Bowman Miao

Generation of multiple farnesoid-X-receptor isoforms through the use of alternative promoters

Bile acid biosynthesis is regulated by both feed-forward and feedback mechanisms involving a cascade of nuclear hormone receptors. Feed-forward regulation of the rate limiting enzyme in bile acid biosynthesis is provided by oxysterols through liver-X-receptor alpha (NR1H3), while feedback regulation is provided by bile acids through farnesoid-X-receptor (FXR) (NR1H4). The Syrian golden hamster provides a useful model for studying lipid metabolism. The hamster metabolizes and transports dietary cholesterol in a similar manner to humans, with the resulting lipid profile being more similar to the human profile than that of other rodent models. Cloning of Fxr from Syrian golden hamster revealed four hamster Fxr splice variants that altered the N-terminal activation domain or the hinge region between the DNA and ligand binding domains. Human genomic sequence and data from hamster Fxr were used to identify and clone a novel human FXR isoform resulting from the use of an alternative promoter. RNA expression analysis indicates that the two human FXR isoforms are differentially expressed in developmental and tissue-specific patterns and are likely to provide a mechanism for cell-specific FXR-dependent transcriptional activity.

Annexin A2 Is a Natural Extrahepatic Inhibitor of the PCSK9-Induced LDL Receptor Degradation

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2−/− mice revealed: i) a ∼1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ∼2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2−/− tissues revealed that the LDLR was decreased by ∼50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9≡LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

Pharmacologic Profile of the Adnectin BMS-962476, a Small Protein Biologic Alternative to PCSK9 Antibodies for Low-Density Lipoprotein Lowering.

Proprotein convertase subtilisin kexin-9 (PCSK9) is an important pharmacological target for decreasing low-density lipoprotein (LDL) in cardiovascular disease, although seemingly inaccessible to small molecule approaches. Compared with therapeutic IgG antibodies currently in development, targeting circulating PCSK9 with smaller molecular scaffolds could offer different profiles and reduced dose burdens. This inspired genesis of PCSK9-binding Adnectins, a protein family derived from human fibronectin-10th-type III–domain and engineered for high-affinity target binding. BMS-962476, an ∼11-kDa polypeptide conjugated to polyethylene glycol to enhance pharmacokinetics, binds with subnanomolar affinity to human. The X-ray cocrystal structure of PCSK9 with a progenitor Adnectin shows ∼910 Å2 of PCSK9 surface covered next to the LDL receptor binding site, largely by residues of a single loop of the Adnectin. In hypercholesterolemic, overexpressing human PCSK9 transgenic mice, BMS-962476 rapidly lowered cholesterol and free PCSK9 levels. In genomic transgenic mice, BMS-962476 potently reduced free human PCSK9 (ED50 ∼0.01 mg/kg) followed by ∼2-fold increases in total PCSK9 before return to baseline. Treatment of cynomolgus monkeys with BMS-962476 rapidly suppressed free PCSK9 >99% and LDL-cholesterol ∼55% with subsequent 6-fold increase in total PCSK9, suggesting reduced clearance of circulating complex. Liver sterol response genes were consequently downregulated, following which LDL and total PCSK9 returned to baseline. These studies highlight the rapid dynamics of PCSK9 control over LDL and liver cholesterol metabolism and characterize BMS-962476 as a potent and efficacious PCSK9 inhibitor.

Effects of the Prosegment and pH on the Activity of PCSK9 EVIDENCE FOR ADDITIONAL PROCESSING EVENTS

PCSK9, a target for the treatment of dyslipidemia, enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes, up-regulating LDL-cholesterol levels. Whereas the targeting and degradation of the PCSK9-LDLR complex are under scrutiny, the roles of the N- and C-terminal domains of PCSK9 are unknown. Although autocatalytic zymogen processing of PCSK9 occurs at Gln152↓, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg46↓ by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ∼4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ∼3-fold higher binding to LDLR in vitro, in ≥4-fold increased activity on cellular LDLR, and faster cellular internalization in endosome/lysosome-like compartments. Finally, swapping the acidic stretch of PCSK9 with a similar one found in the glycosylphosphatidylinositol-anchored heparin-binding protein 1 does not impair PCSK9 autoprocessing, secretion, or activity and confirmed that the acidic stretch acts as an inhibitor of PCSK9 function. We also show that upon short exposure to pH values 6.5 to 5.5, an ∼2.5-fold increase in PCSK9 activity on total and cell surface LDLR occurs, and PCSK9 undergoes a second cleavage at Arg248, generating a two-chain PCSK9-ΔN248. At pH values below 5.5, PCSK9 dissociates from its prosegment and loses its activity. This pH-dependent activation of PCSK9 represents a novel pathway to further activate PCSK9 in acidic endosomes. These data enhance our understanding of the functional role of the acidic prosegment and on the effect of pH in the regulation of PCSK9 activity.

Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor α

The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors. We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET). The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331<WY14643=ciprofibrate<L165041<gemfibrozil. Similarly, for all ligands tested, the rank order of EC(50) for peptide recruitment was CBP<PGC-1<SRC-1. These data suggest that for these LXXLL coactivator peptides, the ligands do not substantially differ in their preferences. Partial agonism was observed with ciprofibrate and PGC-1 and gemfibrozil and CBP giving a lower FRET at saturation than with the other ligands. This suggests that ciprofibrate and gemfibrozil induce a different conformation to the receptor-PGC-1 and receptor-CBP complex, respectively. In cotransfection assays, unexpected differences in potencies and efficacies were observed and the rank order of EC(50)s for activation differed from that predicted by FRET assays. In most cases, the presence of a coactivator peptide led to decrease in the EC(50)s seen in FRET assays compared to the K(i)s observed in binding to receptor only, consistent with the lower EC(50)s obtained in the transfection assays. Our data demonstrate that ligand induced coactivator preferences of PPARalpha contribute to transcription potency and efficacy.

Novel Peroxisome Proliferator-Activated Receptor α Agonists Lower Low-Density Lipoprotein and Triglycerides, Raise High-Density Lipoprotein, and Synergistically Increase Cholesterol Excretion with a Liver X Receptor Agonist

The first generation peroxisome proliferator-activated receptor (PPAR) α agonist gemfibrozil reduces the risk of major cardiovascular events; therefore, more potent PPARα agonists for the treatment of cardiovascular diseases have been actively sought. We describe two novel, potent oxybenzylglycine PPARα-selective agonists, BMS-687453 [N-[[3-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]phenyl]methyl]-N-(methoxycarbonyl)-glycine] and BMS-711939 N-[[5-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]-2-fluorophenyl]methyl]-N-(methoxycarbonyl)-glycine], that robustly increase apolipoprotein (Apo) A1 and high-density lipoprotein cholesterol in human ApoA1 transgenic mice and lower low-density lipoprotein-cholesterol and triglycerides in fat-fed hamsters. These compounds have much lower potency against mouse PPARα than human PPARα; therefore, they were tested in PPARα-humanized mice that do not express murine PPARα but express human PPARα selectively in the liver. We developed hepatic gene induction as a novel biomarker for efficacy and demonstrate hepatic gene induction at very low doses of these compounds. BMS-711939 induces fecal cholesterol excretion, which is further increased upon cotreatment with a liver X receptor (LXR) agonist. It is surprising that this synergistic increase upon coadministration is also observed in mice that express PPARα in the liver only. BMS-711939 also prevented the LXR agonist-induced elevation of serum triglycerides. Such PPARα agonists could be attractive candidates to explore for the treatment of cardiovascular diseases, especially in combination with a suitable LXR agonist.

Discovery of an oxybenzylglycine based peroxisome proliferator activated receptor alpha selective agonist 2-((3-((2-(4-chlorophenyl)-5-methyloxazol-4-yl)methoxy)benzyl)(methoxycarbonyl)amino)acetic acid (BMS-687453).

An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) alpha agonist, with an EC(50) of 10 nM for human PPARalpha and approximately 410-fold selectivity vs human PPARgamma in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPARdelta. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystal structures of the early lead compound 12 and compound 2 in complex with PPARalpha ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPARalpha in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.

Synthesis and structure–activity relationships of 2-aryl-4-oxazolylmethoxy benzylglycines and 2-aryl-4-thiazolylmethoxy benzylglycines as novel, potent PPARα selective activators- PPARα and PPARγ selectivity modulation

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARalpha/gamma dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARalpha versus PPARgamma. Potent, highly selective PPARalpha activators 2a and 2l, as well as PPARalpha activators with significant PPARgamma activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.

Ligand-induced coactivator recruitment to peroxisome proliferator-activated receptorα characterized by fluorescence resonance energy transfer

Publisher Summary This chapter describes the way the affinity of ligands can be measured for peroxisome proliferator-activated receptorα (PPARα) using the fluorescence resonance energy transfer (FRET) assay utilizing a small peptide derived from coactivators containing the LXXLL receptor interaction site. It also compares the results with more traditional methods, such as scintillation proximity assay. The FRET assay has several advantages over conventional assays for determining affinities of PPARα ligands. It is a nonradioactive assay and is more sensitive than the scintillation proximity assay (SPA) for certain ligands, such as gemfibrozil and fenofibric acid. A known, high-affinity (labeled) ligand is not required as in SPA and is therefore ideal for identifying agonist ligands, especially for orphan receptors. The FRET assay can also be performed in the presence of a known agonist to detect receptor antagonists. However, the reagents are relatively expensive. Because the peptide is only a small part of the whole coactivator and the coactivators have multiple receptor interacting domains, the affinities determined with a peptide may be different from that using the whole coactivator protein.

Discovery and Preclinical Evaluation of BMS-711939, an Oxybenzylglycine Based PPARα Selective Agonist

BMS-711939 (3) is a potent and selective peroxisome proliferator-activated receptor (PPAR) α agonist, with an EC50 of 4 nM for human PPARα and >1000-fold selectivity vs human PPARγ (EC50 = 4.5 μM) and PPARδ (EC50 > 100 μM) in PPAR-GAL4 transactivation assays. Compound 3 also demonstrated excellent in vivo efficacy and safety profiles in preclinical studies and thus was chosen for further preclinical evaluation. The synthesis, structure-activity relationship (SAR) studies, and in vivo pharmacology of 3 in preclinical animal models as well as its ADME profile are described.