G-protein coupled receptors: SAR analyses of neurotransmitters and antagonists.

BACKGROUND: From the deductive point of view, neurotransmitter receptors can be divided into categories such as cholinergic (muscarinic, nicotinic), adrenergic (alpha- and beta-), dopaminergic, serotoninergic (5-HT1 approximately 5-HT5), and histaminergic (H1 and H2). Selective agonists and antagonists of each receptor subtype can have specific useful therapeutic applications. For understanding the molecular mechanisms of action, an inductive method of analysis is useful. OBJECTIVE: The aim of the present study is to examine the structure-activity relationships of agents acting on G-protein coupled receptors. METHOD: Representative sets of G-PCR agonists and antagonists were identified from the literature and Medline [P.M. Walsh (2003) Physicians' Desk Reference; M.J. O'Neil (2001) The Merck Index]. The molecular weight (MW), calculated logarithm of octanol/water partition coefficient (C log P) and molar refraction (CMR), dipole moment (DM), E(lumo) (the energy of the lowest unoccupied molecular orbital, a measure of the electron affinity of a molecule and its reactivity as an electrophile), E(homo) (the energy of the highest occupied molecular orbital, related to the ionization potential of a molecule, and its reactivity as a nucleophile), and the total number of hydrogen bonds (H(b)) (donors and receptors), were chosen as molecular descriptors for SAR analyses. RESULTS: The data suggest that not only do neurotransmitters share common structural features but their receptors belong to the same ensemble of G-protein coupled receptor with seven to eight transmembrane domains with their resultant dipoles in an antiparallel configuration. Moreover, the analysis indicates that the receptor exists in a dynamic equilibrium between the closed state and the open state. The energy needed to open the closed state is provided by the hydrolysis of GTP. A composite 3-D parameter frame setting of all the neurotransmitter agonists and antagonists are presented using MW, Hb and mu as independent variables. CONCLUSION: It appears that all neurotransmitters examined in this study operate by a similar mechanism with the G-protein coupled receptors.

Structure-activity relationship: analyses of p-glycoprotein substrates and inhibitors.

OBJECTIVE: A large number of structurally and functionally diverse compounds act as substrates or modulators of p-glycoprotein (p-gp). Some of them possess multiple drug resistance (MDR)-reversing activity, but only a small number of them have entered clinical study. In order to uncover the factors which exert a significant impact on the interaction between substrates/modulators and p-gp, we have performed structure-activity relationship (SAR) analyses, including molecular modelling, two-dimensional (2D) and three-dimensional (3D) parameter-frame-setting analysis, quantitative structure activity relationship (QSAR) analysis among substrates/modulators, as well as clinically promising MDR-reversing agents. METHODS: The physicochemical parameters C log P, CMR and all regression equations were derived by using C log P version 4.0 and the latest CQSAR software, respectively. Molecular modelling and all other parameter calculations were performed by using HyperChem version 5.0 program, after geometry optimization and energy minimization using the AM1 semiempirical method. RESULTS: SAR analyses indicate that MDR reversal activity is correlated with the lipophilicity (C log P), molecular weight (log Mw), longest chain (Nlc) of the molecule and the energy of the highest occupied orbital (Ehomo). In addition, the presence of a basic tertiary nitrogen atom in the structure is also an important contributor to p-gp inhibitory activity. Some separation in space is achieved for different subsets of p-gp substrates and inhibitors using Nlc, C log P and Ehomo as three independent parameters in the 3D-parameter-frame setting. CONCLUSION: A highly effective p-gp modulator candidate should possess a log P value of 2.92 or higher, 18-atom-long or longer molecular axis, and a high Ehomo value, as well as at least one tertiary basic nitrogen atom. The results obtained may be useful in explaining drug-p-gp interactions for different compounds, including drug interactions and the development of new MDR chemosensitizers.

Regulation of the myeloid-cell-expressed human gp91-phox gene as studied by transfer of yeast artificial chromosome clones into embryonic stem cells: suppression of a variegated cellular pattern of expression requires a full complement of distant cis elements.

