Anti-rods and rings autoantibodies can occur in the hepatitis c-naïve population.

INTRODUCTION: The anti-Rods and Rings autoantibody recently described in clinical populations is thought to occur in the setting of hepatitis C treatment, specifically in the context of cytidine triphosphate (CTP) and guanosine triphosphate (GTP) synthetic pathway inhibitors, and is important in its potential impact on response to therapy. This study asks the question: what is the epidemiology of anti-RR autoantibody in the general, non-clinical population? MATERIALS AND METHODS: This is a cross-sectional study using the National Health and Nutrition Examination Survey (NHANES). Immunofluorescence assay for anti-Rods and Rings autoantibody were performed by NHANES labs and the results made publically available. Sample weights were used to calculate the prevalence and distribution of the autoantibody across demographics. A medication profile of the autoantibody positive population was also constructed RESULTS: The study sample consisted of 4738 persons over the age of 12 years. Anti-Rods and Rings autoantibodies were found in 39 persons representing 1.3 million persons in the United States population. 38 of 39 persons with anti-Rods and Rings autoantibody had no prior history of hepatitis C virus infection. A majority of these persons were found to have poly-pharmacy. DISCUSSION: This is the first study to show that anti-RR can occur in the general population without evidence of hepatits C virus infection, and that the majority of persons with anti-RR in the population have no evidence of prior hepatitis C infection. This indicates that there may be another undetermined etiology for anti-rods and rings autoantibodies besides the currently accepted exposure etiology of hepatitis C virus infection and treatment found in clinical studies.

Drug pharmacophores covalently linked to the red cell surface are active without prior release. Drug targeting of renin with a synthetic ligand conjugated to red blood cells.

Red blood cells have been labeled with an anti-renin pharmacophore using the activated labeling agent Boc-Phe-His-ACHPA-Ile-6-NH(CH2)5CO-NHS (4) and the corresponding sulfo-NHS-ester (5). Renin inhibition by labeled cells varies according to the concentrations of 4 or 5 used in the labeling protocols, and with the densities of the red cells employed. Flow cytometry measurements using specific polyclonal antibodies toward the anti-renin pharmacophore confirm that red cells are labeled on their outer surfaces with anti-renin pharmacophores. Inhibitory activity of labeled red cells is clearly associated with the cells themselves, and does not require prior release of an inhibitory entity: renin inhibition increases as a function of the concentration of NHS-ester used to label cells suspended in buffer, and with cell density; on the other hand, the separated supernatant portions of the medium make only minor contributions to the observed inhibitory activities. Renin inhibition also increases with increasing concentrations of ghosts derived from labeled red cells, firmly establishing that activity is intimately associated with cell membranes. Thus, the composite evidence is strongly supportive of inhibitory activity specific to the extracellular surface of red cells, which has been modified by the introduction of anti-renin pharmacophores. This study of inhibitory activity by drug/red blood cell-conjugates represents one of the few examples of a red cell-bound ligand of synthetic origin capable, without prior release, of specifically blocking the activity of its target enzyme. As well, it demonstrates the feasibility of exploiting the activity of covalently bound pharmacophores, free from interference of their carriers, for drug targeting.

Red cell-mediated therapy: opportunities and challenges.

Red blood cells have unique properties that offer attractive therapeutic possibilities. It is proposed that by covalently linking drug pharmacophores to red cell surface proteins, significant enhancement of pharmacokinetics can be achieved. Specific anchoring to erythrocytes can be accomplished in principle, using unique, affinity labeling, combinatorial libraries targeting the surface protein, glycophorin A. The anticipated advantages of red cell-anchored drugs over free drugs, include extended half-lives, controlled volumes of distribution, and multivalent interactions.

Site-directed mutagenesis of Cys324 and Cys331 in human cytosolic phospholipase A2: locus of action of thiol modification reagents leading to inactiviation of cPLA2.

