Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

A genetic analysis of the London strain of rainbow trout.

The London strain of rainbow trout (Oncorhynchus mykiss) was created by interbreeding three other strains of rainbow trout and therefore was expected to have higher levels of genetic variation than other strains of rainbow trout. We examined 129 London strain rainbow trout from Indiana by allozyme electrophoresis to assess levels of genetic variation and to examine the relationship between the London strain and other hatchery strains. When using the same loci to compare with other hatchery strains the London strain showed levels of genetic variation within the range of other hatchery strains: mean heterozygosity of 0.053 (0.031-0.099), 1.27 (1.20-1.60) alleles per locus and 20.0% (20.0-40.0%) of the loci were polymorphic. The London strain is somewhat distinct from other hatchery strains (D=0.009-0.072), in part because of the high frequency of the sIDHP*40 allele.

Using 12-lead ECG and synthesized VCG in detection of right ventricular hypertrophy with terminal right conduction delay versus partial right bundle branch block in the pediatric population.

In pediatric electrocardiogram (ECG) analysis, mild right ventricular hypertrophy (RVH) and especially mild RVH with terminal right conduction delay (RVHtcd) are often confused with partial right bundle branch block (PRBBB). This is problematic for computer ECG analysis algorithms and even for most experienced pediatric cardiologists. This study was designed to achieve better classification of mild RVHtcd and PRBBB by combining the 12-lead synthesized vectocardiogram (VCG) transverse plane measurements with scalar ECG measurements. Pediatric ECGs used in the study were recorded with 15 leads and a 500 Hz sampling rate at the Lucile Salter Packard Children's Hospital, Stanford University Medical Center. Out of 4,200 ECGs collected consecutively over a period of 18 months, 447 RVH, 335 RBBB and 589 Normal were interpreted by expert pediatric cardiologists, and were included in the study. Statistical comparison of ECG and VCG measurements were done in stratified ECG sets (412) that have a visually indistinguishable waveform pattern, 117 RVHtcd, 96 PRBBB and 199 normal, showed significant differences in initial and terminal vectors in the transverse plane. The mean angle of the initial vector was anterior (57.2 degrees +/- 41.8) in the normal group, left anterior in the PRBBB group (34.4 degrees +/- 39.5) and in the RVHtcd group (31.9 degrees +/- 41.0) and. The mean angle of the terminal vector was right anterior (158.3 degrees +/- 36.8) in the PRBBB group, rightward (179.7 degrees +/- 29.9) in the RVHtcd group and right posterior (212.6 degrees +/- 37.8) in the normal group. These are clearly applicable features for a classification algorithm. Significantly improved classification results were obtained from a new algorithm using combined ECG and VCG measurements versus an existing algorithm. The limitation of this study stems from the unavailability of a more reliable gold standard. It may be necessary to used body surface potentials obtained with a large number of electrodes to accurately differentiate the study groups.

Differential effects of pravastatin, simvastatin, and atorvastatin on Ca2+ release and vascular reactivity.

The direct effects of the cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors, on vascular smooth muscle responsiveness were examined by incubation of isolated aorta from normocholesterolemic rats with simvastatin, atorvastatin, or pravastatin. The smooth muscle contractions caused by phenylephrine were progressively inhibited with increasing concentrations of simvastatin. Similarly, atorvastatin at the higher concentration caused decreased responses to phenylephrine. In contrast, incubation with pravastatin had no significant effect at all concentrations studied. In Ca2+-free buffer, the transient contraction caused by phenylephrine, which results from intracellular release of Ca2+, also was inhibited by simvastatin and atorvastatin but not by pravastatin. In cultured rat aortic smooth muscle cells loaded with fura-2, increases in intracellular free-Ca2+ concentration ([Ca2+]i) induced by angiotensin II were markedly inhibited in cells incubated with simvastatin and atorvastatin but not pravastatin. The inhibitory effects of simvastatin and atorvastatin were reversed by mevalonate. These findings demonstrate that inhibition of HMG CoA reductase by using simvastatin and atorvastatin, but not pravastatin, has effects on vascular smooth muscle cell responsiveness that involve alteration of Ca2+ homeostasis through a mevalonate-dependent pathway.

An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

Upregulated synthesis of both apolipoprotein A-I and apolipoprotein B in familial hyperalphalipoproteinemia and hyperbetalipoproteinemia.

