Elucidation of reaction scheme describing malondialdehyde-acetaldehyde-protein adduct formation

Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed animals. Our previous studies have shown that MAA adducts are comprised of two distinct products. One adduct is composed of two molecules of malondialdehyde and one molecule of acetaldehyde and was identified as the 4-methpl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group (MHHDC adduct). The other adduct is a 1:1 adduct of malondialdehyde and acetaldehyde and was identified as the 2-formyl-3-(alkylamino)butanal derivative of an amino group (FAAB adduct). In this study, information on the mechanism of MAA adduct formation was obtained, focusing on whether the FAAB adduct serves as a precursor for the MDHDC adduct. Upon the basis of chemical analysis and NMR spectroscopy, two initial reaction steps appear to be a prerequisite for MDHDC formation. One step involves the reaction of one molecule of malondialdehyde and one of acetaldehyde with an amino group of a protein to form the FAAB product, while the other step involves the generation of a malondialdehyde-enamine. It appears that generation of the MDHDC adduct requires the FAAB moiety to be transferred to the nitrogen of the MDA-enamine. For efficient reaction of FAAB with the enamine to take place, additional experiments indicated that these two intermediates likely must be in positions on the protein of close proximity to each other. Further studies showed that the incubation of liver proteins from ethanol-fed rats with MDA resulted in a marked generation of MDHDC adducts, indicating the presence of a pool of FAAB adducts in the liver of ethanol-fed animals. Overall, these findings show that MDHDC-protein adduct formation occurs via the reaction of the FAAB moiety with a malondialdehyde-enamine, and further suggest that a similar mechanism may be operative in vivo in the liver during prolonged ethanol consumption.

Lipophilic methotrexate conjugates with glucose-lipoamino acid moieties: Synthesis and in vitro antitumor activity

To obtain methotrexate (MTX) derivatives with a balanced hydrolipophilic character, we synthesized a series of conjugates in which the drug was linked to lipoamino acid (LAA)-glucose residues (LAAG-MTX). These conjugates displayed increased solubility in polar media compared with the corresponding LAA-MTX conjugates previously described. In vitro biological testing of LAAG-MTX indicated that the introduction of the sugar moiety decreased the biological activity of these MTX conjugates. The tetradecyl derivative 6b, however, was effective in inhibiting the dihydrofolate reductase activity in vitro and showed an inhibitory effect on human lymphoblastoid cell growth. (C) 2001 Wiley-Liss, Inc.

Lipid, sugar and liposaccharide based delivery systems

Although there are formidable barriers to the oral delivery of biologically active drugs, considerable progress in the field has been made, using both physical and chemical strategies of absorption enhancement. A possible method to enhance oral absorption is to exploit the phenomenon of lipophilic modification and mono and oligosaccharide conjugation. Depending on the uptake mechanism targeted, different modifications can be employed. To target passive diffusion, lipid modification has been used, whereas the targeting of sugar transport systems has been achieved through drugs conjugated with sugars. These drug delivery units can be specifically tailored to transport a wide variety of poorly absorbed drugs through the skin, and across the barriers that normally inhibit absorption from the gut or into the brain. The delivery system can be conjugated to the drug in such a way as to release the active compound after it has been absorbed (i.e. the drug becomes a prodrug), or to form a biologically stable and active molecule (i.e. the conjugate becomes a new drug moiety). Examples where lipid, sugar and lipid-sugar conjugates have resulted in enhanced drug delivery will be highlighted in this review.

Macrocycles mimic the extended peptide conformation recognized by aspartic, serine, cysteine and metallo proteases

It has been previously demonstrated that aspartic, serine, metallo and cysteine proteases bind to their inhibitors and substrate analogues in a single conformation, the saw-tooth or extended beta-strand. Consequently a generic approach to the development of protease inhibitors is the use of constraints that conformationally restrict putative inhibitor molecules to an extended form. In this way the inhibitor is pre-organized for binding to a protease and does not need to rearrange its structure. One constraining device that has proven to be effective for such pre-organization is macrocyclization. This article illustrates the general principle that macrocycles, especially those composed of 3-4 amino acids and usually 13-17 ring atoms, can effectively mimic the extended conformation of short peptide sequences. Such structure-stabilising macrocycles are stable to degradation by proteases, valuable components of potent protease inhibitors, and in many cases they are also bioavailable.

