The mechanism of galactosamine toxicity revisited; a metabonomic study.

1H NMR spectroscopy was used to investigate the metabolic effects of the hepatotoxin galactosamine (galN) and the mechanism by which glycine protects against such toxicity. Rats were acclimatized to a 0 or 5% glycine diet for 6 days and subsequently administered vehicle, galN (500 mg/kg), glycine (5% via the diet), or both galN and glycine. Urine was collected over 12 days prior to administration of galN and for 24 hours thereafter. Serum and liver tissue were sampled on termination, 24 hours post-dosing. The metabolic profiles of biofluids and tissues were determined using high-field 1H NMR spectroscopy. Orthogonal-projection to latent structures discriminant analysis (O-PLS-DA) was applied to model the spectral data and enabled the hepatic, urinary, and serum metabolites that discriminated between control and treated animals to be determined. Histopathological data and clinical chemistry measurements confirmed the protective effect of glycine. The level of N-acetylglucosamine (glcNAc) in the post-dose urine was found to correlate strongly with the degree of galN-induced liver damage, and the urinary level of glcNAc was not significantly elevated in rats treated with both galN and glycine. Treatment with glycine alone was found to significantly increase hepatic levels of uridine, UDP-glucose, and UDP-galactose, and in view of the known effects of galactosamine, this suggests that the protective role of glycine against galN toxicity might be mediated by changes in the uridine nucleotide pool rather than by preventing Kupffer cell activation. Thus, we present a novel hypothesis: that administration of glycine increases the hepatic uridine nucleotide pool which counteracts the galN-induced depletion of these pools and facilitates complete metabolism of galN. These novel data highlight the applicability of NMR-based metabonomics in elucidating multicompartmental metabolic consequences of toxicity and toxic salvage.

NMR of biofluids and pattern recognition: assessing the impact of NMR parameters on the principal component analysis of urine from rat and mouse.

The ability to interpret metabolic responses to toxic insult as expressed in altered urine composition and measured by NMR spectroscopy is dependent upon a database of proton NMR spectra of urine collected from both control and treated animals. Pattern recognition techniques, such as principal component analysis (PCA), can be used to establish whether the spectral data cluster according to a dose response. However, PCA will be sensitive to other variables that might exist in the data, such as those arising from the NMR instrument itself. Thus, studies were conducted to determine the impact that NMR-related variables might impart on the data, with a view towards understanding and minimizing variables that could interfere with the interpretation of a biological effect. This study has focused on solvent suppression methods, as well as instrument-to-instrument variability, including field strength. The magnitude of the NMR-induced variability was assessed in the presence of an established response to the nephrotoxin bromoethanamine. Changes caused by the model toxin were larger and easily distinguished from those caused by using different solvent suppression methods and field strengths.

Metabonomic assessment of vasculitis in rats.

The vasculitides are a heterogeneous group of lesions characterized by inflammation and necrosis of the vascular wall and have proven to be a disconcerting dilemma in the development of several classes of therapeutics. Metabonomics is an emerging technology having great potential for rapid noninvasive assessment of toxicity in vivo and providing identification of peripheral surrogate markers of toxicity. Metabonomic evaluation of CI-1018, a selective type 4 phosphodiesterase inhibitor associated with vasculitis in rats, was undertaken. Two experiments were performed in which CI-1018 was administered for up to 4 d to groups of male Wistar rats at doses up to 3000 mg/kg. Urine was collected from all animals pretest and daily for metabonomic analysis. Eleven of 38 CI-1018-treated animals were found to have vascular injury of varying severity at doses = or > 750 mg/kg. Principal component analysis produced a clear pattern separation among 8 of 11 animals with lesions and 36 of 37 animals without lesions in samples collected on d 3 or 4. These data demonstrate that the metabonomics approach has significant potential for developing a noninvasive method for identifying vasculitis in rats. It remains to be seen if urinary analyte patterns identified in this study are reproducible and whether a biomarker pattern for vasculitis can be established.

Metabonomics: evaluation of nuclear magnetic resonance (NMR) and pattern recognition technology for rapid in vivo screening of liver and kidney toxicants.

