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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 A(2) 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.

Proprotein convertase subtilisin-kexin-9 (PCSK9) inhibition markedly augments the LDL lowering action of statins. The combination is being evaluated for long-term effects on atherosclerotic disease outcomes. However, effects of combined treatment on hepatic cholesterol and bile acid metabolism have not yet been reported. To study this, PCSK9-Y119X mutant (knockout) and wild-type mice were treated with or without atorvastatin for 12 weeks. Atorvastatin progressively lowered plasma LDL in each group, but no differences in liver cholesterol, cholesterol ester, or total bile acid concentrations, or in plasma total bile acid levels were seen. In contrast, atorvastatin increased fecal total bile acids (≈ 2-fold, P < 0.01) and cholesterol concentrations (≈ 3-fold, P < 0.01) versus controls for both PCSK9-Y119X and wild-type mice. All 14 individual bile acids resolved by LC-MS, including primary, secondary, and conjugated species, reflected similar increases. Expression of key liver bile acid synthesis genes CYP7A1 and CYP8B1 were ≈ 2.5-fold higher with atorvastatin in both strains, but mRNA for liver bile acid export and reuptake transporters and conjugating enzymes were not unaffected. The data suggest that hepatocyte cholesterol and bile acid homeostasis is maintained with combined PCSK9 and HMG-CoA reductase inhibition through efficient liver enzymatic conversion of LDL-derived cholesterol into bile acids and excretion of both, with undisturbed enterohepatic recycling.

Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a novel regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss-of-function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2 deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Additional glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs associated with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metabolism across mammals.

The geometrical isomers of 33,34,35-trimethoxy-5,10,15-trimethyl-22,25,30-trioxa-1,19-diazapentacyclo[17.8.51 3,7 .1 8,12 .1 13,17 ]pentatriaconta-3,5,7(33),8,10,12(34),13,15,17(35)-nonaene-2,2,18-dione, C 36 H 44 N 2 O 8 , are intermediates in the synthesis of reduced crypta-hemispherands. Their amide groups are characteristically coplanar with their attached atoms. Consequently, the N atoms in these structures are neither exo nor endo, unlike the N atoms in most other cryptands and crypta-hemispherands. In the isomer formed in smaller amount, (II), the N atoms are about 1 A closer to each other than in the other structure, (I), because the five-atom bridge between these N atoms is so constrained as to draw them together. Structure (I) is more dense than structure (II) by about 9%.

The [1.1]cryptahemispherand host 30,31,32-trimethoxy-5,10,15-trimethyl-22,27-dioxa-1,19-diazapentacyclo-[17.5.5.1 3,7 .1 8,12 .1 13,17 ]dotriaconta-3,5,7(30),8,10,12(31),13,15,17(32)-nonaene forms a crystalline complex, (I), with sodium tetraphenylborate, [Na(C 34 H 44 N 2 O 5 )](C 24 H 20 B), and also forms a coordination complex, (II), with two molecules of BH 3 , μ-(30,31,32-trimethoxy-5,10,15-trimethyl-22,27-dioxa-1,19-diazapentacyclo[17.5.5.1 3,7 .1 8,12 .1 13,17 ]dotriaconta-3,5,7(30),8,10-12(31),13,15,17(32)-nonaene)-N 1 : N 19 -bis(trihydroboron), C 34 H 50 B 2 N 2 O 5 , which crystallizes as a CH 2 Cl 2 hemisolvate. The host of (I) complexes Na + more strongly than it does any other alkali metal ion, consistent with the nearly ideal coordination of Na + reported here. The N atoms, which are only about 2.6 A from the Na + ion, are unusually close to each other in (I), being separated by about 4.63 A. The solvent in the structure of (I) is disordered and unidentifiable. The structure of (II) was determined at 115 K. It contains two independent molecules in the asymmetric unit, each with site symmetry m; they have very similar conformations. The BH 3 groups attached to the N atoms are directed outward from the cavity of each molecule, giving the molecules the uncommon exo-exo conformation. The N atoms in (II) are about 1.3 A farther apart than those in (I). Because the N atoms are tetrasubstituted, (II) is a poor complexer. One -CH 2 CH 2 OCH 2 CH 2 - bridge in each cryptand moiety in (II) adopts a 'crown' conformation, while the other bridge has trans -CH 2 CH 2 - torsion angles. The cavity of the host in (II) is filled, and the conformation is stabilized by typical C-H...O interactions.

