Synthesis and antifeedant properties of N-benzoylphenylisoserinates of Lactarius sesquiterpenoid alcohols.

The esterification of various sesquiterpenoid alcohols of Lactarius origin with N-benzoyl-[2R,3S]-phenylisoserine (side chain of Taxol) produced compounds whose antifeedant properties against storage pests Tribolium confusum, Trogoderma granarium and Sitophylus granarius were measured. The introduction of the taxol side chain in these molecules, in comparison to original compounds, moderately enhanced their antifeedant activities, as well as changed their selectivity of activity towards the test insects.

An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin.

Immunoassay technology is routinely used to measure concentrations of proteins and polypeptides in biological matrices. Increasingly, research efforts have sought to create analogs of human proteins with the aim of improving efficacy or pharmaceutical properties relative to the native protein. Pharmacokinetic assessment of these polypeptide analogs, however, can be greatly confounded by the presence of endogenous native protein. This report describes an immunization and immunoabsorption strategy that was used to create monospecific polyclonal antibodies against analogs of human leptin (LY355101 and LY396623, one and two amino acid changes relative to native human leptin, respectively). Rabbits were immunized with either LY355101 or LY396623. Antisera were screened to determine if any showed increased specificity for the analog relative to native human leptin. Antisera showing increased specificity for the leptin analog were then treated by immunoabsorption against native human leptin, thus depleting human leptin cross-reactivity. The antibodies developed in this process were used in radioimmunoassays. which were validated for use in clinical studies. Both assays proved to be highly specific for LY355101 or LY396623 in the presence of native human leptin. Use of this procedure permitted the measurement of LY355101 and LY396623 pharmacokinetics that were not confounded by the high levels of endogenous human leptin found in obese subjects. This technique has the potential for broad application in the development of assays capable of specifically measuring protein analogs without cross-reactivity to an endogenous substance.

Metabolism of des(64,65)-human proinsulin in the rat. Evidence for the proteolytic processing to insulin.

The metabolism of des(64,65)-human proinsulin was examined in rats after subcutaneous administration. Profiles of circulating insulin-like immunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Both techniques indicated the presence of circulating immunoreactivity having retention characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention characteristics of des(64,65)-human proinsulin. The peaks of immunoreactive material were isolated and their structure determined using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two circulating metabolites were identified. These metabolites co-eluted by reversed-phase high-performance liquid chromatography with human insulin and diarginyl(B31,32)-human insulin and had mass spectra identical to the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at the carboxy side of ArgB32, with the formation of human insulin by the subsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dipeptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin may be mediated in part by circulating insulin-like metabolites.

Mechanisms involved in degradation of human insulin by cytosolic fractions of human, monkey, and rat liver.

The degradation of native and 125I-labeled human insulin (HI) was examined in the cytosolic fraction of human, monkey, and rat liver. The purpose of these studies was to provide a species comparison of the interaction of insulin-degrading enzyme (IDE) and protein disulfide isomerase (PDI) in the degradation of HI. Western-blot analysis with monoclonal antibodies indicated the presence of both IDE and PDI in the cytosolic fraction of human and monkey liver. In contrast, rat liver cytosol contained, detectable levels of IDE only. A species comparison of metabolic profiles was performed by fractionating peptide products with reversed-phase high-performance liquid chromatography. After a 60-min incubation, human liver cytosol degraded unlabeled HI into three major products. Two of these peptides coeluted with the products of the incubation of HI with purified rat liver PDI. The three peptides were isolated and determined by NH2-terminal sequence analysis to be intact A chain, B chain, and des(Phe1)-B chain. Human liver cytosol also formed 125I-A chain and 125I-B chain as major products when specifically labeled 125I-HI isomers were used as substrate. Significant proteolytic degradation was observed only when reactions with human liver cytosol were supplemented with Mn2+. In contrast, monkey and rat liver cytosol proteolytically degraded 125I-HI isomers to small peptide fragments. The rat and monkey metabolic profiles were similar to each other and to that observed with Mn(2+)-supplemented human liver cytosol. Proteolysis in monkey and rat was sensitive to inhibition by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteolytic cleavage of human growth hormone (hGH) by rat tissues in vitro: influence on the kinetics of exogenously administered hGH.