The purpose of this study was to evaluate the feasibility of metabonomics technology for developing a rapid-throughput toxicity screen using 2 known hepatotoxicants: carbon tetrachloride (CCl(4)) and alpha-naphthylisothiocyanate (ANIT) and 2 known nephrotoxicants: 2-bromoethylamine (BEA) and 4-aminophenol (PAP). In addition, the diuretic furosemide (FURO) was also studied. Single doses of CCl(4) (0.1 and 0.5 ml/kg), ANIT (10 and 100 mg/kg), BEA (15 and 150 mg/kg), PAP (15 and 150 mg/kg) and FURO (1 and 5 mg) were administered as single IP or oral doses to groups of 4 male Wistar rats/dose. Twenty-four-h urine samples were collected pretest, daily through Day 4, and on Day 10 (high dose CCl(4) and BEA only). Blood samples were taken on Days 1, 2, and 4 or 1, 4, and 10 for clinical chemistry assessment, and the appropriate target organ was examined microscopically. NMR spectra of urine were acquired and the data processed and subjected to principal component analyses (PCA). The results demonstrated that the metabonomic approach could readily distinguish the onset and reversal of toxicity with good agreement between clinical chemistry and PCA data. In at least 2 instances (ANIT and BEA), PCA analysis suggested effects at low doses, which were not as evident by clinical chemistry or microscopic analysis. Furosemide, which had no effect at the doses employed, did not produce any changes in PCA patterns. These data support the contention that the metabonomic approach represents a promising new technology for the development of a rapid throughput in vivo toxicity screen.

Design of a potent combined pseudopeptide endothelin-A/endothelin-B receptor antagonist, Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of its pharmacokinetic and spectral properties.

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

Metabolic disposition of the cognition activator tacrine in rats, dogs, and humans. Species comparisons.

The metabolic fate of tacrine [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate (THA)] was examined in rats, dogs, and humans. After administration of single oral doses of [14C]THA to rats, dogs, and humans, drug-derived material was well absorbed, with urinary excretion being the predominant route of radiolabel elimination. Metabolic profiling of plasma and urine from rats, dogs, and humans showed THA to be extensively metabolized with marked species differences in quantitative amounts of metabolites observed. Plasma profiles were similar to respective urinary profiles in all three species. Present in profiles of urine from rats were 1-hydroxy (OH)-THA (major), 2-OH-THA, and 4-OHA-THA, and unchanged THA. Also observed were trace amounts of more polar metabolites, presumably arising from sequential metabolism. Metabolic profiling of dog urine also showed 1-OH-THA to be the major metabolite, with trace amounts of the 2-OHA-THA and 4-OH-THA regioisomers and THA excreted. In dog urine, more of the radioactivity was associated with polar metabolites, including 1,3-dihydroxy-THA and a dihydrodiol metabolite. Human urinary metabolic profiles were more similar to that in dogs than in rats, with no single metabolite constituting > 10% of urinary radioactivity. Present in human urine were phenol glucuronide metabolites, of which 7-OH-THA was identified as an aglycone. Relevance of the marked quantitative differences in THA metabolism between rats, dogs, and humans to species differences in THA hepatotoxic potential remains to be established.

Biophysical characterization of zinc ejection from HIV nucleocapsid protein by anti-HIV 2,2'-dithiobis[benzamides] and benzisothiazolones.

HIV nucleocapsid protein (NCp7) has been suggested as a possible target for 2,2'-dithiobis-[benzamide] and benzisothiazolone agents that inhibit viral replication in infected cells (Rice et al. Science 1995, 270, 1194-1197). The solution behavior of these compounds and the mechanistic events leading to removal of Zn from HIV nucleocapsid protein in vitro has been studied by electrospray ionization mass spectrometry, 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy. We demonstrate that (1) Zn ejection is accompanied by formation of covalent complexes formed between the 2,2'-dithiobis[benzamide] monomers and Cys residues of Zn-depleted NCp7, (2) the rate of Zn ejection is faster for the C-terminal Zn finger and slower for the N-terminal finger, (3) Zn ejection results in a loss of structural integrity of the NCp7 protein, and (4) there is no appreciable interaction between a nonreactive isostere of the lead 2,2'-dithiobis[benzamide] and NCp7 in buffered aqueous solution. These findings are discussed in terms of the mechanism of action of Zn ejection by aromatic 2,2'-dithiobis[benzamides].

Identification of a 3-hydroxylated tacrine metabolite in rat and man: metabolic profiling implications and pharmacology.

Discrepancies in urinary metabolic profiles in rats administered tacrine (1) suggested the presence of an unidentified metabolite of 1. Chromatographic methods were developed that allowed isolation of a metabolite fraction containing both 1-hydroxytacrine (2) and an unknown metabolite from rat urine. Mass spectral analysis indicated this metabolite to be a monohydroxylated derivative, which upon two dimensional COSY NMR analysis could be assigned as 3-hydroxytacrine (4). This structural assignment was confirmed by independent synthesis of 4. Compound 4 was also identified as a human urinary metabolite of 1. Biologically, 4 was found to have in vitro human red blood cell acetylcholinesterase inhibitory activity similar to that of 2 and 4-hydroxytacrine (5) and approximately 8-fold less than that of 1. These results underscore the need to conduct rigorous structural identification studies, especially in cases where isomeric metabolites are possible, in assessing the accuracy of chromatographic profiling techniques.

