Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological ...inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)−N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.
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IJS, KILJ, NUK, PNG, UL, UM
Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL ...cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.
OBJECTIVE—The goal of this study was to understand the molecular basis of how the amino acid substitution C112R that distinguishes human apolipoprotein (apo) E4 from apoE3 causes the more ...proatherogenic plasma lipoprotein–cholesterol distribution that is known to be associated with the expression of apoE4.
APPROACH AND RESULTS—Adeno-associated viruses, serotype 8 (AAV8), were used to express different levels of human apoE3, apoE4, and several C-terminal truncation and internal deletion variants in C57BL/6 apoE-null mice, which exhibit marked dysbetalipoproteinemia. Plasma obtained from these mice 2 weeks after the AAV8 treatment was analyzed for cholesterol and triglyceride levels, as well as for the distribution of cholesterol between the lipoprotein fractions. Hepatic expression of apoE3 and apoE4 induced similar dose-dependent decreases in plasma cholesterol and triglyceride to the levels seen in control C57BL/6 mice. Importantly, at the same reduction in plasma total cholesterol, expression of apoE4 gave rise to higher very low-density lipoprotein–cholesterol (VLDL-C) and lower high-density lipoprotein–cholesterol levels relative to the apoE3 situation. The C-terminal domain and residues 261 to 272 in particular play a critical role, because deleting them markedly affected the performance of both isoforms.
CONCLUSIONS—ApoE4 possesses enhanced lipid and VLDL-binding ability relative to apoE3, which gives rise to impaired lipolytic processing of VLDL in apoE4-expressing mice. These effects reduce VLDL remnant clearance from the plasma compartment and decrease the amount of VLDL surface components available for incorporation into the high-density lipoprotein pool, accounting for the more proatherogenic lipoprotein profile (higher VLDL-C/high-density lipoprotein–cholesterol ratio) occurring in apoE4-expressing animals compared with their apoE3 counterparts.
Pharmacia Corporation, Kalamazoo, Michigan 49001
Submitted 21 September 2003
; accepted in final form 9 October 2003
Thiazolidinediones address underlying causes of type 2 diabetes, although their ...mechanism of action is not clearly understood. The compounds are thought to function as direct activators of the nuclear receptor PPAR (peroxisome proliferator-activated receptor- ), although pioglitazone, the weaker agonist of the two thiazolidinediones now in clinical use, seems to have more useful effects on circulating lipids. We have used tritiated pioglitazone and a photoaffinity cross-linker to identify a novel binding site in mitochondria. A saturable binding site for 3 Hpioglitazone was solubilized from the membranes with CHAPS and migrated as a large complex by size exclusion chromatography. The binding correlated with a <17-kDa protein (m17), marked by a photoaffinity cross-linker, in both subcellular location and selectivity of competition by analogs. The protein was isolated and identified by mass spectrometry analysis and NH 2 -terminal sequencing. Three synthetic peptides with potential antigenic properties were synthesized from the predicted nontransmembrane sequence to generate antibodies in rabbits. Western blots show that this protein, which we have termed "mitoNEET," is located in the mitochondrial fraction of rodent brain, liver, and skeletal muscle, showing the identical subcellular location and migration on SDS-PAGE as the protein cross-linked specifically by the thiazolidinedione photoprobe. The protein exists in low levels in preadipocytes, and expression increases exponentially in differentiated adipocytes. The synthetic protein bound to solid phase associated with a complex of solubilized mitochondrial proteins, including the trifunctional -oxidation protein. It is possible that thiazolidinedione modification of the function of the mitochondrial target may contribute to lipid lowering and/or antidiabetic actions.
insulin sensitizers; thiazolidinediones; photoaffinity probes; novel site of action; mitoNEET
Address for reprint requests and other correspondence: J. R. Colca, Pfizer St. Louis, 700 Chesterfield Pkwy, Chesterfield, MO 63198 (E-mail: jerry.r.colca{at}pfizer.com ).
Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action ...has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
OBJECTIVE—The present study is a comparative investigation of cellular lipid mobilization and efflux to lipid-free human apoA-I and apoA-IMilano, reconstituted high-density lipoprotein (rHDL) ...particles containing these proteins and serum isolated from mice expressing human apoA-I or apoA-IMilano.