A family was identified with vertical transmission through three generations with simultaneous increases of apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol, which we have designated familial hyperalphalipoproteinemia and hyperbetalipoproteinemia (HA/HBL). Affected patients develop xanthomas and coronary artery disease (CAD). HA/HBL apoA-I and LDL-apoB were isolated and characterized. The in vivo kinetics of radiolabeled apoA-I and LDL-apoB were evaluated in two HA/HBL probands and three controls. Structural and metabolic characterization showed normal apoA-I and LDL-apoB. The kinetics of metabolism of HA/HBL apoA-I in the HA/HBL subjects showed that elevated apoA-I levels were solely due to an increased synthesis rate (15.2 to 17.6 mg/kg/d v 11.1 to 11.4 mg/kg/d) with a normal apoA-I residence time in plasma (4.2 to 5.4 days v 5.1 to 5.3 days). The elevation of LDL-apoB levels resulted from both an increased synthetic rate (16.6 to 22.9 mg/kg/d v 12.3 to 13.8 mg/kg/d) and a prolonged residence time (3.3 to 3.8 days v 1.4 to 1.9 days). In addition, we evaluated another HA/HBL proband of an unrelated family with HA/HBL to confirm the kinetic data. LDL-receptor binding studies of HA/HBL fibroblasts showed normal binding, uptake, and degradation of LDL isolated from a normolipemic control. The serum concentration of the cholesterol ester transfer protein (CETP) was normal in the studied probands. An apoB 3500 and apoB 3531 mutant, respectively, was ruled out by polymerase chain reaction (PCR). In conclusion, the site of the molecular defect in HA/HBL subjects may be involved in the coordinate regulation of metabolism for both LDL and HDL.

Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. A key unresolved question is whether the MTP-mediated step is rate limiting. To address this, a unique experimental strategy was used that allowed the in situ modulation and measurement of MTP triglyceride transfer activity. In order to accomplish this, an irreversible photoaffinity inhibitor, BMS-192951, was designed and synthesized. When incubated with purified MTP and irradiated with UV light at 360 nm, BMS-192951 inhibits triglyceride transfer by covalently binding to the protein. HepG2 cells were treated with either increasing concentrations of BMS-192951 (0-15 microM) with 5 min of ultraviolet irradiation, or 3.0 microM BMS-192951 with various lengths (0-15 min) of ultraviolet irradiation. Microsomal extracts were prepared exhaustively dialyzed to remove unbound inhibitor, and assayed for MTP-mediated triglyceride transfer activity. BMS-192951 was shown to reduce MTP activity in both a dose- and UV exposure time-dependent fashion. Measurement of apoB concentration in the media showed that apoB secretion was reduced in proportion to the in situ inhibition of MTP activity, while no change was observed in apoA-I secretion. Experiments performed in McArdle RH-7777 rat hepatoma cells and primary rat hepatocytes gave nearly identical results; the decrease in apoB secretion was proportional to the decrease in MTP activity. These results indicate that MTP-mediated lipid transfer is limiting in the assembly and secretion of apoB-containing lipoproteins in hepatic cells under the conditions tested.

Erdheim-Chester disease: low low-density lipoprotein levels due to rapid catabolism.

We have identified a 44-year-old patient with symmetrically excessive xanthomatosis, called Erdheim-Chester disease (ECD), and simultaneously decreased levels of low-density lipoprotein (LDL) cholesterol. Clinically, this patient presents lipoidgranulomatosis of numerous long and flat bones with involvement of the liver, spleen, pericardium, pleura, thyroid, skin, conjunctiva, and gingiva. However, the patient does not have any signs of atherosclerosis. So far, the underlying defect has not been elucidated. We performed a LDL-apolipoprotein B (apoB) kinetic study in the ECD patient and a normal control to determine the etiology of the low LDL level in ECD. LDL was isolated from both subjects, radioiodinated with either 131I or 125I, and injected simultaneously into the ECD patient and the normal control. Normal and ECD LDL was catabolized at the same rate after injection into the control subject (fractional catabolic rate [FCR], 0.43/d and 0.46/d, respectively). Therefore, LDL isolated from an ECD subject is metabolically normal. In contrast, autologous LDL injected into the ECD subject showed a markedly increased catabolism (FCR, 0.69/d) compared with that in the control subject (FCR, 0.43/d). This is the first report about increased catabolism of LDL cholesterol in a patient.

Inhibition of cholesterol synthesis by squalene synthase inhibitors does not induce myotoxicity in vitro.

The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previous in vitro study, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-induced in vitro myotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis ([3H]leucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis ([14C]acetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-induced in vitro myotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that depletion of metabolites of geranylgeranyl pyrophosphate, and not inhibition of cholesterol synthesis, is the primary cause of HMG CoA reductase-induced myotoxicity.