Three-dimensional structure analysis of mu-agatoxins: further evidence for common motifs among neurotoxins with diverse ion channel specificities.

We report the solution structure of mu-agatoxin-I (mu-Aga-I) and model structures of the closely related mu-agatoxin-IV (mu-Aga-IV) which were isolated from venom of the American funnel web spider, Agelenopsis aperta. These toxins, which modify the kinetics of neuronal voltage-activated sodium channels in insects, are C-terminally amidated peptides composed to 36 amino acids, including four internal disulfide bonds. The structure of mu-Aga-I was determined by NMR and distance geometry/molecular dynamics calculations. Structural calculations were carried out using 256 interresidue NOE-derived distance restraints and 25 angle restraints obtained from vicinal coupling constants. The peptide contains eight cysteines involved in disulfide bonds, the pairings of which were uncertain and had to be determined from preliminary structure calculations. The toxin has an average rmsd of 0.89 A for the backbone atoms among 38 converged conformers. The structure consists of a well-defined triple-stranded beta-sheet involving residues 7-9, 20-24, and 30-34 and four tight turns. A homologous peptide, mu-Aga-IV, exhibited two distinct and equally populated conformations in solution, which complicated spectral analysis. Analysis of sequential NOE's confirmed that the conformers arose from cis and trans peptide bonds involving a proline at position 15. Models were developed for both conformers based on the mu-Aga-I structure. Our structural data show that the mu-agatoxins, although specific modifiers of sodium channels, share common secondary and tertiary structural motifs with phylogenetically diverse peptide toxins targeting a variety of channel types. The mu-agatoxins add voltage-sensitive sodium channel activity to a growing list of neurotoxic effects elicited by peptide toxins which share the same global fold yet differ in their animal origin and ion channel selectivity.

The solution structure of omega-Aga-IVB, a P-type calcium channel antagonist from venom of the funnel web spider, Agelenopsis aperta.

The 48 amino acid peptides omega-Aga-IVA and omega-Aga-IVB are the first agents known to specifically block P-type calcium channels in mammalian brain, thus complementing the existing suite of pharmacological tools used for characterizing calcium channels. These peptides provide a new set of probes for studies aimed at elucidating the structural basis underlying the subtype specificity of calcium channel antagonists. We used 288 NMR-derived constraints in a protocol combining distance geometry and molecular dynamics employing the program DGII, followed by energy minimization with Discover to derive the three-dimensional structure of omega-Aga-IVB. The toxin consists of a well-defined core region, comprising seven solvent-shielded residues and a well-defined triple-stranded beta-sheet. Four loop regions have average backbone rms deviations between 0.38 and 1.31 A, two of which are well-defined type-II beta-turns. Other structural features include disordered C- and N-termini and several conserved basic amino acids that are clustered on one face of the molecule. The reported structure suggests a possible surface for interaction with the channel. This surface contains amino acids that are identical to those of another known P-type calcium channel antagonist, omega-Aga-IVA, and is rich in basic residues that may have a role in binding to the anionic sites in the extracellular regions of the calcium channel.

Design and structure-activity relationships of C-terminal cyclic neurotensin fragment analogues.

Neurotensin (NT) is a linear tridecapeptide with a broad range of central and peripheral pharmacological effects. The C-terminal hexapeptide of NT (NT8-13) has been shown to possess similar properties to NT itself, and in fact, an analogue of NT8-13 (N alpha MeArg8-Lys-Pro-Trp-Tle-Leu13, Tle = tert-leucine) has been reported to possess central activity after peripheral administration. Cyclic derivatives of this hexapeptide were synthesized by a combination of solution and solid-phase peptide synthetic methodologies, and several analogues had low nanomolar binding affinity for the NT receptor. In particular, cyclo[Arg-Lys-Pro-Trp-Glu]-Leu (cyclized between the alpha amine of Arg and the gamma carboxylate of Glu) possessed 16 nM NT receptor affinity and was determined to be an agonist in vitro. 1H-NMR and 13C-edited 1H-NMR spectroscopy were performed on this and related cyclic analogues to help identify structural properties which may be important for receptor recognition. These cyclic peptides represent novel molecular probes to further investigate NT receptor pharmacology, as well as to advance our understanding of the structure-conformation relationships of NT and to help establish a working basis for additional pharmacophore mapping studies.