The potassium complex of the [2.1]cryptahemispherand 33,34,35-trimethoxy-5,10,15-trimethyl-22,25,30-trioxa-1, 19-diazapentacyclo [17.8.5.1 3,7 .1 8,12 .1 13,17 ] pentatriaconta-3,5,7(33),8,10,12(34),13,15,17(35)-nonaene, crystallizes from ethyl acetate with a thiocyanate counterion ([K(C 36 H 48 N 2 O 6 )]SCN}. The space group is Pnma. The longer cryptand-like bridge is disordered, departing from mirror symmetry because it is constrained to fold unsymmetrically by the geometry of the rest of the host. Five of the O atoms and the two N atoms of the host, as well as the N of the SCN- ion, are coordinated by the K + ion. The sixth O atom of the host is at 3.211 (3) A from the K + ion, about 0.5 A farther than the closest O atoms.

Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.

Two pharmacologically distinct CRF receptors are distributed in different brain regions and peripheral tissues. Studies suggest that CRF(1) receptors play an important role in mediating the anxiety provoking effects of CRF. In contrast, far less functional information is available on CRF(2) receptors. Therefore, we conducted dose response studies using antisauvagine-30 (anti-SVG-30, 0-20 microg, 20-min pretreatment, i.c.v.), a potent CRF(2) peptide antagonist, and tested rats in three models of anxiety - the conditioned freezing, the elevated plus maze, and the defensive-withdrawal test. Anti-SVG-30 produced a significant dose-dependent reduction in conditioned freezing. In the elevated plus maze test, administration of anti-SVG-30 effectively increased the number of entries and time spent in the open arms. In the defensive-withdrawal test, anti-SVG-30 treatment facilitated exploratory activity in a large illuminated open field. Thus, in all three animal models, administration of anti-SVG-30 was consistent in producing an anxiolytic-like behavioral effect. In addition, a dose of anti-SVG-30 (10 microg) that produced anxiolytic-like behavior had no significant effects on locomotor activity measured in an automated activity box. This latter finding suggests that antagonism of CRF(2) receptors is not associated with a non-specific increase in behavioral movements. These results provide evidence that, in addition to CRF(1) receptors, CRF(2) receptors may play an important role in the mediation of anxiety behavior.

Corticotropin releasing factor (CRF) is an important regulator of the endocrine, behavioral, autonomic and immune responses to stress. Two high affinity CRF receptors have been identified, which are distributed in distinct anatomical regions. CRF(1) receptors have been relatively well characterized and antagonists to this receptor effectively block stress-induced behaviors in rodents. The function of CRF(2) receptors, which are highly expressed in limbic brain regions, is less well understood. Therefore, an antisense oligonucleotide approach was used to study the role of CRF(2) receptors in the lateral septum in rats. An antisense oligonucleotide directed against the CRF(2) receptor mRNA reduced expression of CRF(2) receptors by 60--80%. In shock-induced freezing tests, animals administered the antisense oligonucleotide exhibited a significant reduction in freezing duration. However, pain sensitivity and locomotor activity were unaltered. A four-base mismatch of the antisense sequence had no significant effects on CRF(2) receptor density and on freezing behavior. These data support the involvement of CRF(2) receptors in fear conditioning. CRF(1) receptor antagonists also reduce freezing in this test. Additional studies to determine the effects of simultaneous inhibition of both receptor subtypes show that rats receiving both CRF(2) receptor antisense oligonucleotide and CRF(1) receptor antagonist froze significantly less than animals treated with either agent alone. These results provide additional evidence for the role of CRF(2) receptors in mediating the stress-induced actions of endogenous CRF.