Human cytosolic phospholipase A2 contains two cysteines, cyS324 and cyS331, chemical modification of which using thiol modifying reagents abolishes the activity of the enzyme [Li et al. (1994) Biochemistry 33, 8594-8603]. To verify the functional importance of the two cysteine residues, site-directed mutagenesis has been used to create six mutations at positions 324 and 331. The mutant enzymes include C324A, C331A, C324Q, C331Q, C324R, and C331S. Complete loss of activity is observed for C331Q, whereas the other mutants have retained varying degrees of activity. These results show that neither CyS324 nor CyS311 is catalytically essential for the enzyme activity. Further chemical modification studies of the mutant enzymes by thiol-specific reagents suggest that modification of Cys331 is responsible for the complete loss of the enzyme activity. The possible roles of Cys324 and Cys331 are discussed.

Acute accidental lindane ingestion in toddlers.

Lindane toxicity has been reported to occur mostly by way of dermal exposure. Cases of ingestion in which blood levels have been determined are rare. We present three such cases, along with a comparison of cases in the literature with respect to blood level half-lives and correlation with signs of toxicity. Emergency physicians can prevent acute ingestion by educating patients on the proper use of lindane and by selecting less toxic scabicidal agents.

Inactivation of a cytosolic phospholipase A2 by thiol-modifying reagents: cysteine residues as potential targets of phospholipase A2.

The cytosolic phospholipase A2 (cPLA2) from the human monocytic cell line U937 contains nine cysteine residues and is subject to oxidation. Iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid) were used to explore the susceptibility of cysteine residues to thiol modification agents as outlined in Schemes 2 and 3. In the absence of thiol reducing agents such as DTT, cPLA2 takes up only 2.8 equiv of [1-14C]iodoacetamide at pH 8.03/37 degrees C. With DTT present, cPLA2 is in its fully reduced form, and 4-5 equiv of acetamide are taken up without altering enzyme activity to give IA-cPLA2. A single equivalent of DTNB suffices to inactivate IA-cPLA2, giving a TNB-labeled enzyme, with the loss of activity correlating with release of an equivalent of 5-thio-2-nitrobenzoate. The TNB-labeled enzyme is quite stable up to 33 degrees C; enzyme activity is recoverable with DTT, even after this disulfide-enzyme adduct is incubated with iodoacetamide at pH 9.5, conditions that inactivate the free enzyme. At pH 9.5/37 degrees C, a single equivalent of 14C-labeled iodoacetamide is incorporated by IA-cPLA2 concomitant with complete loss of enzyme activity. Amino acid analysis of the 14C-labeled enzyme indicates that only cysteine residues are labeled. Lys-C digestion of labeled enzyme with 2 M guanidine at pH 8.0 yields a 40-mer peptide. Amino acid sequencing establishes that the label resides primarily in Cys324, although Cys331 is also labeled. These results identify a region of the enzyme that is susceptible to labeling by group modification reagents and may represent a suitable target for small molecule inhibitors.

In vivo inhibition of cathepsin B by peptidyl (acyloxy)methyl ketones.

Peptidyl (acyloxy)methyl ketones, previously established as potent irreversible inhibitors of the cysteine proteinase cathepsin B in vitro, were investigated and optimized for their inhibitory activity in vivo. Incorporation of polar or charged functional groups in the inhibitor structure afforded effective cathepsin B inhibition, following dosing to rats. The most effective inhibitor, Z-Phe-Lys-CH2OCO-(2,4,6-Me3)Ph (8), was found to give ED50 values of 18 mg/kg po (orally) and 5.0 mg/kg ip (intraperitoneally) at 4-5 h postdose, and 2.4 mg/kg sc (subcutaneously) at 24 h postdose, for liver cathepsin B inhibition (measured ex vivo). The subcutaneous route of administration of (acyloxy)methyl ketone 8 also provided potent cathepsin B inhibition in certain peripheral tissues (e.g., ED50 1.0 mg/kg for skeletal muscle, 0.1 mg/kg for heart). These investigations demonstrate that peptidyl (acyloxy)methyl ketones such as 8 have promise as tools for the characterization of in vivo biochemical processes and as therapeutic agents.

Potent inactivation of cathepsins S and L by peptidyl (acyloxy)methyl ketones.