The 13C chemical shifts of amino acids in aqueous solution containing organic solvents: application to the secondary structure characterization of peptides in aqueous trifluoroethanol solution.

The 13C chemical shifts for all of the protonated carbons of the 20 common amino acid residues in the protected linear pentapeptide Gly-Gly-X-Gly-Gly have been obtained in water at low pH as well as in aqueous solution containing 10, 20 and 30% acetonitrile or trifluoroethanol. Dioxane was used as an internal reference and its carbon chemical shift value was found to be 66.6 ppm relative to external TMS in water. Comparison of the different referencing methods for 13C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate. In the present study, external TSP was chosen to define the 0 ppm of the 13C chemical shift scale. When the difference in referencing the dioxane carbon resonance is taken into account, the carbon chemical shift values of the amino acids in aqueous solution are similar to those previously reported (Richarz and Wüthrich (1978) Biopolymers, 17, 2133-2141; Howarth and Lilley (1979) Prog. NMR Spectrose., 12, 1-40). The pentapeptides studied were assumed to be in a random coil conformation and the measured 13C chemical shifts were used as reference values to correlate carbon chemical shifts with the secondary structure of two well-characterized peptides, bombesin and the 1-29 amino acid fragment of Nle27 human growth hormone-releasing factor. In both cases, the C alpha chemical shifts exhibited a characteristic positive deviation from the random coil values, which indicates the presence of alpha-helices.

Stereochemistry of 3-deoxyoctulosonate 8-phosphate synthase.

(Z)- and (E)-[3-2H]phosphoenolpyruvate were prepared chemically by the reductive deuteration of (Z)- and (E)-3-bromophosphoenolpyruvate, respectively, and were converted into 3-deoxyoctulosonate 8-phosphates deuterated at the C-3 position by incubation with unlabeled D-arabinose 5-phosphate in the presence of the enzyme, 3-deoxyoctulosonate 8-phosphate synthase (EC4.1.2.16) purified from Escherichia coli K-12 containing the plasmid pMW101. Analysis of the stereochemistry of the two 3-deoxyoctulosonate 8-phosphates deuterated at the C-3 position by 1H NMR showed that the (Z)-[3-2H]phosphoenolpyruvate had produced [3-2H]-3-deoxyoctulosonate 8-phosphate of predominantly the 3S configuration and that the E isomer had given predominantly (3R)-[3-2H]-3-deoxyoctulosonate 8-phosphate. The 3-deoxyoctulosonate 8-phosphate synthase reaction is therefore stereospecific with respect to the C-3 of phosphoenolpyruvate. The results indicate a si face attack from the C-3 of phosphoenolpyruvate, a result identical to that reported for 3-deoxyheptulosonate 7-phosphate synthase (EC 4.1.2.15), an enzyme catalyzing an identical aldol-type condensation, except that it takes place between phosphoenolpyruvate and D-erythrose 4-phosphate. The stereochemistry with respect to the face of the carbonyl of the attacked aldehyde, in both 3-deoxyoctulosonate 8-phosphate synthase and 3-deoxyheptulosonate 7-phosphate synthase, is re. On the basis of the results of the studies reported herein, the presence of a transient methyl group at the C-3 of phosphoenolpyruvate as part of the reaction mechanism seems unlikely.

Structure and properties of omega-agatoxin IVB, a new antagonist of P-type calcium channels.

A new peptide antagonist of voltage-activated calcium channels was purified from venom of the funnel web spider, Agelenopsis aperta. This 48-amino acid peptide, omega-agatoxin (omega-Aga)-IVB, was found to be a potent (Kd, approximately 3 nM) blocker of P-type calcium channels in rat cerebellar Purkinje neurons but had no activity against T-type, L-type, or N-type calcium channels in a variety of neurons. The calcium channel-blocking properties of omega-Aga-IVB were similar to those of another toxin, omega-Aga-IVA, which has 71% amino acid identity with omega-Aga-IVB. The 10-fold greater abundance of omega-Aga-IVB in venom allowed structural studies using NMR spectroscopy. The three-dimensional structure derived from NMR data resulted in a proposed disulfide bond configuration for the peptide. Although omega-Aga-IVB has fewer basic and more acidic residues than does omega-Aga-IVA, the two toxins show conservation of positively charged residues in a mid-peptide region that is predicted to form one face of the omega-Aga-IVB molecule. This region may be crucial for high affinity binding to the P-type calcium channel. In contrast, the amino termini of the two toxins have different charges and seem unlikely to be involved in binding to the channel.

Synthesis and pharmacological evaluation of hexahydrofluorenamines as noncompetitive antagonists at the N-methyl-D-aspartate receptor.

