Stability of low concentrations of guanine-based antivirals in sucrose or maltitol solutions.

Three guanine-based antiviral drugs, entecavir, lobucavir, and acyclovir showed degradation in presence of sucrose in ready-to-use solutions held at 50 degrees C, with more degradation at pH 4 than at pH 6 or 7. LC/MS analysis of the solutions showed isomeric adducts of the drugs and reducing sugars. Sucrose, a disaccharide and a non-reducing sugar, was the source of monosaccharides, the reducing sugars. Sucrose showed pH-dependent hydrolysis at 50 degrees C into two monosaccharides, fructose and glucose, with more sucrose hydrolyzing at pH 4 than pH 6 or 7. Additionally, the three drugs showed pH-dependent degradation at 50 degrees C in fructose and glucose solutions with the following rank order: pH 7>pH 6>pH 4. This indicated that the increased degradation of the drugs in sucrose solutions at pH 4 was mainly due to more hydrolysis of sucrose into fructose and glucose compared to pH 6 or 7, and subsequent reactions of the fructose and glucose with the drugs. Based on structures of the major degradants, it is proposed that the main cause of the degradation was nucleophilic addition of the primary amine group of the drugs to the carbonyl group of the fructose and glucose. This reaction was facilitated as the solution pH increased from 4 to 7. All the drugs showed satisfactory stability regardless of the storage temperature or solution pH in maltitol, an alternate sweetener. The free aldehyde or ketone group in maltitol precursors is reduced to a hydroxyl group after the hydrogenation process making maltitol less susceptible to nucleophilic addition.

Molecular heterogeneity of urinary proteins in wild house mouse populations.

Major urinary proteins (MUPs) from the urine of individual wild mice were characterized using electrospray ionization mass spectrometry (ESI-MS) and compared to MUPs from the urine of inbred mice. The wild mice showed considerable variation between individuals in the expression of a group of MUPs with similar masses. Some individuals excreted MUPs of unique molecular mass whilst some failed to express MUPs seen commonly in the other individuals. All the wild individuals contained proteins not previously observed in inbred mice. Urine from one individual was fractionated using anion exchange chromatography prior to analysis by ESI-MS. By analysing urine from inbred samples under the same conditions it was possible to relate, using mass and net charge in solution, MUPs from the wild sample to the MUPs that have been observed previously in inbred strains. This has allowed tentative identification of some MUPs from the wild mouse. The effect of collection history of urine from wild mice was also investigated. ESI-MS analysis of MUPs in a faecally contaminated sample showed the loss of a C-terminal tripeptide when compared to an uncontaminated sample from the same mouse, consistent with the presence of a specific endopeptidase. Similarly a sample of pooled urine provided by twelve individuals trapped from the same population showed evidence of loss of the C-terminal dipeptide.

First direct evidence for lipid/protein conjugation in oxidized human low density lipoprotein.

It has been postulated that lipids incorporated in atherosclerotic plaques are derived from the uptake of oxidized low density lipoprotein (LDL) by a macrophage-bound receptor. In vitro studies of LDL oxidation have established that reactive lipids are formed and that the exposure of native LDL to these products leads to modified protein with physical properties similar to oxidized LDL. Here we describe the application of highly specific tandem mass spectrometric techniques to the first characterization of lipid-modified LDL by demonstrating the addition of 4-hydroxy-2-nonenal to histidine residues of apolipoprotein B-100, following oxidation of LDL. The modified residues have been assigned to specific locations that have been previously shown to reside on the surface of the LDL particle.

The proteolytic specificity of the natural enediyne-containing chromoproteins is unique to each chromoprotein.

BACKGROUND: Enediyne chromoproteins are potent antitumor antibiotic agents. They consist of a labile nine-membered enediyne chromophore non-covalently associated with a stabilizing acidic polypeptide. Studies in vitro on three members of this superfamily of natural products--kedarcidin, maduropeptin and neocarzinostatin--demonstrated that their chromophores cleave DNA at sites specific to each chromophore. Recently, we showed that these chromoproteins possess proteolytic activity against histones in vitro, with histone H1 as a preferred substrate. Based on these results, we speculated that this selective proteolytic activity may be important in vivo in the delivery of the enediynes intact to the DNA in chromatin. RESULTS: We show here that each chromoprotein generates a unique set of H1 fragments as revealed by gel analyses of the H1 cleavage reaction products. To probe the observed cleavage specificity, we synthesized a 24-amino-acid peptide representing a basic region of histone H1. This model peptide was incubated individually with similar concentrations of the kedarcidin, neocarzinostatin and maduropeptin chromoproteins as well as the kedarcidin apoprotein. The reaction products were analyzed by electrospray liquid chromatography/mass spectrometry. Our results indicate that all proteins cleave the peptide at selected backbone amides, and that these sites vary according to the chromoprotein used. Moreover, the kedarcidin apoprotein appears to be less specific than the kedarcidin chromoprotein complex. CONCLUSIONS: The small size, unique architecture and very acidic nature of the enediyne chromoproteins are highly unusual. These natural products exhibit the dual functionalities of specific DNA cleavage and selective proteolytic activity. This observation adds to the fascinating properties of these molecules and suggests that it is possible not only to design small moieties to cleave DNA but also to conceive of small proteins to deliver these moieties intact to defined areas of chromatin.