Peptidyl (acyloxy)methyl ketones (Z-Aa-Aa-CH2-O-CO-R), a new class of irreversible inhibitors whose chemical reactivity can be modulated by varying the substitution pattern of the carboxylate leaving group, are shown to be extremely potent inactivators of the lysosomal cysteine proteinases cathepsin L and cathepsin S. The highest k2/Ki values measured were found to exceed 10(6) M-1s-1 for both cathepsin L and cathepsin S. The rate of inactivation can be controlled by varying the dipeptidyl moiety or the carboxylate leaving group, with the second-order rate constants for both enzymes found to be strongly dependent on the pKa values of the leaving group. The specificities of the cathepsins S and L reveal a different selectivity towards the nature of substitution of the aryl P' leaving group of the inhibitor. This new inhibitor class opens the possibility of the design of selective and specific inhibitors for lysosomal cysteine proteinases.

Solid-state 13C NMR study of a transglutaminase-inhibitor adduct.

We have used solid-state 13C NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-halo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assumption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazole ring. The tetrahedral intermediate formed could then break down with the loss of the halide group and the subsequent formation of a stable imino thioether adduct. We have compared the 13C CPMAS spectra of the chloro-, bromo-, and (ethylthio)dihydroisozazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensitive to the nature of the heteroatom. Subtraction of the natural-abundance 13C solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad peak at 156 ppm. The chemical shift of this peak is very close to that observed for a model 3-ethylthio compound and suggests the formation of a stable imino thioether enzyme adduct. Similar results were obtained for lyophilized enzyme adducts and for frozen solutions of the enzyme adduct in the absence and presence of Ca2+. We have also compared these results with those obtained by solution NMR on an aqueous solution of the enzyme-inhibitor complex. The 13C-labeled C-3 resonance was not observed in this case.

Comparative behaviour of calpain and cathepsin B toward peptidyl acyloxymethyl ketones, sulphonium methyl ketones and other potential inhibitors of cysteine proteinases.

Peptidyl acyloxymethyl ketones, previously established as potent inactivators of the lysosomal cysteine proteinase cathepsin B, were evaluated against smooth-muscle calpain, a member of the family of Ca(2+)-dependent cysteine proteinases. Only modest rates of time-dependent inhibition could be achieved, even with peptidyl affinity groups optimized for calpain and linked to a carboxylate leaving group of very low pKa [2,6-(CF3)2PhCOO-, pKa 0.58]. Selective inactivation of cathespin B versus calpain was consistently observed with this type of inhibitor. Examination of other potential inhibitors revealed a rank order of potency against calpain to be: peptidyl sulphonium methyl ketones > fluoromethyl ketones, diazomethyl ketones >> acyloxymethyl ketones, an order which differs sharply from that found for cathespin B.

Irreversible inhibition of transglutaminases by sulfonium methylketones: optimization of specificity and potency with omega-aminoacyl spacers.

Sulfonium methylketones, of structure Cbz-Phe-NH(CH2)nCOCH2S+ (CH3)2, n > 2, are specific and potent inactivators of transglutaminases. The length of the -(CH2)n-spacer moiety, n = 1-5, is a critical determinant for both the specificity and potency of the inactivator. The dipeptidyl analog Cbz-Phe-Gly-(CH2)nS+ (CH3)2, n = 1, is a more powerful inactivator of the thiol proteinase cathepsin B, k/K > 3 x 10(5) M-1 min-1, than of transglutaminases, ki(app)/Ki(app) < 1.5 x 10(4) M-1 min-1. In contrast, the gamma-aminobutyryl analog, n = 3, is a very potent transglutaminase inactivator with ki(app)/Ki(app) = 3.1 x 10(6) M-1 min-1, but does not inactivate cathepsin B. In cell studies, the gamma-aminobutyryl and epsilon-aminohexyl analogs inhibited the transglutaminase-mediated process of ionophore-induced cross-linked envelope formation by human malignant keratinocytes and the order of potency was related to that found for enzyme inhibition. The sulfonium methylketones, in equilibrium with the resonance stabilized ylides, are chemically inert towards glutathione under ambient conditions demonstrating the potential utility of this novel class of transglutaminase inhibitors for the study of enzyme inhibition in cellular environments.

Nature of papain products resulting from inactivation by a peptidyl O-acyl hydroxamate.