The noncompetitive (PCP) site of the N-methyl-D-aspartate (NMDA) receptor complex has been implicated in a number of pathologies, including the etiology of ischemic stroke. Recent testing has shown that cis-1,2,3,4,9,9a-hexahydro-N-methyl-4aH-fluoren-4a-amine (1), a rigid analog of PCP, is a potent antagonist at this site (IC50 = 30 nM for displacement of [3H]TCP). On the basis of this finding, a number of derivatives encompassing variations in stereochemistry, amine substitution and position, aromatic and aliphatic ring substitution, and heteroatom ring substitution have been prepared to explore the structure-activity relationships around this ring system. All compounds were evaluated for their PCP receptor affinity; potent compounds were also tested in vitro (cultured neurons) and in vivo (prevention of NMDA-induced lethality in mice). The present hexahydrofluorenamines demonstrated a wide range of potencies, with optimal affinity concentrated in analogs containing a heteroatom (sulfur) in the B ring (IC50 of 11 nM versus [3H]TCP for 16b), methyl substitution on the amine, and R stereochemistry at the 4a position. No significant improvement in affinity was seen with aromatic ring substitution. Aliphatic ring substitution, large amine substituents, and alterations in the position of amine substitution on the ring system resulted in a loss of potency. To explore the effect of simultaneous hydrogen bonding with a putative receptor atom from two directions, the 2-hydroxymethyl derivatives were prepared. This substitution resulted in a loss in receptor binding affinity. Molecular modeling, X-ray, and NMR studies have been used to determine an optimal conformation of the hexahydrofluoreneamines at the receptor site.

Design, synthesis and solution structure of a renin inhibitor. Structural constraints from NOE, and homonuclear and heteronuclear coupling constants combined with distance geometry calculations.

A macrocyclic renin inhibitor was designed using molecular modeling and a model of human renin. The synthesized molecular displayed poor binding affinity. To investigate the reasons for the observed inactivity, the structure of the compound has been studied by NMR spectroscopy and distance geometry. Structural constraints for distance geometry calculations were derived from nuclear Overhauser effects and homonuclear and heteronuclear three bond coupling constants. Homonuclear coupling constants were measured directly from the resolution-enhanced proton spectra and heteronuclear coupling constants were measured from the natural abundance 15N- and 13C-edited TOCSY experiments. One phi angle was determined uniquely by this method and two were reduced to two possible values each. By using a statistical analysis of 400 structures generated with distance geometry, two families of structures were found to be consistent with the NMR data. The solution structures so derived were different from the originally designed structure, including an internal hydrogen bond. This provides a possible explanation for the lack of effectiveness of this compound.

The solution structure of a cyclic endothelin antagonist, BQ-123, based on 1H-1H and 13C-1H three bond coupling constants.

A cyclic pentapeptide endothelin antagonist, cyclo(dTrp-dAsp-Pro-dVal-Leu), recently reported (K. Ishikawa et al., 13th Am. Pept. Symp., Cambridge MA, 1991) has been studied by NMR spectroscopy and molecular modeling. A stable structure has been determined without the use of nuclear Overhauser effects and is based primarily on homonuclear and heteronuclear three bond coupling constants. The 13C-edited TOCSY experiment is demonstrated at natural abundance and approximately 30 mM peptide concentrations. Three bond 13C-1H coupling constants obtained by this method are shown to reduce the ambiguity in phi angle determination which exists when only interproton coupling constants are used. Three out of four phi angles were determined uniquely by this method and the fourth was reduced to two possible values. The proline phi angle was determined to be -78 degrees based on the 3JH alpha, H beta and 3JH alpha, H beta coupling constants. Comparison of amide proton temperature dependence, chemical shifts and vicinal proton coupling constants in a 20% acetonitrile/80% water solvent mixture and in (CD3)2SO indicates that the structure is similar in both solvents.

The conformation of endothelin-1 in aqueous solution: NMR-derived constraints combined with distance geometry and molecular dynamics calculations.

The aqueous solution conformation of the bicyclic, 21 amino acid vasoconstrictor peptide, endothelin-1, has been determined using two dimensional NMR and a combination of distance geometry and molecular dynamics. The dominant structural feature is a helical region between Lys9 and Cys15 characterized by strong NHi-NHi+1 NOEs and several long range NOEs spanning 3 to 5 residues. Solvent inaccessibility and possible hydrogen bonding in the Cys3-Cys11 loop is suggested by the temperature independence of the chemical shifts of several amide protons. There is no evidence for association of the C-terminal hexapeptide with the bicyclic region.