Identifying the full repertoire of cis elements required for gene expression in mammalian cells (or animals) is challenging, given the moderate sizes of many loci. To study how the human gp91-phox gene is expressed specifically in myeloid hematopoietic cells, we introduced yeast artificial chromosome (YAC) clones and derivatives generated in yeast into mouse embryonic stem cells competent to differentiate to myeloid cells in vitro or into mouse chimeras. Fully appropriate regulation was recapitulated with a 130-kb YAC containing 60 and 30 kb of 5' and 3' flanking sequences, respectively. Immunodetection of human gp91-phox protein revealed uniform expression in individual myeloid cells. The removal of upstream sequences led to decreased overall expression which reflected largely a variegated pattern of expression, such that cells were either "on" or "off," rather than pancellular loss of expression. The proportion of clones displaying marked variegation increased with progressive deletion. DNase I mapping of chromatin identified two hypersensitive clusters, consistent with the presence of multiple regulatory elements. Our findings point to cooperative interactions of complex regulatory elements and suggest that the presence of an incomplete set of elements reduces the probability that an open chromatin domain (or active transcriptional complex) may form or be maintained in the face of repressive influences of neighboring chromatin.

Hormone therapy and phytoestrogens.

As ageing progresses the levels of sex hormones decrease in the human body. In the male population, the decrease or absence of testosterone leads to decreased strength and stamina, thin bones and a low sex drive (1). In the female population, the immediate symptoms of menopause include irregular periods, painful sexual intercourse due to vaginal dryness, hot flushes and night sweats (2). Lack of oestrogen also leads to the risk of developing osteoporosis and cardiovascular diseases. In this report, the authors will mainly discuss the effects of hormone therapy (HT) in menopausal women. Available current clinical data on the effects of calcium supplementation with and without HT, exercise, exercise plus calcium and exercise with HT on bone loss are presented. The effects of transdermal and oral oestrogen therapy (OT) on serum lipids are discussed. Commercially-available HT products, their indications, dosages, contra-indications, side-effects and drug interactions are compared. Alternative therapies for menopausal symptoms with Chinese traditional herbs, and a comparison of the molecular structures of phytoestrogens with estradiol and diethylstilbestrol are examined (3, 4). A list of medicinal herbs and foods reported to elicit an oestrogenic response in animals is compiled.

Preventing potential drug interactions in community pharmacy.

With ongoing debate on health care reform including improved pharmaceutical care, there is much current concern about drug interactions and their prevention. Many patients visit more than one doctor for their different diseases and receive more than one drug at a time, and often doctors are unaware of all the medications their patients are taking and the risks to which their patients are exposed when treated with multiple drugs. Pharmacists in the community setting or hospital are the most accessible health care providers able to intervene when faced with potential drug interactions that may occur during patients' multiple drug therapy. A few selected examples of potential drug-drug interactions and interventions instituted are presented in this paper. Possible mechanisms for the drug interactions are also discussed. It is hoped that more documentation of pharmacists' involvement in such interventions will demonstrate the true value of pharmaceutical care.

Cloning of human microtubule-associated protein 1B and the identification of a related gene on chromosome 15.

We report here the complete cloning and sequencing of human microtubule associated protein 1B (MAP1B). Comparisons to mouse and partial rat MAP1B sequence indicate that this gene is extremely well conserved, with 91 and 90% identity, respectively. The entire human MAP1B genomic region has been isolated and the genomic organization determined. The gene includes seven exons, and the third exon contains sequence not represented in mouse or rat MAP1B. This sequence, labeled 3A, is present at the 5' end of an alternative transcript that is expressed at approximately 1/10th the level of the full-length transcript. By comparisons of human MAP1B with the sequence databases, we have identified a MAP1B-related gene that is probably the human homologue of rat MAP1A. This gene is expressed at high levels in brain and spinal cord and much lower levels in muscle and maps to the long arm of human chromosome 15.

Construction of a yeast artificial chromosome contig spanning the spinal muscular atrophy disease gene region.

