PLP and GABA Trigger GabR-mediated Transcription Regulation in Bacillus Subtilis via External Aldimine Formation

The Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of γ-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5′-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the “active site” in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic γ-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O–PLP–AFPA complex and Asp-AT–PLP–AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP

Organic synthesis combined with molecular modeling: A powerful approach to map the functional topography of dopamine and serotonin receptors

Previous molecular modeling studies of dopamine and serotonin receptor agonists docked into their receptors have produced mixed results, due mainly to the use of homology models based on the published crystal structure of inactive bovine rhodopsin. With the recently published structure of the more closely related β2 adrenergic receptor, whose endogenous ligands are also biogenic monoamine neurotransmitters, we have been able to develop an in silico-activated model of this G protein-coupled receptor (GPCR) based on Molecular Dynamics (MD) simulations of the membrane-bound and solvated protein. From this system, improved homology models of the serotonin 5-HT2A and dopamine D1 receptors have been created that appear well suited for the qualitative and quantitative study of agonist properties. For the dopamine D1 receptor, the models were employed to: (1) elucidate the catechol hydrogen bonding network using the selective agonist A-77,636 and two analogs; (2) probe the steric space at the bottom of the binding site with analogs of dihydrexidine (DHX) methylated at the 7 and 8 positions; and (3) investigate the binding of analogs of DHX with substitutions at the pendant aromatic ring. In the case of the 5-HT2A receptor, the binding of different classes of ligands was studied, including phenylalkylamines, tryptamines, and ergolines. For the latter, the observations were used to determine the region of the binding pocket occupied by an 8-diethylamide group. Additionally, free energy of binding calculations using the Linear Interaction Energy (LIE) method were performed, obtaining a model that showed a squared correlation coefficient of 0.7018, with an associated error of 4.56 kJ/mol–1 . Validation of this model was performed with an independent test set, which showed a squared correlation coefficient of 0.7080. Notably, electrostatic interaction energies were not significant for this model. Finally, the models were used to study the binding profiles of newly synthesized ligands. During the course of this investigation, two sets of ligands were synthesized. The first group, targeted toward the D1 receptor, involved replacing the catechol group of DHX with a pyrazole or an aminopyrimidine ring, based on the structures of quinpirole and quinelorane. Only the pyrazole compound could be synthesized, and it showed poor binding affinity (18.3 μM), probably due to the incorrect orientation of the polar pyrazole proton. The second group of compounds, designed as 5-HT2A agonists, consisted of piperidine-based rigid analogs of a superpotent N-benzylphenethylamine. The (S,S)-2,6-disubstituted piperidine analog retained most of the affinity (2.5 nM) of the original ligand (0.19 nM), and showed dramatically increased selectivity for the 5-HT 2A over the 5-HT2C receptor of up to 128-fold, making it the most selective 5-HT2A agonist synthesized to date. Molecular modeling revealed that the rigidified N-benzyl group occupied the same general location in the 5-HT2A binding site as the original flexible compound. A better understanding of the subtle details of the activation process, as well as the interactions of various agonists with these receptors, will lead to new insights into the basis of their action, laying the groundwork for the design of more efficacious ligands with potential therapeutic and research applications

Mechanism of Inactivation of γ‑Aminobutyric Acid Aminotransferase by (1<i>S</i>,3<i>S</i>)‑3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1<i>S</i>,3<i>S</i>)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5′-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (<b>22</b>) and two other precursors of this compound (<b>20</b> and <b>21</b>). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (<b>20</b>), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general

Extensive Rigid Analogue Design Maps the Binding Conformation of Potent <i>N</i>‑Benzylphenethylamine 5‑HT_2A Serotonin Receptor Agonist Ligands

Based on the structure of the superpotent 5-HT_2A agonist 2-(4-bromo-2,5-dimethoxyphenyl)-<i>N</i>-[(2-methoxyphenyl)­methyl]­ethanamine, which consists of a ring-substituted phenethylamine skeleton modified with an <i>N</i>-benzyl group, we designed and synthesized a small library of constrained analogues to identify the optimal arrangement of the pharmacophoric elements of the ligand. Structures consisted of diversely substituted tetrahydroisoquinolines, piperidines, and one benzazepine. Based on the structure of (<i>S</i>,<i>S</i>)-<b>9b</b>, which showed the highest affinity of the series, we propose an optimal binding conformation. (<i>S</i>,<i>S</i>)-<b>9b</b> also displayed 124-fold selectivity for the 5-HT_2A over the 5-HT_2C receptor, making it the most selective 5-HT_2A receptor agonist ligand currently known

Extensive Rigid Analogue Design Maps the Binding Conformation of Potent <i>N</i>‑Benzylphenethylamine 5‑HT_2A Serotonin Receptor Agonist Ligands

Based on the structure of the superpotent 5-HT_2A agonist 2-(4-bromo-2,5-dimethoxyphenyl)-<i>N</i>-[(2-methoxyphenyl)­methyl]­ethanamine, which consists of a ring-substituted phenethylamine skeleton modified with an <i>N</i>-benzyl group, we designed and synthesized a small library of constrained analogues to identify the optimal arrangement of the pharmacophoric elements of the ligand. Structures consisted of diversely substituted tetrahydroisoquinolines, piperidines, and one benzazepine. Based on the structure of (<i>S</i>,<i>S</i>)-<b>9b</b>, which showed the highest affinity of the series, we propose an optimal binding conformation. (<i>S</i>,<i>S</i>)-<b>9b</b> also displayed 124-fold selectivity for the 5-HT_2A over the 5-HT_2C receptor, making it the most selective 5-HT_2A receptor agonist ligand currently known

The relationship between effectiveness and costs measured by a risk-adjusted case-mix system: multicentre study of Catalonian population data bases

<p>Abstract</p> <p>Background</p> <p>The main objective of this study is to measure the relationship between morbidity, direct health care costs and the degree of clinical effectiveness (resolution) of health centres and health professionals by the retrospective application of Adjusted Clinical Groups in a Spanish population setting. The secondary objectives are to determine the factors determining inadequate correlations and the opinion of health professionals on these instruments.</p> <p>Methods/Design</p> <p>We will carry out a multi-centre, retrospective study using patient records from 15 primary health care centres and population data bases. The main measurements will be: general variables (age and sex, centre, service [family medicine, paediatrics], and medical unit), dependent variables (mean number of visits, episodes and direct costs), co-morbidity (Johns Hopkins University Adjusted Clinical Groups Case-Mix System) and effectiveness.</p> <p>The totality of centres/patients will be considered as the standard for comparison. The efficiency index for visits, tests (laboratory, radiology, others), referrals, pharmaceutical prescriptions and total will be calculated as the ratio: observed variables/variables expected by indirect standardization.</p> <p>The model of cost/patient/year will differentiate fixed/semi-fixed (visits) costs of the variables for each patient attended/year (N = 350,000 inhabitants). The mean relative weights of the cost of care will be obtained. The effectiveness will be measured using a set of 50 indicators of process, efficiency and/or health results, and an adjusted synthetic index will be constructed (method: percentile 50).</p> <p>The correlation between the efficiency (relative-weights) and synthetic (by centre and physician) indices will be established using the coefficient of determination. The opinion/degree of acceptance of physicians (N = 1,000) will be measured using a structured questionnaire including various dimensions. Statistical analysis: multiple regression analysis (procedure: enter), ANCOVA (method: Bonferroni's adjustment) and multilevel analysis will be carried out to correct models. The level of statistical significance will be p < 0.05.</p