Identification of the serine residue at the active site of the herpes simplex virus type 1 protease.

Herpes simplex virus type 1 (HSV-1) encodes a protease that is essential for proteolytic processing of itself and of the nucleocapsid-associated protein, ICP35 (infected cell protein 35) (Liu, F., and Roizman, B. (1991) J. Virol. 65, 5149-5156). Inhibitor studies indicated that the HSV-1 protease is sensitive to the serine protease inactivator diisopropyl fluorophosphate (DFP). Inactivation is irreversible and dependent on time and concentration of DFP. Loss of activity correlates linearly with the incorporation of [3H]DFP. Analysis of completely inactivated protease by mass spectrometry indicated a stoichiometry of 1 DFP/protease. In order to identify the specific residue modified by DFP, the protease was labeled with [3H]DFP and subsequently digested with trypsin or chymotrypsin. The peptides resulting from each digestion were separated by reverse phase HPLC, and the radioactivity was recovered in a single peak. Mass spectrometric studies and sequencing analysis by Edman degradation identified Ser-129 as the residue modified by DFP. This residue and the region in which it is found is highly conserved among the herpes viral proteases. These data demonstrate that HSV-1 protease is a serine protease and that Ser-129 is the active site nucleophile.

In vitro activity of the herpes simplex virus type 1 protease with peptide substrates.

Herpes simplex virus type-1 (HSV-1) protease is responsible for proteolytic processing of itself and the virus assembly protein ICP35 (infected cell protein 35). Two proteolytic processing sites within the protease have recently been identified between Ala247 and Ser248 and between Ala610 and Ser611. In this report we demonstrate that peptides corresponding to each of these cleavage sites are substrates for recombinant HSV protease-glutathione S-transferase fusion protein in vitro by high performance liquid chromatography analysis of cleavage reactions. Analysis of the products by fast atom bombardment-mass spectrometry confirmed that cleavage occurred at the expected position between the Ala and Ser residues of the substrate. Peptide cleavage was linear with respect to time and enzyme concentration and proceeded optimally at pH 8.0. A peptide that spans Ala99/Ser100 of the protease but does not correspond to a naturally occurring cleavage site was not a substrate for the protease in vitro confirming that sequence elements outside the conserved dipeptide sequence are required for substrate recognition and cleavage. Identification of P5-P8' as the minimal substrate peptide for the Ala610/Ser611 cleavage site revealed a requirement for residues flanking the conserved core P4-LVNA/S-P1' in substrate recognition and hydrolysis. Kinetic analysis with peptide P5-P8' yielded a Km of 190 microM and kcat of 0.2 min-1. Experiments with a panel of class-specific protease inhibitors were consistent with the protease being a member of the general class of serine proteases.

Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus.

Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K(+)-ATPase in humans. We have purified a bovine hypothalamic Na+/K(+)-ATPase inhibitory factor (HIF) by using affinity chromatography combined with HPLC. This inhibitor has a molecular weight of 584 as determined by ion-spray mass spectrometry, making it isobaric with ouabain. Glycosidase treatment or acid hydrolysis of HIF released only L-rhamnose, the hexose isomer found in ouabain, as detected by chiral GC/MS. Additionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabain. HIF and its aglycone were indistinguishable from ouabain and ouabagenin, respectively, by reversed-phase HPLC retention times. However, derivatization with naphthoylimidazole followed by HPLC revealed different retention times for naphthoylation products of HIF and ouabain. Subsequent CD spectroscopy on isolated naphthoylation products of HIF and ouabain confirmed that they were different. This study provides chromatographic and spectroscopic evidence that ouabain and HIF are isomeric cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.

Control of peptide disulfide regioisomer formation by mixed cysteine-penicillamine bridges. Application to endothelin-1.

While incorporation of penicillamine residues (Pen; beta,beta-dimethyl cysteine) into a peptide can cause dramatic changes in biological activity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the beta-methyls as a synthetic device to control the production of disulfide isomers. That is, oxidation of a peptide containing an equal number of Cys and Pen residues should predominantly form products which contain mixed Cys-Pen disulfides. Endothelin (ET) is a 21 amino acid peptide which contains Cys at positions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been reported to produce a 3:1 ratio of the natural 1-15, 3-11 to the unnatural 1-11, 3-15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products containing Cys-Pen disulfides. Random oxidation (air, aqueous NH4OH) of the tetrathiols of [Pen1,11, Nle7]-ET-1 or [Pen3,15, Nle7]-ET-1 produced the correct 1-15, 3-11 isomer in > 12:1 and > 22:1 ratios, respectively. Oxidation of the tetrathiol of [Pen1,15, Nle7]-ET-1 favored the unnatural 1-11, 3-15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product as a result of the driving force for penicillamine mixed disulfide formation. Disulfide isomers were identified using ion-spray mass spectrometry in conjunction with enzymatic and acid hydrolysis. [Pen1,11, Nle7]-ET-1 was a partial agonist at the ETA receptor (EC50 = 7.5 nM in rabbit carotid artery rings; Kd = 4.5 nM in rat A10 cell membranes) while [Pen3,15, Nle7]-ET-1 (EC50 = 0.9 nM; Kd = 0.7 nM) was a full agonist with similar potency to ET-1.

