamine-labeled phosphatidylethanolamine, a phospholipid that is not readily taken up by cells.
  • the use of short-chain precursors obviates the requirement for the phospholipid transfer protein in the media.
  • NBD-cholesterol ester can be reconstituted into LDL. The LDL can efficiently deliver this lipid to cells via the LDL receptor pathway.
  • the NBD-cholesterol esters are hydrolyzed in the lysosomes, resulting in NBD-cholesterol that can now be transported back to the plasma membrane and efflux from the cell.
  • the efflux can be monitored by the aforementioned FRET assay in which NBD transfers its fluorescence resonance energy to the rhodamine-phosphatidylethanoline acceptor.
  • FRET assays are available to identify AIPs that mosulate ABCA1 activity and thus identify targets for drug screening for agents that interact with such AIPs.
  • Test compounds identified as having activity with AIPs that regulate ABCA1 activity in any of the above-described assays are subsequently screened in any available animal model system, including, but not limited to, pigs, rabbits, and WHAM chickens, for their ability to modulate lipid metabolism. Test compounds are administered to these animals according to standard methods.
  • the present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent.
  • identifying an agent i.e., the therapeutic agents identified according to the assay procedures disclosed herein
  • said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent.
  • the present invention specifically contemplates a situation whereby a user of an assay of the invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the invention.
  • information i.e., information as to structure, dosage, etc
  • the user of the assay may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with said code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the. compounds having a particular modulating activity (for example, the code number with the corresponding results).
  • This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.
  • AIPs may also regulate the ability of ABCA1 to facilitate cholesterol and/or phospholipid efflux.
  • the cholesterol/phospholipid efflux assay measures the ability of cells to transfer cholesterol to an extracellular acceptor molecule and is dependent on ABCA1 function. In this procedure, cells are loaded with radiolabeled cholesterol by any of several biochemical pathways (Marcil et al., Arterioscler. Thromb. Vase. Biol. 19:159-169, 1999). Cholesterol efflux is then measured after incubation for various times (typically 0 to 24 hours) in the presence of HDL3 or purified ApoAI. Cholesterol efflux is determined as the percentage of total cholesterol in the culture medium after various times of incubation. ABCA1 biological activity is associated with increased efflux of phospholipid and cholesterol while decreased levels of ABCA1 are associated with decreased efflux.
  • AIPs regulating ABCA1 activity are identified and used as targets for large scale screening efforts identifying drugs with potential lipid- regulating activity. It is significant that AIPs are expressed in the liver because the ABCA1 expression level is high in the liver.
  • AIPs The relative level of expression of AIPs in different cells varies considerably. Because AIPs are expressed at lower levels than ABCA1 protein, they have a high ABCA1 :AIP ratio, for example, in HepG2 and CaCo-2 cells. Thus, the ratios of ABCA1 :LimD1 and ABCA KIAA0528 in HepG2 and CaCo-2 cells (see Table 1) demonstrate physiologically important points of interaction for purposes of ABCA1 regulation and subsequent regulation of cholesterol and phospholipid metabolism. Conversely, the ratios for other AIPs may be greater than 100.
  • LimD1 and KIAA0528 offer highly advantageous methods of screening potentially valuable lipid regulatory agents. More importantly, the LimD1 and KIAA0528 proteins have not been be identified in yeast two-hybrid screens and therefore do not appear to be indiscriminant protein binding agents (as opposed to chaperones, which tend to bind to a host of proteins).
  • the cells of choice may be engineered to express ABCA1 in any desired levels, along with a selected AIP, or combination of AIPs.
  • Such engineering includes genetic engineering, which may be accomplished by any of the methods already known to those skilled in the art and which need not be described in detail herein. However, these may include such diverse methods as utilizing plasmids containing the genes of choice and then transfecting the cells with said plasmids, or may include infecting cells with retroviral vectors comprising the desired genes.
  • ABCA1 is expressed at very high levels in HepG2 and CaC02 cells.
  • the data are expressed as the increase in ABCA1 RNA levels relative to HeLa cells. Therefore there is 56 and 146 times more ABCA1 in HepG2 and CaC02 cells, respectively, than in HeLa cells. In contrast, there is only three times more and 0.9 times less ABCA1 in fibroblasts and THP1 macrophages, respectively, relative to HeLa cells (data not shown). Most of the data is summarized in Table 1 below.
  • One way to examine the effect of LIMD1 and KIAA0528 and other AIPs on ABCA1 functions is by carrying out transient transfection for modulating (increase) the levels of each protein. If cholesterol and/or phospholipid efflux is increased by transient transfection of a given AIP, then compound screening programs will be focused on identifying an agonist for that AIP. Alternatively, if efflux is decreased by transient transfection of an AIP, then an antagonist to that AIP will be identified in order to achieve increased efflux capacity.

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Abstract

La présente invention concerne des tests de criblage à haut rendement d'agents capables de moduler le métabolisme des lipides. On détecte ces agents à leur capacité de moduler l'activité de protéines interagissant avec la protéine exprimée par l'ABCA1 et modulant l'activité de cette dernière (appelée CERP-cholesterol efflux regulating protein-, ou protéine de régulation de l'efflux de cholestérol). L'invention se rapporte à des procédés permettant de traiter des troubles du métabolisme des lipides, en particulier les troubles impliquant des concentrations élevées de phospholipides ou de cholestérol.
PCT/CA2002/000489 2001-04-12 2002-04-12 Test de criblage d'agents modulant l'activite de la proteine abca1 Ceased WO2002084301A2 (fr)

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US28342401P 2001-04-12 2001-04-12
US60/283,424 2001-04-12

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WO2002084301A2 true WO2002084301A2 (fr) 2002-10-24
WO2002084301A3 WO2002084301A3 (fr) 2003-06-05

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002101392A3 (fr) * 2001-06-08 2003-07-10 Xenon Genetics Inc Procedes de traitement de troubles des systemes nerveux et de reproduction
WO2004113572A1 (fr) * 2003-06-21 2004-12-29 Ewha University Industry Collaboration Foundation Composition destinee a l'identification par criblage d'un medicament antihypertenseur, contenant un gene tctp de mammifere ou son produit proteique, et procede d'identification d'un medicament antihypertenseur au moyen de ladite composition
EP2505660A4 (fr) * 2009-11-27 2013-01-02 Japan Science & Tech Agency Méthode de criblage d'un agent de traitement de l'hyperlipémie

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60035163T2 (de) * 1999-03-15 2008-02-21 University Of British Columbia, Vancouver Abc1 polypeptide und verfahren und reagenzien zur modulation des cholesterolgehalts

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002101392A3 (fr) * 2001-06-08 2003-07-10 Xenon Genetics Inc Procedes de traitement de troubles des systemes nerveux et de reproduction
WO2004113572A1 (fr) * 2003-06-21 2004-12-29 Ewha University Industry Collaboration Foundation Composition destinee a l'identification par criblage d'un medicament antihypertenseur, contenant un gene tctp de mammifere ou son produit proteique, et procede d'identification d'un medicament antihypertenseur au moyen de ladite composition
EP2505660A4 (fr) * 2009-11-27 2013-01-02 Japan Science & Tech Agency Méthode de criblage d'un agent de traitement de l'hyperlipémie
US9090932B2 (en) 2009-11-27 2015-07-28 Japan Science And Technology Agency Method for screening of therapeutic agent for hyperlipemia

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