Ring flexibility within tricyclic antidepressant drugs

The internal flexibility of the central seven-membered ring of a series of tricyclic antidepressant drugs (TCAs), imipramine {l}, amitriptyline {2}, doxepin {3}, and dothiepin {4}, has been investigated by H-1 and C-13 nuclear magnetic (NMR) techniques. Two dynamic processes were examined: ring inversion and bridge flexing. H-1 NMR lineshape analysis was used to obtain ring inversion barriers for 2-4. These studies yielded energy barriers of 14.3, 16.7, and 15.7 +/- 0.6 kcal/mol for the hydrochloride salts of doxepin, dothiepin, and amitriptyline, respectively. The barriers for the corresponding free bases were lower by 0.6 kcal/mol on average. (CT1)-C-13 relaxation measurements were used to determine the degree of bridge flexing associated with the central seven-membered ring for all four compounds. By fitting the T-1 data to a two-state jump model, lifetimes and amplitudes of rapid bridge flexing motions were determined. The results show that imipramine has the fastest rate of bridge flexing, followed by amitriptyline, doxepin, and dothiepin. The pharmacological profiles of the TCAs are complex and they interact with many receptor sites, resulting in numerous side effects and a general lack of understanding of their precise mode of action in different anxiety-related disorders. They all have similar three-dimensional structures, which makes it difficult to rationalize their differing relative potency in different assays/clinical settings. However, the clear finding here that there are significantly different degrees of internal mobility suggests that molecular dynamics should be an additional factor considered when trying to understand the mode of action of this clinically important family of molecules. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:713-721, 2001.

Radioresistant Burkitt's lymphoma cells exhibit defective Mapk signalling

Using a pair of isogenic Burkitt's lymphoma cell lines, one of which is sensitive (BL30A) and the other resistant (BL30K) to apoptosis induced by ionising radiation and exogenous ceramide, we investigated mitogen-activated protein kinase (MAPK) signalling to determine which members of this kinase family are involved in the apoptotic process in these cells. We have previously shown that BL30A cells produce ceramide after irradiation and that this does not occur in BL30K cells (Michael et at. [1997] Cancer Res 57:3600-3605). We show that p38 MAPK is activated transiently in both cells after ionising radiation. On the of her hand, although JNK is rapidly activated in both cells, this activation is only transient in the resistant cells, whereas in the sensitive cells the activation is sustained. Addition of exogenous ceramide resulted in only a transient activation of INK in both cells. Interestingly, ERK activity was decreased in BL30A cells after ceramide treatment, whereas no such decrease occurred in the resistant cells. Treatment of BL30A cells with phorbol ester before irradiation, which blocks the increase in ceramide and apoptosis, also prevents the sustained increase in JNK activity. At the same time, ERK activity is increased. Our results suggest that p38 MAPK is not required for apoptosis signalling in response to ionising radiation in Burkitt's lymphoma cells and that sustained activation of JNK is necessary for apoptosis in these cells. These results also support the hypothesis that a balance between JNK and ERK activity determines cell fate after exposure to ceramide or ionising radiation. In addition, our results suggest different signalling pathways from exogenous ceramide and radiation, supporting the concept of different intracellular pools of active ceramide. Drug Dev. Res. 52:534-541, 2001. (C) 2001 Wiley-Liss, Inc.

Alpha-conotoxins: Nicotinic acetylcholine receptor antagonists as pharmacological tools and potential drug leads

Alpha-Conotoxins are small disulfide rich peptides from the venoms of marine cone snails. They target specific nicotinic acetylcholine receptor (nAChR) subtypes with high affinity and potency and are therefore valuable as neurophamacological probes and potential drug leads. This article gives a general overview of the chemical and biological features of alpha -conotoxins, including their pharmacology, binding interactions and structure. A detailed analysis of recently reported three-dimensional structures from members of different subfamilies of the alpha -conotoxins, including those with 3/5, 4/3, 4/6 and 4.7 spacings of their two intracysteine loops is given. The structures are generally well defined and represent useful frameworks for the display of amino acid residues to target molecules.

