Byron H. Arison

Inferences about the conformation of somatostatin at a biologic receptor based on NMR studies

Abstract Examination of two diastereomeric analogs of somatostatin differing in stereochemistry at the tryptophan residue has revealed a high field resonance in the d -Trp isomer which is assigned to the γ-methylene of Lys 9 . The extent of correlation of this shift with biologic activity for a series of analogs of somatostatin is discussed. From comparison of close analogs, it is suggested that the biologically active conformation of somatostatin at the receptor controlling insulin release is not the major conformation of this hormone in solution. It is suggested that the conformation of somatostatin at this receptor resembles more closely the solution conformation of analogs having tryptophan in the d -configuration. This latter conformation places the Trp 8 -Lys 9 side chains in close proximity, thus shifting the γ-methylene protons of Lys 9 upfield.

Identification of cytochrome P4503A4 as the major enzyme responsible for the metabolism of ivermectin by human liver microsomes

1. Ivermectin was extensively metabolized by human liver microsomes to at least 10 metabolites. The structure of many of them (mostly hydroxylated and demethylated) was determined by 1H-NMR and LC/MS. 2. To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on ivermectin metabolism. TAO, a specific inhibitor of cytochrome P4503A4, was the most potent inhibitor, inhibiting the total metabolism as well as formation of each metabolite. Metabolism was also inhibited by an anti-human cytochrome 3A4 antibody by 90%. 3. When ivermectin was incubated with microsomes from cells expressing CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 at 4 mg/ml protein concentrations, metabolic activity was only detected with the microsomes containing CYP3A4. The metabolic profile from cDNA-expressed CYP3A4 microsomes was qualitatively similar to that from human liver microsomes. 4. Thus, cytochrome P4503A4 is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes.

Structure-function studies on the cyclic peptide MT-II, lactam derivative of α-melanotropin

The alanine-substituted and the retro, enantio, and retro-enantio analogs of MT-II, a potent agonist at melanocortin (MC) receptors, were prepared by solid-phase synthesis and evaluated for their ability to bind and activate human MC3, MC4, and MC5 receptors. Replacement of His with Ala resulted in [Ala6]-MT-II with affinity and agonist potency at human MC3, MC4, and MC5 receptors similar to MT-II. Substitution of Arg with Ala gave compound 100-fold less potent than MT-II, but replacement of Phe or Trp with Ala led to inactive compounds (at the micromolar concentrations). The significant drop of potency of the retro, enantio, and retro-enantio analogs of MT-II, demonstrated a crucial role of side-chain topology, and to a lesser degree, of peptide backbone in interactions of MT-II with the melanocortin receptors. The nuclear magnetic resonance analysis of MT-II suggested involvement of Phe and Arg residues in H-bonds stabilizing the bent conformations of the peptide backbone.

Carnitine and glucuronic acid conjugates of pivalic acid

The [1-14C]pivaloyloxyethyl ester of methyldopa administered to man and cynomolgus monkeys resulted in the elimination in the urine of 14C-pivalic acid metabolites. Pivaloyl glucuronide and pivaloyl carnitine were identified as the major radioactive urinary metabolites in monkey urine and human urine, respectively. N.m.r. analysis indicated that pivaloyl carnitine had a cyclic structure. Although the role of carnitine is in the transport of fatty acids across mitochondrial membranes, it may also function in the conjugation of carboxylic acid xenobiotics in humans.

Neolignans from PIPER futokadsura

Abstract The structures of three new neolignans, kadsurenone, kadsurin A and kadsurin B, isolated from Piper futokadsura were determined by chemical and spectral analysis and X-ray diffraction study. Their biological activities are reported.

Drug residue formation from ronidazole, a 5-nitroimidazole. V. Cysteine adducts formed upon reduction of ronidazole by dithionite or rat liver enzymes in the presence of cysteine

When ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reduced by either dithionite or rat liver microsomal enzymes in the presence of cysteine, ronidazole-cysteine adducts can be isolated. Upon reduction with dithionite ronidazole can react with either one or two molecules of cysteine to yield either a monosubstituted ronidazole-cysteine adduct substituted at the 4-position or a disubstituted ronidazole-cysteine adduct substituted at both the 4-position and the 2-methylene position. In both products the carbamoyl group of ronidazole has been lost. The use of rat liver microsomes to reduce ronidazole led to the formation of the disubstituted ronidazole-cysteine adduct. These data indicate that upon the reduction of ronidazole one or more reactive species can be formed which can bind covalently to cysteine. The proposed reactive intermediates formed under these conditions may account for the observed binding of ronidazole to microsomal protein and the presence of intractable drug residues in the tissues of animals treated with this compound. They may also account for the mutagenicity of this compound in bacteria.