HMG CoA reductase inhibitor-induced myotoxicity: pravastatin and lovastatin inhibit the geranylgeranylation of low-molecular-weight proteins in neonatal rat muscle cell culture.

In previous studies, inhibition of cholesterol synthesis by HMG CoA reductase inhibitors (HMGRI) was associated with myotoxicity in cultures of neonatal rat skeletal myotubes, and rhabdomyolysis in rats, rabbits, and humans in vivo. In vitro myotoxicity was directly related to HMGRI-induced depletion of mevalonate, farnesol, and geranylgeraniol, since supplementation with these intermediate metabolites abrogated the toxicity. Both farnesol and geranylgeraniol are required for the posttranslational modification, or isoprenylation, of essential regulatory proteins in mammalian cells. The objective of the present study was to measure changes in protein isoprenylation in cultured neonatal rat skeletal muscle cells exposed for 24 hr to increasing concentrations of pravastatin or lovastatin. Proteins were labeled with [3H]mevalonate, [3H]farnesyl pyrophosphate (FPP), or [3H]geranylgeranyl pyrophosphate (GGPP), and then separated by SDS-PAGE and quantitated by scintillation counting and densitometry of autoradiographs. Mevalonate and FPP labeling of the majority of proteins increased in a concentration-dependent manner, even at concentrations greater than 2 microM lovastatin and 25 microM pravastatin that completely inhibited cholesterol synthesis. In contrast, mevalonate and FPP labeling of three protein bands with molecular weights of 26.6, 27.7, and 28.9 kDa was markedly inhibited at concentrations higher than 1 microM lovastatin and 400 microM pravastatin, which inhibited protein synthesis and disrupted myotube morphology after longer exposures in a previous study. In contrast, these proteins were equally well labeled by GGPP at all HMGRI concentrations tested, suggesting that isoprenylation of the 26.9-, 27.8-, and 28.9-kDa proteins requires geranylgeraniol. The results of this study indicate that HMGRI-induced myotoxicity is most likely related to reduced posttranslational modification of specific regulatory proteins by geranylgeraniol.

Will genetics really revolutionize the drug discovery process?

Genetics has played only a modest role in drug discovery, but new technologies will radically change this. Whole genome sequencing will identify new drug discovery targets, and emerging methods for the determination of gene function will increase the ability to select robust targets. Detection of single nucleotide polymorphisms and common polymorphisms will enhance the investigation of polygenic diseases and the use of genetics in drug development. Oligonucleotide arraying technologies will allow analysis of gene expression patterns in novel ways.

Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step.

The microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of the lipoproteins containing apolipoprotein B (apoB): very low density lipoproteins and chylomicrons. Evidence indicates that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poor primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles. To determine if either step is mediated by MTP, a series of clonal cell lines stably expressing apoB-53 and MTP was established in non-lipoprotein-producing HeLa cells. MTP activity in these cells was approximately 30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis. Despite having robust levels of triglyceride and phospholipid synthesis, these cell lines, as exemplified by HLMB53-59, secreted >90% of the apoB-53 on relatively lipid-poor particles in the density range of 1.063-1.21 g/ml. These results suggested that coexpression of MTP and apoB only reconstituted the first but not the second step in lipoprotein assembly. To extend this observation, additional studies were carried out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined. Treatment of these cells with the MTP photoaffinity inhibitor BMS-192951 before pulse labeling with [35S]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly. After a 30-min [35S]methioneine/cysteine pulse labeling and 120 min of chase, all of the nascent apoB-48 was observed to have a density of high density lipoproteins (1.063-1.21 g/ml), indicating that only the first step of lipoprotein assembly had occurred. The addition of oleic acid to the cell culture media activated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period. Inactivation of MTP after completion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion. Thus, inhibition of MTP did not hinder the addition of bulk core lipid to the primordial lipoprotein precursor particles, indicating that MTP is not required for the second step of apoB-48 lipoprotein assembly.

An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

Dominant expression of type III hyperlipoproteinemia. Pathophysiological insights derived from the structural and kinetic characteristics of ApoE-1 (Lys146-->Glu).