Mass spectrometry has been used to provide insights into the mechanism of inhibition of cysteine proteases by a hydroxylamine derivative, CBZ-Phe-Gly-NH-O-CO-(2,4,6-Me3)Ph. An oxidized form of papain resulting from the incubation of the enzyme with the peptidyl hydroxamate in the absence of a reducing agent has been identified as a sulfinic acid. The presence of a covalent enzyme-inhibitor complex of molecular mass consistent with a sulfenamide adduct of papain could also be detected by this method. Implications on the mechanism of inactivation of cysteine proteases by peptidyl hydroxamates are discussed.

Peptidyl (acyloxy)methyl ketones and the quiescent affinity label concept: the departing group as a variable structural element in the design of inactivators of cysteine proteinases.

(Acyloxy)methyl ketones, of general structure Z-[AA2]-[AA1]-CH2OCOAr, are potent inactivators of the cysteine proteinase cathepsin B. These reagents have been designed as affinity labels in which the dipeptidyl moiety serves as an affinity group (complementary to the S1 and S2 sites of the enzyme), while the (acyloxy)methyl ketone unit (-COCH2OCOR), containing a weak leaving group in the form of a carboxylate nucleofuge, functions as the potentially reactive entity that labels the enzyme. The inhibition is time dependent, active site directed, and irreversible. The apparent second-order rate constant kinact/Kinact, which characterizes the inhibition of cathepsin B by this series, spans several orders of magnitude and in certain cases exceeds 10(6) M-1 s-1. The activity of this series of inhibitors was found to be exquisitely sensitive to the nature of the carboxylate leaving group as well as the affinity group. A strong dependence of second-order inactivation rate on leaving group pKa was uncovered for Z-Phe-Ala (acyloxy)methyl ketones [log(k/K) = 1.1 (+/- 0.1) X pKa + 7.2 (+/- 0.4); r2 = 0.82, n = 26]. Heretofore in constructing affinity labels the choice of leaving group was quite restricted. The aryl carboxylate group thus offers considerable variation as a design element in that both its binding affinity and reactivity can be controlled by substituent effects. Specific peptidyl (acyloxy)methyl ketones thus represent prime examples of highly potent, chemically stable enzyme inhibitors with variable structural elements in both the affinity and departing groups.

Design and synthesis of 4H-3,1-benzoxazin-4-ones as potent alternate substrate inhibitors of human leukocyte elastase.

4H-3,1-Benzoxazin-4-ones are alternate substrate inhibitors of the serine proteinase human leukocyte elastase (HL elastase) and form acyl enzyme intermediates during enzyme catalysis. We have synthesized a large variety of benzoxazinones using specific methods that have been adapted to achieve the pattern of ring substitution dictated by theoretical considerations. The results of the inhibition of HL elastase by 175 benzoxazinones are reported herein with reference to hydrophobicity constants D, alkaline hydrolysis rates kOH-, inhibition constants Ki, and their component acylation and deacylation rate constants, kon and koff, respectively. The ranges for the compounds are considerable; alkaline hydrolysis rates and kon span 6, koff covers 5, and ki spans 8 orders of magnitude. Multiple regression on this large data set has been used to isolate the contributions of electronic and steric effects, as well as other factors specific to compound stability and elastase inhibition. Essentially, a simple electronic parameter is sufficient to account for almost all the variance in the alkaline hydrolysis data, indicating that electronic factors are the major determinants of this type of benzoxazinone reactivity. Factors that significantly enhance the potency of benzoxazinones I are R5 alkyl groups and electron withdrawal by R2. Bulk in R7 and R8 and compound hydrophobicity are not significant, but substitution in R6 is highly unfavorable as are substituents linked via carbon to C2. The physiochemical factors that underlie these trends in Ki are further analyzed in terms of equations that describe kon and koff. A conclusion that emerges is that chemically stable, potent benzoxazinone inhibitors of HL elastase with inhibition constants in the nanomolar range can be designed with (1) R5 alkyl groups to inhibit enzyme-catalyzed deacylation, (2) small alkyl substituents linked via heteroatoms to C2 to enhance acylation and limit deacylation rates, and (3) strongly electron-donating groups at C7 to stabilize the oxazinone ring to nucleophilic attack. Thus, 2-(isopropylamino)-5-n-propyl-7-(dimethylamino)benzoxazinone 95 has kOH = 0.01 M-1 s-1, which extrapolates to a half-life at pH 7.4 of over 8.5 years, and 2-ethoxy-5-ethylbenzoxazinone 38 has Ki = 42 pM.