A Tethered Porphyrin Dimer with π Overlap of a Single Pyrrole Ring

The special pair of the bacterial photosystem has been modeled with a porphyrin dimer (the partial structure is shown). As with the natural system, only one pyrrole ring from each monomer subunit participates in π overlap.

4 -DEOXY-4 -AMINOAVERMECTINS WITH POTENT BROAD SPECTRUM ANTIPARASITIC ACTIVITIES

Abstract Reductive amination of 4″-oxo-5-O-tert-butyldimethylsilyl-avermectins with sodium cyanoborohydride and ammonium acetate gave an epimeric mixture of 4″-deoxy-4″-amino analogs with the epimeric, axial 4″-β-amino derivative as the major component. Acylation of the amino substituent gave highly active broad spectrum antiparasitic compounds, as determined in a sheep anthelmintic assay. 4″-Epi-acetylamino-4″-deoxyavermectin B 1 ( 12 ) was selected for further antiparasitic studies and is currently under development as a novel avermectin endectocide.

Further studies on the biosynthesis of the avermectins

SummaryThe biosynthesis of avermectins was studied further inStreptomyces avermitilis MA5502 by feeding experiments with labeled precursors.13C-NMR analysis of the compounds biosynthesized from [2-13C]acetate, [1,2-13C2]acetate, [3-13C]propionate and [2,3-13C2]propionate confirmed that the aglycone of avermectins is made from seven intact acetate and five propionate units. Feeding experiments with [1-13C]2-methylbutyrate and [1-13C]isobutyrate have shown that 2-methylbutyrate and isobutyrate are immediate precursors of the starter units of the polyketide chains of avermectin ‘a’ and ‘b’ components, respectively. The3H/14C doublelabeling experiments suggest that the two oleandrose moieties are derived from glucose.

Structure-function relationships of rat liver CYP3A9 to its human liver orthologs: site-directed active site mutagenesis to a progesterone dihydroxylase.

CYP3A9 is an estrogen-inducible ortholog of human liver CYP3A4 with 76.5% sequence identity to CYP3A4. Unlike CYP3A4, it is a very poor testosterone 6beta- and 2beta-hydroxylase, but a relatively better catalyst of progesterone monohydroxylation largely at 6beta, 16alpha, and 21 positions with negligible 6beta, 21-dihydroxylation. We reasoned that such differences in substrate catalyses must be due to differences in the active site architecture of each CYP3A enzyme. Indeed, alignment of CYP3A4 substrate recognition sites (SRSs) with the corresponding regions of CYP3A9 sequence revealed that of the 22 fully divergent residues, 4 reside in SRS regions [P107N (SRS-1), M371G (SRS-5), and L479K and G480Q (SRS-6)]. Accordingly, we substituted these and other divergent CYP3A9 SRS residues with the corresponding residues of CYP3A4 and/or CYP3A5. Our findings of the influence of these site-directed mutations of the CYP3A9 active site on its catalysis of testosterone and three other established but structurally different CYP3A substrates (progesterone, imipramine, and carbamazepine) are described. These findings revealed that some mutations (N107P, N107S, V207T, G371M, and Q480G) not only improved the ability of CYP3A9 to hydroxylate testosterone at the 6beta and 2beta positions, but also converted it into a robust progesterone 6beta, 21-dihydroxylase. The latter in the case of CYP3A9N107P was accompanied by a shift from sigmoidal to hyperbolic enzyme-substrate kinetics. In contrast, the catalytic potential of CYP3A9 mutants K206N, K206S, M240V, and K479L/Q480G was either relatively unchanged or negligible to nonexistent. Together these findings attest to the unique substrate-active site fit of each CYP3A enzyme.