The mouse aspartyl beta-hydroxylase gene (Asph, BAH) has been cloned and characterized. The mouse BAH gene spans 200 kilobase pairs of genomic DNA and contains 24 exons. Of three major BAH-related transcripts, the two largest (6,629 and 4,419 base pairs) encode full-length protein and differ only in the use of alternative polyadenylation signals. The smallest BAH-related transcript (2,789 base pairs) uses an alternative 3' terminal exon, resulting in a protein lacking a catalytic domain. Evolutionary conservation of this noncatalytic isoform of BAH (humbug) is demonstrated in mouse, man, and Drosophila. Monoclonal antibody reagents were generated, epitope-mapped, and used to definitively correlate RNA bands on Northern blots with protein species on Western blots. The gene for mouse junctin, a calsequestrin-binding protein, was cloned and characterized and shown to be encoded from the same locus. When expressed in heart tissue, BAH/humbug preferably use the first exon and often the fourth exon of junctin while preserving the reading frame. Thus, three individual genes share common exons and open reading frames and use separate promoters to achieve differential expression, splicing, and function in a variety of tissues. This unusual form of exon sharing suggests that the functions of junctin, BAH, and humbug may be linked.

The importance of finding good antisense sequences cannot be underestimated. Poor inhibition of the targeted protein can compromise the final outcome of an antisense experiment, making it difficult to arrive at a definitive understanding of the function of the protein of interest. In antisense therapeutics, identification of potent sequences becomes even more important. RNA mapping greatly increases the odds of finding active sequences. When antisense sequences are selected randomly or by gene walking, a substantial number of the oligonucleotides have little to no activity. In contrast, oligonucleotides selected by RNA mapping typically produce an antisense inhibition of greater than 50%. Oligonucleotides targeted to 60% of the accessible sites in the 5′ portion of the multidrug resistance transcript inhibited P-glycoprotein function with high potency.5 In the angiotensin type 1 receptor system, oligonucleotides to the eight accessible sites examined inhibited AT1 receptor binding by at least 70% inhibition.6 The RNA mapping assay, which is based on standard molecular techniques, therefore provides an easy and reliable method for potent antisense sequence selection.

Multiple, diverse sites in the coding region of the angiotensin type-1 receptor mRNA were targeted with 2'-deoxyribonucleotide antisense oligonucleotides (ONs). The uptake of 1 microM concentration of these ONs into Chinese hamster ovary cells was facilitated by the use of cationic liposomes. The antisense sequences reduced binding of 125I-angiotensin II by 57-73%, while mismatch ONs and reverse sequence ONs produced little reduction in receptor binding. These reductions in AT1 receptor binding were accompanied by comparable decreases in AT1 receptor mRNA levels. Furthermore, mRNA cleavage fragments corresponding in size to 3'-cleavage fragments were observed with two of the antisense ONs, consistent with the involvement of an RNase H-type enzyme. When 2'-methoxyribonucleotide analogs of these same sequences were tested, AT1 receptor mRNA levels were unchanged even though small reductions in AngII binding were observed. Antisense effects seen with these 2'-methoxyribonucleotide sequences may have arisen through a translational arrest mechanism. Direct comparisons between 2'-deoxyribonucleotide analogs and their 2'-methoxyribonucleotide counterparts show that antisense effects are significantly larger when they are mediated through an RNase H-type mechanism. 2'-methoxyribonucleotide sequences were most effective when they were directed against the translation initiation codon.

