Ian S. Blagbrough

Arthropod toxins as leads for novel insecticides: An assessment of polyamine amides as glutamate antagonists

In the search for new toxins, preferably with new sites of action, the polyamine amides represent a new class of compounds with potential as insecticides and as pharmaceutical agents due to their antagonism of ligand-gated cation channels. In particular, they are potent antagonists of the L-glutamate receptors of insect skeletal muscle. In this paper, we report on synthetic studies to produce hybrid analogues based upon the argiotoxin spider toxins and philanthotoxin-433 which is obtained from a solitary, parasitic wasp. We speculate upon possible modes and sites of action for these antagonists and we discuss their potential as insecticides and in the possible treatment of ischaemic damage. The synthesis and characterization of 4-hydroxyphenylpropanoylspermine is reported and the locust muscle biological assay is described. Using this pharmacological screen, structure-activity relationships have been determined in our laboratories. These are reviewed in the light of the current literature. Voltage clamp studies of the synthetic analogue philanthotoxin-343 and the effects of this polyamine amide on glutamate receptors expressed in Xenopus oocytes are outlined. In conclusion, a description of our current ideas and understanding of the many sites and modes of action of the polyamine amides, based both upon our own studies and also upon those recently reported, is presented.

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-Sörensens buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.

Kynurenine aminotransferase activity in human liver: identity with human hepatic C-S lyase activity and a physiological role for this enzyme.

The C-S lyase enzymes are responsible for the generation of mutagenic and cytotoxic metabolites via aberrant drug-metabolising pathways in mammalian tissues. We have examined human hepatic cytosolic, mitochondrial and microsomal fractions for evidence of C-S lyase activity. The cytosolic enzyme was purified using fast protein liquid chromatography over FFQ Sepharose, Mono P and Superose 12. An homogeneous protein (monitored by SDS-PAGE) was obtained following purification, and an 11-fold increase in C-S lyase specific activity was observed. The molecular weight of the enzyme was found to be 37 kDa in denaturing conditions, 82.3 kDa in non-denaturing conditions, and the C-S lyase activity was shown to co-purify with kynurenine aminotransferase activity when the transaminase activity of the enzyme was examined with kynurenine as the substrate.

Antagonism of Insect Muscle Glutamate Receptors — with Particular Reference to Arthropod Toxins

A model of the cation-selective channel gated by the quisqualatesensitive glutamate receptor (G1uR) of locust muscle is described. This model is based, in part, upon the vertebrate, nicotinic acetylcholine receptor channel with which the G1uR channel has many properties in common. The GluR channel is c. 140A in length and an integral part of the proposed tetrameric glutamate receptor protein. It has an outer vestibule, 30A in diameter at its extracellular face, which narrows at the channel gate to a c. 45A long ion-selectivity filter terminating on the cytoplasmic face of the G1uR. The channel is lined by fixed -ve charges to which permeating cations bind and has a hydrophobic pocket just external to the selectivity filter. Chlorisondamine, an organic cation with a maximum dimension of 9.8A, passes through the channel at high membrane potentials. This fixes the minimum dimension of the selectivity filter at c. 10A. The argiotoxins and philanthotoxin should permeate the channel, but perhaps only at high membrane potentials, but the high affinity of these flexible, polycationic molecules for the fixed charges on the channel wall makes them potent open channel blockers. If these toxins compete with divalent cations for the fixed charges on membrane proteins, then competition between Joro spider toxin for Ca2+-bindings sites on the crustacean muscle GluR could cause closed channel block since Ca2+ is a requirement for channel gating in this system, but not for the locust GluR. Interactions with acidic groups of membrane phospholipids may lead to changes in membrane flexibility which could account for some of the effects of these toxins on GluR.

Isomerisation of ω-alkenyl substituted cyclohexane-1,3-dione enol derivatives using rhodium catalysis. A practical synthesis of substituted resorcinols

Treatment of the ω-alkenyl substituted cyclohexane-1,3-dione enol derivatives (1), (11), and (13) with rhodium trichloride trihydrate leads to the corresponding resorcinol derivatives (2), (12), and (15) respectively. By contrast, the RhCl3.3H2O catalysed isomerisations of the related enol ethers (5) and (9) instead produce the dienones (6) and (10) respectively.

Human renal C-S lyase : structure-activity relationships of cytosolic and mitochondrial enzymes

The enzyme cysteine conjugate /.I-lyase (C-S lyase, EC 4.4.1.13) from rat renal tissue has been identified as a transaminase: glutamine transaminase K (GTK) [l]. This is one member of a family of enzymes which effect a transamination reaction on amino acid substrates. Other members of this family include glutamine transaminase L (GTL, isolated from rat liver, but which has a structure-activity profile broadly overlapping that of GTK), kynurenine aminotransferase (KAT), and aspartate aminotransferase. Rat hepatic cytosolic C-S lyase co-purifies as kynureninase, an enzyme which is involved in anthranilic acid biosynthesis [2], but antibodies raised against this purified protein did not crossreact with the renal cytosolic enzyme (i.e. with GTK). In one study with human hepatic cytosolic C-S lyase [3], kynurenine was not a substrate for the enzyme. These workers did not establish whether the C-S lyase enzyme had a physiological role, and if it co-purified with a protein which had been previously characterized. We have recently established that human hepatic cytosolic C-S lyase co-purifies as an aminotransferase: KAT [4]. In this article, we discuss the structureactivity relationships displayed by human C-S lyases obtained from both the cytosolic and mitochondrial sub-cellular fractions of kidney, liver and lung tissue. Elsewhere

Cysteine Conjugate Toxicity in a Human Cell Line: Correlation with C-S Lyase Activity in Human Hepatic Tissue

C-S lyase enzymes catalyse the generation of mutagenic and/or cytotoxic thiols from cysteine conjugated xenobiotics. These cysteine conjugates are produced subsequent to glutathione conjugations as a metabolic step in the mercapturic acid pathway, traditionally thought of as a pathway solely associated with detoxification. Human Chang liver (HCL) cells were challenged with a range of cysteine conjugates demonstrated to be substrates for human hepatic C-S lyases. The cellular toxicity of these compounds was determined and it was observed that the rank order of substrate toxicity obtained for the HCL cells followed the rank order of C-S lyase activity of the substrates in a freshly isolated mitochondrial fraction of human tissue. The presence of C-S lyase activity was also established in this cell line.

THE SYNTHESIS AND ENZYMATIC DEGRADATION OF DEXTRANMETHOTREXATE CONJUGATES

Dextran T40 was oxidized with 17% aqueous sodium periodate solution (Malaprade reaction). Schiff base formation with diamines including 1,3-diamino-2-propanoI (Shih et al 1988) and 1,3-diaminopropane, and polyamines e.g. spermine followed by sodium borohydride reduction afforded amino-modified dextrans. Biodegradable amide linkages were introduced by a carbodiimide (ECDI) catalysed reaction between the amino-modified dextran pol mer and the carboxylic acid groups of the L-glutamic acid residue in dTX (in 40mM aqueous sodium hydrogen carbonate solution, pH 7.4). The incorporation of MTX was achieved most satisfactorily with 1,3-diamin0-2-propanol.