Development of response-selective agonists of human C5a anaphylatoxin: Conformational, biological, and therapeutic considerations

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

Oxidizing species in the mechanism of cytochrome P450

This review discusses the mechanisms of oxygen activation by cytochrome P450 enzymes, the possible catalytic roles of the various iron-oxygen species formed in the catalytic cycle, and progress in understanding the mechanisms of hydrocarbon hydroxylation, heteroatom oxidation, and olefin epoxidation. The focus of the review is on recent results, but earlier work is discussed as appropriate. The literature through to February 2002 is surveyed, and 175 referenced are cited.

Antitubercular constituents from the hexane fraction of Morinda citrifolia Linn. (Rubiaceae)

A crude ethanol extract and hexane fraction from Morinda citrifolia Linn. (Rubiaceae) show antitubercular activity. The major constituents of the hexane fraction are E-phytol, cycloartenol, stigmasterol, beta-sitosterol, campesta-5,7,22-trien-3beta-ol and the ketosteroids stigmasta-4-en-3-one and stigmasta-4-22-dien-3-one. E-Phytol, a mixture of the two ketosteroids, and the epidioxysterol derived from campesta-5,7,22-trien-3beta-ol all show pronounced antitubercular activity. Copyright (C) 2002 John Wiley Sons, Ltd.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Evaluating Australia's National Medicines Policy using geographical mapping

Background: There has been a proliferation of quality use of medicines activities in Australia since the 1990s. However, knowledge of the nature and extent of these activities was lacking. A mechanism was required to map the activities to enable their coordination. Aims: To develop a geographical mapping facility as an evaluative tool to assist the planning and implementation of Australia's policy on the quality use of medicines. Methods: A web-based database incorporating geographical mapping software was developed. Quality use of medicines projects implemented across the country was identified from project listings funded by the Quality Use of Medicines Evaluation Program, the National Health and Medical Research Council, Mental Health Strategy, Rural Health Support, Education and Training Program, the Healthy Seniors Initiative, the General Practice Evaluation Program and the Drug Utilisation Evaluation Network. In addition, projects were identified through direct mail to persons working in the field. Results: The Quality Use of Medicines Mapping Project (QUMMP) was developed, providing a Web-based database that can be continuously updated. This database showed the distribution of quality use of medicines activities by: (i) geographical region, (ii) project type, (iii) target group, (iv) stakeholder involvement, (v) funding body and (vi) evaluation method. At September 2001, the database included 901 projects. Sixty-two per cent of projects had been conducted in Australian capital cities, where approximately 63% of the population reside. Distribution of projects varied between States. In Western Australia and Queensland, 36 and 73 projects had been conducted, respectively, representing approximately two projects per 100 000 people. By comparison, in South Australia and Tasmania approximately seven projects per 100 000 people were recorded, with six per 100 000 people in Victoria and three per 100 000 people in New South Wales. Rural and remote areas of the country had more limited project activity. Conclusions: The mapping of projects by geographical location enabled easy identification of high and low activity areas. Analysis of the types of projects undertaken in each region enabled identification of target groups that had not been involved or services that had not yet been developed. This served as a powerful tool for policy planning and implementation and will be used to support the continued implementation of Australia's policy on the quality use of medicines.

Syntheses of polycationic dendrimers on lipophilic peptide core for complexation and transport of oligonucleotides

Synthesis of novel polycationic lipophilic peptide core(s) was accomplished and these agents successfully transfected human retinal pigment epithelium cells with ODN1 upon complexation with the oligonucleotide. The level of transfection was indirectly measured by the decreased production of the protein hVEGF (human vascular endothelial growth factor) in comparison to the transfection agent cytofectin GSV(TM). (C) 2002 Elsevier Science Ltd. All rights reserved.

Lipoamino acid-based adjuvant carrier system: Enhanced immunogenicity of group a streptococcal peptide epitopes

Lipoamino acid-based synthetic peptides (lipid core peptides, LCP) derived from the type-specific and conserved region determinants of group A streptococci (GAS) were evaluated as potential candidate sequences in a vaccine to prevent GAS-associated diseases, including rheumatic heart, disease and poststreptococcal acute glomerulonephritis. The LCP peptides had significantly enhanced immunogenicity as compared with the monomeric peptide epitopes. Furthermore, the peptides incorporated into the LCP system generated epitope-specific antibodies without the use of any conventional adjuvant.