We have determined the time course, the spatial spread in brain tissue, and the intracellular distribution of biotin- and fluorescein-labeled phosphorothioate oligodeoxynucleotides (ODNs) following single injections into the rat striatum or the lateral ventricle. These time and space parameters were correlated with the ability of c-fos phosphorothioate antisense ODNs to suppress the induction of Fos protein by cocaine. A rapid and dose-dependent tissue penetration of labeled ODNs was observed following either intrastriatal or intraventricular injections of a constant sample volume. Inspection of tissue sections by confocal microscopy uncovered a distinct change in the intracellular disposition of labeled ODNs during the 24 h post-injection period. At 1, 6 and 12 h, the vast majority of the fluorescent signal was confined to the interstitial spaces throughout the zone penetrated by ODNs. Neuronal nuclei displayed faint labeling along the outer portion of the nucleus at 1 and 6 h post-injection. At these time-points, ODNs were not detected in the cytoplasm. By 16 h, ODNs were barely detectable in the extracellular space and absent from neuronal nuclei. Instead, ODNs were seen in large cytoplasmic granules of neurons throughout the tissue zone penetrated by the ODNs. Experiments with intrastriatal injections of antisense ODNs to c-fos mRNA revealed Fos suppression between 3 and 12 h, but not at 16 and 24 h. This combined analysis has revealed that (1) restricted tissue penetration by ODNs limits their antisense effects on protein expression, and (2) depletion of extracellular ODNs and sequestration of c-fos antisense ODNs into large intracellular granules coincides with the loss of their biological activity.

There is increasing evidence that phosphorothioate oligonucleotides infused into the brain can cause a host of undesired side effects which compromise the antisense experiment. In studies on the corticotropin releasing factor type-2 receptor, several phosphorothioate oligonucleotides administered intraventricularly produced significant weight loss in rats. Four different phosphodiester and phosphorothioate oligonucleotide analogs were examined to identify molecules which could eliminate these side effects while maintaining good potency for antisense inhibition. Of these, chimeric oligonucleotides consisting of a mixed phosphodiester–phosphorothioate backbone, and having 2′-methoxyribonucleotide modifications in 60% of the oligonucleotide were the most optimal. Rats treated with these chimeric oligonucleotides gained weight at rates identical to that of saline-treated controls. In addition, the antisense oligonucleotide but not the mismatch control sequence reduced corticotropin releasing factor type-2 receptor binding of 125 iodo-sauvagine in the lateral septum by 40–60% after 5 daily injections. Increasing the dosing period to 9 days reduced receptor binding by 78%. Reductions in protein binding were accompanied by comparable reductions in the in situ hybridization signal of the corticotropin releasing factor type-2 receptor mRNA. However, when an oligonucleotide analog incapable of supporting ribonuclease H activity was used, neither protein nor RNA binding levels were changed compared to saline-treated controls. These results suggest that ribonuclease H or enzymes with similar activity are critical to the antisense inhibition observed in the lateral septum.

Antisense experiments are often complicated by the lack of reliable methods for selecting effective antisense sequences. Chimeric oligodeoxynucleotide (ODN) libraries and ribonuclease H (RNase H) were used to identify regions on the 1253 nucleotide angiotensin type-1 receptor (AT1) mRNA that are accessible to hybridization with antisense ODNs. Phosphorothioate antisense ODNs targeted against accessible sites reduced AT1 receptor levels by at least 50% in cell culture. ODNs to 4 sites produced a 70% to 80% reduction. In contrast, most sequences targeted between accessible sites were ineffective. When injected into the brains of rats, ODNs targeted to accessible sites reduced AT1 (by 65%) but not AT2 receptor levels. Additionally, AT1 receptor function as measured by agonist-induced water intake, was significantly attenuated in these rats. ODNs directed between accessible sites were ineffective at suppressing water intake. RNA mapping can be applied to any RNA target to facilitate selection of multiple, active antisense sequences for cell culture and in vivo experiments.

Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor alpha. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NF kappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

Antisense oligonucleotides can vary significantly and unpredictably in their ability to inhibit protein synthesis. Libraries of chimeric oligonucleotides and RNase H were used to cleave and thereby locate sites on human multidrug resistance-1 RNA transcripts that are relatively accessible to oligonucleotide hybridization. In cell culture, antisense sequences designed to target these sites were significantly more active than oligonucleotides selected at random. This methodology should be generally useful for identification of potent antisense sequences. Correlation between oligonucleotide activity in the cell culture assay and in an in vitro RNase H assay supports the proposed role of the enzyme in the mechanism of antisense suppression in the cell.

The association constants (K/sub a/, M/sup -1/) and free energies of binding (-..delta..G/sup 0/), kcal mol/sup -1/) have been measured at 25 /sup 0/C in CDCl/sub 3/ saturated with D/sub 2/O for cryptahemispherands, cryptands, and diazahemispherand binding the alkali metal picrate salts. Methods were used in which complexes whose K/sub a/ values had been determined were equilibrated with hosts of unknown values. The equilibrium points were determined by /sup 1/H NMR spectral methods. The cryptahemispherands as a class were found to be more powerful complexing agents than the cryptands. The two classes exhibited comparable ion selectivities. Plots of the -..delta..G/sup 0/ values of the cryptands in CDCl/sub 3/-D/sub 2/O vs. those in 95% CH/sub 3/OH-5% H/sub 2/O were linear. The -..delta..G/sup 0/ values for eight different sets of binding partners in CDCl/sub 3/-D/sub 2/O were found to be 5.2 +/- 0.4 kcal mol/sup -1/ higher than the corresponding eight values in CH/sub 3/OH-H/sub 2/O. The results are discussed in terms of the relative states of preorganization for binding and desolvation of the various host classes.

It is well known that the dynamic response rate of the Severinghaus-type CO2 electrode is improved significantly in some cases by addition of the enzyme carbonic anhydrase to the electrode assembly. Hysteresis in the response rate also is reduced. Experimental data and modelling results indicate that catalysis of the CO2 hydration reaction in the bulk of the bicarbonate layer (the Nernst film) is not responsible for the improved response behavior, Evidence is presented to show that catalysis in the electrostatic double layer region at the glass electrode surface is a possible explanation. This proposed phenomenon may have widespread implications for the optimal design of analytical devices, commercial processes involving electrochemical phenomena, and may also provide insight into electrobiologi-cal processes.

Syntheses and crystal structures are reported for a new class of hosts, their complexes, and their precursors. The cryptahemispherands are composed of molecular modules that are half spherand and half cryptand. They were synthesized by the reactions of diacid chloride and cyclic diamines to produce diamides, reduction of which gave the desired hosts. Cryptahemispherands form a variety of complexes with the alkali metal cations. Crystal structures were determined for the isomeric diamides for the hydroborane complex, and for alkali cation complexes.

Etude des reactions de transfert d'acyle entre le perchlorate de nitro-4' phenoxycarbonyl-1 ethyle et des nucleophiles derives d'hexaaza [4.4.3] metacyclophane

Twelve- and sixteen-residue peptides have been designed to form tetrameric alpha-helical bundles. Both peptides are capable of folding into amphiphilic alpha-helices, with leucyl residues along one face and glutamyl and lysyl residues along the opposite face. Four such amphiphilic alpha-helices are capable of forming a noncovalently bonded tetramer. Neighboring helices run in antiparallel directions in the design, so that the complex has 222 symmetry. In the designed tetramer, the leucyl side chains interdigitate in the center in a hydrophobic interaction, and charged side chains are exposed to the solvent. The designed 12-mer (ALPHA-1) has been synthesized, and it forms helical aggregates in aqueous solution as judged by circular dichroic spectroscopy. It has also been crystallized and characterized by x-ray diffraction. The crystal symmetry is compatible with (but does not prove) the design. The design can be extended to a four-alpha-helical bundle formed from a single polypeptide by adding three peptide linkers.