METHODS AND RESULTS—Cholesterol and phospholipid efflux to these acceptors was measured in cell systems designed to assess the contributions of ATP-binding cassette A1 (ABCA1), scavenger receptor type BI (SRBI), and cellular lipid content to cholesterol and phospholipid efflux. Acceptors containing the Milano variant of apoA-I showed no functional increase in lipid efflux in all assays when compared with wild-type apoA-I. In fact, in some systems, acceptors containing the Milano variant of apoA-I promoted significantly less efflux than the acceptors containing wild-type apoA-I (apoA-Iwt). Additionally, intracellular cholesteryl ester hydrolysis in macrophage foam cells was not different in the presence of either apoA-IMilano or apoA-Iwt.
CONCLUSION—Collectively these studies suggest that if the Milano variant of apoA-I offers greater atheroprotection than wild-type apoA-I, it is not attributable to greater cellular lipid mobilization.
Quantitative assessment of bile acids in biological matrixes is of growing interest, primarily due to hepatic toxicity resulting from drug interactions with the bile salt export pump. Nevertheless, ...many bile acids demonstrate poor fragmentation in MS, making conventional MS/MS not a good match for their selective quantitation in biological matrices.
The current study was designed to evaluate the feasibility of simultaneous quantitation of 19 bile acids using HRMS coupled to UHPLC separation with minimal instrument optimization. An effective chromatography was developed using an Agilent Zorbax(®) Eclipse XDB-C18 column (1.8 µm, 50 x 2.1 mm internal diameter), achieving separation of 19 compounds in 10 min. Excellent assay reproducibility was demonstrated, with two sets of standard curves, run 42 days apart.
The results show that LC-HRMS is a viable platform for high throughput bioanalysis of bile acids especially in a drug-discovery setting.
The reliable detection of ligand and analyte binding is of significant importance for the field of medical diagnostics. Recent advances in proteomics and the rapid expansion in the number of ...identified protein biomarkers enhance the need for reliable techniques for their identification in complex samples. Surface plasmon resonance imaging (SPRi) provides label-free detection of this binding process in real-time. This chapter details the fabrication of an SPR imaging instrument and its use in analyzing molecular binding interactions with the use of a high-density microfluidic SPRi chip, capable of multiplexed analysis as well as various immobilization chemistries. Controlled recovery of bound biomarkers is demonstrated to enable their identification using mass spectrometry. Finally, activated leukocyte cell adhesion molecule (ALCAM), a protein biomarker associated with a variety of cancers, is identified from human crude cell lysates using the microfluidic surface plasmon resonance imaging (SPRi) instrument.
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FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
► A system established resulted in high expression level of an alpha7 type chimerical receptor. ► The receptor was purified in either an agonist bound form or an antagonist bound form. ► Purified ...receptor has been confirmed to be the intact mature receptor by mutiple methods.
Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT
3 chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT
3 receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies, and tau protein kinase II is reported to play a role in the pathogenesis of ...Alzheimer's disease. Recently, we reported that tau protein kinase II (cdk5/p20)‐phosphorylated human tau inhibits microtubule assembly, and tau protein kinase II (cdk5/p20) phosphorylation of microtubule‐associated tau results in dissociation of phosphorylated tau from the microtubules and tubulin depolymerization. In the studies reported here, a combination of mass spectrometric techniques was used to study the phosphorylation of human recombinant tau by recombinant tau protein kinase II (cdk5/p20) in vitro. The extent of phosphorylation was determined by measuring the molecular mass of phosphorylated tau using mass spectrometry. Reaction of human recombinant tau with tau protein kinase II (cdk5/p20) resulted in the formation of two major species containing either five or six phosphate groups. The specific amino acid residues phosphorylated were determined by analyzing tryptic peptides by tandem mass spectrometry via either MALDI/TOF post‐source decay or by electrospray tandem mass spectrometry. Based on these experiments, we conclude that tau protein kinase II (cdk5/p20) can phosphorylate human tau at Thr181, Thr205, Thr212, Thr217, Ser396 and Ser404.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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