The presence of several endogenous molecular forms of human GH (hGH), including proteolytically cleaved two-chain forms, has been proposed to be related to the diverse biological activity of hGH. The present study characterized hGH degradation in the rat to determine how peripheral metabolism may influence the kinetics and pharmacology of exogenously administered hGH. In vitro studies indicated that hGH was proteolytically degraded by thyroid gland and skeletal muscle, but not liver and kidney homogenates. The proteolytic activity, localized to the 9000 x g pellet fraction, was characterized as a chymotrypsin-like serine protease using class-specific inhibitors. N-Terminal sequencing of hGH peptides formed by the thyroid gland and skeletal muscle indicated that cleavage sites were almost exclusively at Tyr/Phe-Xaa bonds, with similar points of cleavage observed in the two tissues. Immunoreactive two-chain forms of hGH were also formed. The two-chain molecules had similar cleavage sites, but differed in apparent mol wt when analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To understand the potential significance of two-chain product formation, we compared the kinetics and degradation of hGH with those of a synthetic two-chain derivative of hGH (Des-1-8,135-145; 2-CAP). The in vitro tissue distribution of 2-CAP proteolysis was the same as that for hGH. The fragmentation pattern of 2-CAP was less complex when analyzed by reverse phase HPLC. The major peptide fragments formed from 2-CAP were chromatographically similar to those formed from hGH. The plasma kinetics of 2-CAP were compared to those of hGH with a RIA using polyclonal antiserum to hGH. After im and sc administration of 2-CAP (125 micrograms/kg), the area under the plasma concentration curve was 3.2- and 4.5-fold greater, respectively, than after administration of hGH (125 micrograms/kg). Both compounds had a greater area under the curve by the im than the sc route. 2-CAP had 2- to 3-fold greater bioavailability than hGH by the im and sc routes. Plasma from rats treated 30 min earlier with hGH im was immunoextracted and analyzed by Western blotting. A circulating immunoreactive fragment was detected which had similar electrophoretic mobility as a two-chain hGH product formed during the in vitro incubations of hGH with skeletal muscle and thyroid gland homogenates. The results indicate that hGH is proteolytically processed in peripheral tissue homogenates, with the formation of two-chain products. The greater bioavailability of 2-CAP suggests that metabolism of hGH to two-chain forms may influence the in vivo kinetics of hGH.

The lead structure in cardiac glycosides is 5 beta, 14 beta-androstane-3 beta 14-diol.

The purpose of the present study was to determine the lead structure in cardiac glycosides at the receptor level, i.e. the minimal structural requirement for specific and powerful receptor recognition. Accordingly 73 digitalis-like acting steroids were characterized as to the concentration effecting half-maximum inhibition of Na,K-ATPase from human cardiac muscle under standardized turnover conditions. Since the Ki value equaled the apparent KD value, K'D was expressed in terms of the apparent standard Gibbs energy change delta G degrees' of steroid interaction with Na,K-ATPase. This allowed the use of the extrathermodynamic approach as a rational way of correlating in a quantitative manner, the potency and structure of the various steroidal compounds. The results of the present analysis taken in conjunction with relevant findings reported in the literature, favour the following conclusions. Cassaine, canrenone, prednisolone- and progesterone-3,20-bisguanylhydrazone, and chlormadinol acetate are compounds that are not congeneric with digitalis. The butenolide ring of cardenolides or the analogous side-chains at C17 beta of 5 beta, 14 beta-androstane-3 beta, 14-diol are not pharmacophoric substructures, but merely amplifiers of the interaction energy of the steroid lead. All modifications of the structure, geometry and spatial relationship between the steroid nucleus and butenolide side chain of digitoxigenin all at once weaken the close fit interaction with the steroid and butenolide binding subsites of the enzyme in such way that the cardenolide derivatives interact with the receptor binding site area in whatever orientation that will minimize the Gibbs energy of the steroid-receptor-solvent system. The "butenolide carbonyl oxygen distance model" (Ahmed et al. 1983) for the interpretation of the differences in potency of the cardenolide derivatives describes the change in interaction energy through structural modification as a function of the entire molecule. 5 beta, 14 beta-androstane-3 beta, 14-diol, the steroid nucleus of cardiac glycosides of the digitalis type, is the minimum structure for specific receptor recognition and the key structure for inducing protein conformational change and thus Na,K-ATPase inhibition. It is also the structural requirement for maximum contributions of the butenolide substituent at C17 beta and the sugar substituent at C3 beta-OH to the overall interaction energy, i.e. this steroid nucleus is the lead structure.(ABSTRACT TRUNCATED AT 400 WORDS)