Type III hyperlipoproteinemia is characterized by delayed chylomicron and VLDL remnant catabolism and is associated with homozygosity for the apoE-2 allele. We have identified a kindred in which heterozygosity for an apoE mutant, apoE-1 (Lys146-->Glu), is dominantly associated with the expression of type III hyperlipoproteinemia. DNA sequence analysis of the mutant apoE gene revealed a single-point mutation that resulted in the substitution of glutamic acid (GAG) for lysine (AAG) at residue 146 in the proposed receptor-binding domain of apoE. The pathophysiological effect of this mutation was investigated in vivo by kinetic studies in the patient and six normal subjects, and in vitro by binding studies of apoE-1 (Lys146-->Glu) to LDL receptors on human fibroblasts and to heparin. The kinetic studies revealed that apoE-1 (Lys146-->Glu) was catabolized significantly slower than apoE-3 in normals (P < 0.005). In the proband, the plasma residence times of both apoEs were substantially longer and the production rate of total apoE was about two times higher than in the control subjects. ApoE-1 (Lys146-->Glu) was defective in interacting with LDL receptors, and its ability to displace LDL in an in vitro assay was reduced to 7.7% compared with apoE-3. The affinity of apoE-1 (Lys146-->Glu) to heparin was also markedly reduced compared with both apoE-2 (Arg158-->Cys) and apoE-3. These abnormal in vitro binding characteristics and the altered in vivo metabolism of apoE-1 (Lys146-->Glu) are proposed to result in the functional dominance of this mutation in the affected kindred.

Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein particles.

The mechanism of assembly of lipoprotein particles in the lumen of the endoplasmic reticulum is an important but poorly understood biological problem. A knowledge of this process is of great practical importance because possession of elevated levels of lipoproteins is one of the major risk factors for the development of atherosclerosis. This review describes a major advance in the delineation of the mechanisms involved in the assembly and secretion of apolipoprotein-B-containing lipoproteins: the demonstration of a requirement for microsomal triglyceride transfer protein.

A 30-amino acid truncation of the microsomal triglyceride transfer protein large subunit disrupts its interaction with protein disulfide-isomerase and causes abetalipoproteinemia.

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the multifunctional enzyme, protein disulfide-isomerase, and a unique large, 97 kDa, subunit. It is found as a soluble protein within the lumen of the endoplasmic reticulum of liver and intestine and is required for the assembly of very low density lipoproteins and chylomicrons. Mutations in MTP which result in an absence of MTP function have been shown to cause abetalipoproteinemia. Here, the gene encoding the MTP 97-kDa subunit of an abetalipoproteinemic subject, which we have previously demonstrated lacks MTP activity and protein (Wetterau, J. R., Aggerbeck, L. P., Bouma, M.-E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay, G., Rader, D. J., and Gregg, R. E. (1992) Science 258, 999-1001), was isolated and sequenced. A nonsense mutation, which predicts the truncation of the protein by 30 amino acids, was identified. To investigate if this apparently subtle change in MTP could explain the observed absence of MTP, protein disulfide-isomerase was co-expressed with either the normal or mutant MTP 97-kDa subunit in Sf9 insect cells using a baculovirus expression system. Although there were high levels of expression of both the normal and mutant forms of the MTP 97-kDa subunit, only the normal subunit was able to form a stable, soluble complex with protein disulfide-isomerase. These results indicate that the carboxyl-terminal 30 amino acids of the MTP 97-kDa subunit plays an important role in its interaction with protein disulfide-isomerase.

Microsomal triglyceride transfer protein. Specificity of lipid binding and transport.

Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein that is required for the assembly and secretion of very low density lipoproteins by the liver and chylomicrons by the intestine. To further elucidate the nature of the lipid molecule binding and transport site on MTP, we have studied the relative rates at which MTP transports different lipid species. Assay conditions were chosen in which there were minimal changes in the physical properties of the substrate membranes so that transfer rates would reflect MTP-lipid interactions at a membrane surface. Lipid transport rates decreased in order of triglyceride > cholesteryl ester > diglyceride > cholesterol > phosphatidylcholine. Changes in the hydrophobic nature of a lipid molecule by the addition of a fatty acid, modulated the ability of MTP to transport it. Addition of one acyl chain from diglyceride to triglyceride, lysophosphatidylcholine to phosphatidylcholine, or cholesterol to cholesteryl ester increased the rate of MTP-mediated transport 10-fold. In contrast, the lipid transport rate was insensitive to the changes in the structure or charge of the polar head group on phospholipid substrates. Zwitterionic, net negative, or net positive charged phospholipid molecules were all transported at a comparable rate. The ability of MTP to transport lipids is strongly correlated to the binding of these lipids to MTP. Thus, MTP has a specific preference for binding and transporting nonpolar lipid compared with phospholipids, and within a class of lipid molecules, a decrease in polarity increases its tendency to be transported.