Clinical features and management of intoxication due to hallucinogenic drugs.

Hallucinogenic drugs are unique in that they produce the desired hallucinogenic effects at what are considered non-toxic doses. The hallucinogenic drugs can be categorised into 4 basic groups: indole alkaloid derivatives, piperidine derivatives, phenylethylamines and the cannabinols. The drugs reviewed include lysergic acid diethylamide (LSD), phencyclidine (PCP), cocaine, amphetamines, opiates, marijuana, psilocybin, mescaline, and 'designer drugs.' Particularly noteworthy is that each hallucinogen produces characteristic behavioural effects which are related to its serotonergic, dopaminergic or adrenergic activity. Cocaine produces simple hallucinations, PCP can produce complex hallucinations analogous to a paranoid psychosis, while LSD produces a combination of hallucinations, pseudohallucinations and illusions. Dose relationships with changes in the quality of the hallucinatory experience have been described with amphetamines and, to some extent, LSD. Flashbacks have been described with LSD and alcohol. Management of the intoxicated patient is dependent on the specific behavioural manifestation elicited by the drug. The principles involve differentiating the patient's symptoms from organic (medical or toxicological) and psychiatric aetiologies and identifying the symptom complex associated with the particular drug. Panic reactions may require treatment with a benzodiazepine or haloperidol. Patients with LSD psychosis may require an antipsychotic. Patients exhibiting prolonged drug-induced psychosis may require a variety of treatments including ECT, lithium and l-5-hydroxytryptophan.

A new class of mechanism-based inhibitors of transglutaminase enzymes inhibits the formation of cross-linked envelopes by human malignant keratinocytes.

A series of tyrosinamidomethyl dihydrohaloisoxazole compounds, designed as mechanism-based inhibitors of bovine epidermal transglutaminase enzyme, was examined for effects on the formation of cross-linked envelopes by human SCC-9 malignant keratinocytes. Compounds inhibited ionophore-induced envelope formation in a manner that reflected their capacity to inhibit transglutaminase activity. Preincubation and inhibitor wash-out studies indicated that the inhibitor must be present at the time of cell activation by ionophore in order to inhibit envelope formation. The stereospecific nature of the inhibitory activity of these compounds on both transglutaminase activity and cross-linked envelope formation makes this class of compounds an important tool in the study of transglutaminase-mediated events at the cellular level.

Peptidyl O-acyl hydroxamates: potent new inactivators of cathepsin B.

Peptidyl O-acyl hydroxamates having appropriate active-site recognition features are very potent time-dependent inhibitors of the cysteine proteinase cathepsin B. The inhibition is irreversible, and the inactivation rate is strongly dependent on peptide structure and correct positioning of the P1 amino acid carbonyl group. Lipophilic O-acyl groups provide the most rapid inactivators, as exemplified by the inhibitor O-mesitoyl N-benzyloxycarbonyl-L-phenylalanyl-L-alanine hydroxamate (kmax/Ki = 640,000 M-1s-1).

Inhibition of cathepsin B by peptidyl aldehydes and ketones: slow-binding behavior of a trifluoromethyl ketone.

Inhibition of the cysteine proteinase cathepsin B by a series of N-benzyloxycarbonyl-L-phenylalanyl-L-alanine ketones and the analogous aldehyde has been investigated. Surprisingly, whereas the aldehyde was found to be almost as potent a competitive reversible inhibitor as the natural peptidyl aldehyde, leupeptin, the corresponding trifluoromethyl ketone showed comparatively weak (and slow-binding) reversible inhibition. Evaluation of competitive hydration and hemithioketal formation in a model system led to a structure-activity correlation spanning several orders of magnitude in both cathepsin B inhibition constants (Ki) and model system equilibrium data (KRSH,apparent).