Structure-activity studies of endothelin leading to novel peptide ETA antagonists.

With the goal of producing receptor antagonists, numerous monocyclic and bicyclic endothelin analogs were prepared and tested for vasoconstrictor activity, receptor affinity and functional antagonist activity. Bis-penicillamine endothelin analogs containing Ala or Asn at position 18 were functional antagonists, with Ki values of 20-40 nM but KB values of about 1 microM (e.g., [Pen1,11, Nle7, Ala18]-endothelin-1, Ki = 42 nM, KB = 1.2 microM). While these peptides are antagonists at the ETA receptor, they appear to be at least partial agonists at another receptor subtype.

Isolation and identification of lysophosphatidylcholines as endogenous modulators of thromboxane receptors.

Inhibition of thromboxane receptor radioligand binding to human platelet membranes has been employed as the basis for a radioreceptor assay designed to measure thromboxane receptor binding activity in samples of biological fluids. This method was used during phase 1 clinical evaluation of the thromboxane receptor antagonist SQ 30,741. Frequently, baseline plasma samples as well as plasma samples from placebo-treated subjects showed significant inhibition of radioligand binding in the radioreceptor assay, suggesting the presence of endogenous thromboxane receptor ligands. This receptor binding activity was stable and could be monitored in blood from normal volunteers using a modification of the radioreceptor assay. In order to identify the substance responsible for the observed activity, the activity present in pooled bovine blood was isolated and evaluated by a combination of FAB/MS, 1H-NMR, 13C-NMR and co-injection with reference standards on HPLC. Several endogenous thromboxane receptor ligands were identified as L-alpha-lysophosphatidylcholine (LPC) species. One major species, palmitoyl-LPC, contracted isolated rat aortic spirals, and these contractions could be delayed or prevented, but not reversed by the thromboxane receptor antagonist SQ 29,548. Palmitoyl-LPC slightly potentiated aortic contractions induced by the thromboxane receptor agonist, U-46,619, and diminished in a concentration-dependent manner the antagonism by SQ 29,548 of contractile responses to U-46,619. These findings are consistent with a potential for LPC species to bind and activate thromboxane receptors.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. II. Structure elucidation.

Fermentation of Dactylosporangium sp. (ATCC 53693) produces a mixture of tetracycline derivatives from which several related tetracycline glycosides, the dactylocyclines, were isolated and their structures determined. The most abundant glycoside in initial fermentations was found to be dactylocycline A. Each glycoside proved to be acid sensitive and readily hydrolyzed to a common aglycone, dactylocyclinone. While the aglycone was cross resistant with tetracycline, the dactylocyclines proved active against certain tetracycline-resistant organisms.

Lanomycin and glucolanomycin, antifungal agents produced by Pycnidiophora dispersa. II. Structure elucidation.

Two novel antifungal agents, lanomycin and glucolanomycin, as well as a biologically inactive degradation product, lanomycinol, were isolated from liquid fermentations of Pycnidiophora dispersa. All three compounds share an E,E,E-triene appended to a pyran ring. Lanomycin contains a glycine ester and glucolanomycin possesses a glucose unit attached to the glycine nitrogen. The structures, including absolute stereochemistry, were determined by spectroscopic analysis and partial chemical degradation. Both of the glycine containing compounds show activity against several pathogenic fungi in vitro.

Degradation of brain natriuretic peptide by neutral endopeptidase: species specific sites of proteolysis determined by mass spectrometry.

Brain natriuretic peptide (BNP) from 3 different species was cleaved by neutral endopeptidase (NEP) and the products separated by HPLC. The newly formed products were identified by fast atom bombardment or nebulizer-assisted electrospray mass spectrometry to elucidate the sites of proteolysis. Porcine BNP was cleaved at the Arg8-Leu9 and Ser14-Leu15 bonds. Rat BNP was cleaved at the Arg23-Leu24 and Arg30-Leu31 bonds. Human BNP was cleaved at the Pro2-Lys3, Met4-Val5 and Arg17-Leu18 bonds. The Cys-Phe bond which is present in all species of BNP is not cleaved by NEP.

Janthinocins A, B and C, novel peptide lactone antibiotics produced by Janthinobacterium lividum. II. Structure elucidation.

The structures of janthinocins A, B and C, three novel macrocyclic peptide lactone antibiotics isolated from fermentations of Janthinobacterium lividum, were determined. The janthinocins are of particular interest because they contain three amino acid residues that have not previously been reported in natural products: Each contains erythro-beta-hydroxy-D-leucine while janthinocins A and B also contain beta-hydroxytryptophan and beta-ketotryptophan, respectively.