The childhood spinal muscular atrophies (SMAs) are the most common, serious neuromuscular disorders of childhood second to Duchenne muscular dystrophy. A single locus for these disorders has been mapped by recombination events to a region of 0.7 centimorgan (range, 0.1-2.1 centimorgans) between loci D5S435 and MAP1B on chromosome 5q11.2-13.3. By using PCR amplification to screen yeast artificial chromosome (YAC) DNA pools and the PCR-vectorette method to amplify YAC ends, a YAC contig was constructed across the disease gene region. Nine walk steps identified 32 YACs, including a minimum of seven overlapping YAC clones (average size, 460 kb) that span the SMA region. The contig is characterized by a collection of 30 YAC-end sequence tag sites together with seven genetic markers. The entire YAC contig spans a minimum of 3.2 Mb; the SMA locus is confined to roughly half of this region. Microsatellite markers generated along the YAC contig segregate with the SMA locus in all families where the flanking markers (D5S435 and MAP1B) recombine. Construction of a YAC contig across the disease gene region is an essential step in isolation of the SMA-encoding gene.

A mutant antigen-presenting cell defective in antigen presentation expresses class II MHC molecules with an altered conformation.

Ag presentation by APC to class II MHC-restricted T cells involves a sequence of events: 1) intracellular processing of protein Ag into immunogenic peptides, 2) specific binding of peptides to class II MHC molecules, and then 3) transport of the MHC-peptide complexes to the plasma membrane. The critical event in the activation of T cells by APC is the recognition of MHC-associated antigenic determinants by the TCR/CD3 complex. In this report we describe the isolation and characterization of a mutant APC with a defect in an intracellular process that results in its inability to form MHC-peptide complexes for recognition by T cells. The mutant APC cannot present many different protein Ag with both I-A and I-E molecules but is able to present processing-independent peptides. The functional defect in the mutant APC is not caused by either a decrease in expression or a structural mutation in class II MHC molecules. Further, there is no mutation in the invariant chain (li) and it displays a normal kinetics of association and dissociation from the class II MHC molecules during biosynthesis. Although the mutation is not in the genes encoding for the class II MHC molecules or li, the mutant APC expresses class II MHC molecules with distinct serological epitopes suggestive of an altered conformation. Pulse-chase experiments suggest that a conformational difference between I-Ad molecules of wild-type and mutant cells occurs after the class II molecules exit from the endoplasmic reticulum but while they are still associated with li. The mutant cell produces few compact (SDS-resistant) class II heterodimers. This mutant APC provides a tool for studying the cell biology of Ag processing and presentation.

Fine-mapping of the spinal muscular atrophy locus to a region flanked by MAP1B and D5S6.

The microtubule-associated protein 1B (MAP1B) locus has been mapped in close proximity to spinal muscular atrophy (SMA) on chromosome 5q13. We have identified a second microsatellite within a MAP1B intron, which increases the heterozygosity of this locus to 94%. Two unambiguous recombination events establish MAP1B as a closely linked, distal flanking marker for the disease locus, while a third recombinant establishes D5S6 as the proximal flanking marker. The combination of key recombinants and linkage analysis place the SMA gene in an approximately 2-cM interval between loci D5S6 and MAP1B. Physical mapping and cloning locate MAP1B within 250 kb of locus D5S112. The identification and characterization of a highly polymorphic gene locus tightly linked to SMA will facilitate isolation of the disease gene, evaluation of heterogeneity, and development of a prenatal test for SMA.

Mapping of human microtubule-associated protein 1B in proximity to the spinal muscular atrophy locus at 5q13.

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.

Structure side-effect sorting of drugs. VI. Ototoxicities.

From a literature survey, over 130 (about 7.8%) drugs and chemicals have been associated with ototoxicities. The major classes are basic aminoglycoside and other antibiotics, anti-inflammatory drugs, antimalarials, beta-blockers, antineoplastic agents, heavy metals, diuretics, some topical agents and various miscellaneous drugs. Possible mechanisms of action are presented and discussed. These include inhibition of protein synthesis, the glycolytic cycle, the TCA cycle, energy utilization, energy generation and the respiratory system within the mitochondria membrane of the hair cell, and also alteration of the permeability of the endolymphatic membrane or alteration of the excretion system for the basic aminoglycosides in the lateral wall of the membranous cochlea. The relative rank order of ototoxicity and reactivity toward mucopolysaccharides of five aminoglycosides is found to be related to the number of basic groups in each molecule.