ABSTRACT
BACKGROUND: LXRs (Liver X Receptor alpha and beta) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds. OBJECTIVE: Blood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators. METHODS: Transcript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRalpha, LXRbeta, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated ex vivo with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623. RESULTS: A single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRalpha and LXRbeta, and all cell types significantly increased ABCA1 and ABCG1 expression upon ex vivo LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription. CONCLUSION: Peripheral blood cells express LXRalpha and LXRbeta, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.
Subject(s)
Blood Cells/metabolism , DNA-Binding Proteins/agonists , Receptors, Cytoplasmic and Nuclear/agonists , Transcription, Genetic , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Administration, Oral , Animals , Anticholesteremic Agents/administration & dosage , Anticholesteremic Agents/pharmacology , Biomarkers , Blood Cells/drug effects , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Liver X Receptors , Orphan Nuclear Receptors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic/drug effectsABSTRACT
A structure-based approach was used to optimize our new class of quinoline LXR modulators leading to phenyl acetic acid substituted quinolines 15 and 16. Both compounds displayed good binding affinity for LXRbeta and LXRalpha and were potent activators in LBD transactivation assays. The compounds also increased expression of ABCA1 and stimulated cholesterol efflux in THP-1 cells. Quinoline 16 showed good oral bioavailability and in vivo efficacy in a LDLr knockout mouse model for lesions.
Subject(s)
Anticholesteremic Agents/chemical synthesis , Atherosclerosis/drug therapy , DNA-Binding Proteins/agonists , Phenylacetates/chemical synthesis , Quinolines/chemical synthesis , Receptors, Cytoplasmic and Nuclear/agonists , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/biosynthesis , Animals , Anticholesteremic Agents/chemistry , Anticholesteremic Agents/pharmacology , Binding Sites , Biological Availability , Cell Line , Cholesterol/metabolism , DNA-Binding Proteins/genetics , Drug Stability , Female , Humans , In Vitro Techniques , Ligands , Liver X Receptors , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Orphan Nuclear Receptors , Phenylacetates/chemistry , Phenylacetates/pharmacology , Protein Structure, Tertiary , Quinolines/chemistry , Quinolines/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Structure-Activity Relationship , Transcriptional ActivationABSTRACT
Integrin alphaIIb/beta3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the alphaIIb/beta3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of alphaIIb. Using alphaIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin alpha2 tail, beta3 tail and lamin, but showed a weak binding to the alphaV tail which shares the highest homology with alphaIIb tail among the integrin alpha family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and alphaIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to alphaIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with alphaIIb and may play a critical role in alphaIIb/beta3-mediated platelet function.
Subject(s)
Cytoplasm/metabolism , Platelet Membrane Glycoprotein IIb/metabolism , Triose-Phosphate Isomerase/metabolism , Amino Acid Sequence , Base Sequence , Blood Platelets/metabolism , DNA Primers , Humans , Mutagenesis, Site-Directed , Reverse Transcriptase Polymerase Chain Reaction , Substrate Specificity , Triose-Phosphate Isomerase/chemistryABSTRACT
The liver X receptors (LXRalpha and LXRbeta), ligand-activated transcription factors, belong to the superfamily of nuclear hormone receptors and have been shown to play a major role in atherosclerosis by modulating cholesterol and triglyceride metabolism. In this report, we describe a novel LXR target, the adipocyte fatty acid binding protein (aP2), which plays an important role in fatty acid metabolism, adipocyte differentiation and atherosclerosis. While LXR agonists induce aP2 mRNA expression in human monocytes (THP-1 cells) and macrophages in a time- and concentration-dependent manner, they have no effect on aP2 expression in human adipocytes. The increase in aP2 mRNA level was additive when THP-1 cells were treated with LXR and PPARgamma agonists. Also, an RXR agonist induced aP2 expression in these cells. While no additive effect was observed with LXR and RXR agonists, additive effects were observed with RXR and PPARgamma agonists. GW9662, a potent PPARgamma antagonist, inhibited PPARgamma-induced aP2 expression without affecting LXR-mediated aP2 expression indicating the induction is mediated directly through LXR activation. Analysis of human aP2 promoter revealed a potential LXR response element (LXRE). Gel shift data showed that the LXRalpha/RXRalpha heterodimer bound to the LXRE motif in aP2 promoter in vitro in a sequence-specific manner. Deletion and mutation analyses of the proximal aP2 promoter confirm that this is a functional LXRE. These data indicate for the first time that human macrophage aP2 promoter is a direct target for the regulation by LXR/RXR heterodimers.
Subject(s)
DNA-Binding Proteins/agonists , DNA-Binding Proteins/metabolism , Fatty Acid-Binding Proteins/genetics , Gene Expression Regulation/drug effects , Macrophages/drug effects , Macrophages/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism , Sulfonamides/pharmacology , 5' Flanking Region/genetics , Adipocytes/cytology , Adipocytes/drug effects , Alitretinoin , Base Sequence , Binding Sites , Cell Differentiation/drug effects , Cell Line , Dimerization , Drug Synergism , Fatty Acid-Binding Proteins/metabolism , Humans , Hydrocarbons, Fluorinated , Liver X Receptors , Molecular Sequence Data , Orphan Nuclear Receptors , PPAR gamma/agonists , Protein Binding/drug effects , Receptors, Retinoic Acid/metabolism , Response Elements , Sequence Deletion , Thiazolidinediones/pharmacology , Tretinoin/pharmacologyABSTRACT
A series of phenyl acetic acid based quinolines was prepared as LXR modulators. An SAR study in which the C-3 and C-8 positions of the quinoline core were varied led to the identification of two potent LXR agonists 23 and 27. Both compounds displayed good binding affinity for LXRbeta and LXRalpha, and increased expression of ABCA1 in THP-1 cells. These two compounds also had desirable pharmacokinetic profiles in mice and displayed in vivo efficacy in a 12-week Apo E knockout mouse lesion model.