An early evaluation of the HISTO SPOT® AB ID Class I & II test in cardiothoracic transplant patients.

The HISTO SPOT® AB ID assay (BAG Diagnostics GmbH) is a novel single antigen HLA Class I & II antibody definition test used with the MR.SPOT® processor. We compared this assay with Luminex® -based assays to assess its potential application in defining unacceptable antigens for transplantation in patients awaiting transplants with cardiothoracic organs. A cohort of 40 sensitized cardiothoracic patients were identified, and one sample was selected from each patient. The required screening was based on the patients' antibody profiles (Class I, n = 17, Class II, n = 11, Class I & II, n = 12). Samples were screened with LABScreen™ Single Antigen (SAg), LIFECODES® LSA™, HISTO SPOT® AB ID, and an acid modified LABScreen™ SAg test for detecting antibodies against denatured HLA. Results indicated that HISTO SPOT® AB ID had reduced sensitivity (68% for Class I; 69% for Class II). When compared to LABScreen™ and LIFECODES® , HISTO SPOT® AB ID failed to detect Luminex® -defined antibodies with median fluorescence intensity (MFI) ranging from 1114 to 24,489. The HISTO SPOT® AB ID panel used in the study had reduced antigen representation compared with Luminex® -based assays which further compromised its capacity for antibody detection and definition. Further work is needed to evaluate the clinical relevance of these differences between the performance of HISTO SPOT® and Luminex® -based methods.

Synthesis and SAR study of 4-arylpiperidines and 4-aryl-1,2,3,6-tetrahydropyridines as 5-HT2C agonists.

A series of substituted 4-arylpiperidines and a smaller family of 4-aryl-1,2,3,6-tetrahydropyridines were synthesized and their biological activity at the 5-HT(2C) receptor studied to determine whether either series showed noteworthy agonist activity. Structure-activity relationships were developed from the performed receptor binding assays and functional studies, and the results of the analysis are presented herein.

Antiviral agents 3. Discovery of a novel small molecule non-nucleoside inhibitor of hepatitis B virus (HBV).

The discovery of a small molecule non-nucleoside inhibitor of Hepatitis B Virus is described. During our work on conocurvone derived naphthoquinone 'trimers' for the treatment of HIV, we discovered a potent inhibitor 9 of Hepatitis B Virus in an antiviral screen. During attempts to resynthesis 9 for proof of concept studies, we altered the synthesis in order to attempt to reduced side reactions and difficult to remove by-products. As a result we discovered a small molecule 19 that also was a potent inhibitor of HBV. Importantly, this small molecule inhibitor of Hepatitis B Virus is also an inhibitor of Hepatitis B Virus resistant to 3TC, a bench mark of nucleoside analogues active in the treatment of Hepatitis B Virus. The development of 19 as an agent to treat HBV infections is discussed.

Homology modeling and docking evaluation of aminergic G protein-coupled receptors.

We report the development of homology models of dopamine (D(2), D(3), and D(4)), serotonin (5-HT(1B), 5-HT(2A), 5-HT(2B), and 5-HT(2C)), histamine (H(1)), and muscarinic (M(1)) receptors, based on the high-resolution structure of the beta(2)-adrenergic receptor. The homology models were built and refined using Prime. We have addressed the required modeling of extracellular loop 2, which is often implicated in ligand binding. The orthosteric sites of the models were optimized using induced fit docking, to allow for side-chain flexibility, and the resulting receptor models have been evaluated using protein validation tools. Of the nine homology models developed, six models showed moderate to good enrichment in virtual screening experiments (5-HT(2A), 5-HT(1B), D(2), 5-HT(2C), D(3), and M(1)). The 5-HT(2A) receptor displayed the highest enrichment in virtual screening experiments with enrichment factors of 6.1, 6.9, and 5.9 at 2, 5, and 10%, respectively, of the screened database. However, three of the models require further refinement (5-HT(2B), D(4), and H(1)), due to difficulties in modeling some of the binding site residues as well as the extracellular loop 2. Our effort also aims to supplement the limited number of tested G protein-coupled receptor homology models based on the beta(2) crystal structure that are freely available to the research community.

Antiviral agents 2. Synthesis of trimeric naphthoquinone analogues of conocurvone and their antiviral evaluation against HIV.

The synthesis of a new series of conocurvone analogues is presented that explores the importance of the pyran rings of conocurvone, their degree of unsaturation as well as the role of alkoxy functionalities as pyran ring replacements, for the inhibition of the HIV-1 integrase (IN) enzyme. Difficulties in synthesising a trimeric naphthoquinone where the central quinone bears a peri-dihydropyran ring was attributed to distortion of the electrophilic dihaloquinone successfully utilised in the past. Increased electron density could also be a factor in reducing reactivity. The desired central dihydropyran bearing trimeric naphthoquinone was successfully synthesised by using a more reactive bromo-tosyloxyquinone intermediate. A maleimide derivative, where the central quinone between the pendant hydroxyquinones was replaced, was successfully synthesised and although it exhibited comparable enzyme inhibitory activity it had negligible HIV inhibitory cellular activity. Compounds were assessed for activity in both in vitro assays using purified recombinant HIV-1 IN and demonstrated superior or comparable activity to conocurvone derivatives previously reported.

A Review of the Important Role of CYP2D6 in Pharmacogenomics.

Cytochrome P450 2D6 (CYP2D6) is a critical pharmacogene involved in the metabolism of ~20% of commonly used drugs across a broad spectrum of medical disciplines including psychiatry, pain management, oncology and cardiology. Nevertheless, CYP2D6 is highly polymorphic with single-nucleotide polymorphisms, small insertions/deletions and larger structural variants including multiplications, deletions, tandem arrangements, and hybridisations with non-functional CYP2D7 pseudogenes. The frequency of these variants differs across populations, and they significantly influence the drug-metabolising enzymatic function of CYP2D6. Importantly, altered CYP2D6 function has been associated with both adverse drug reactions and reduced drug efficacy, and there is growing recognition of the clinical and economic burdens associated with suboptimal drug utilisation. To date, pharmacogenomic clinical guidelines for at least 48 CYP2D6-substrate drugs have been developed by prominent pharmacogenomics societies, which contain therapeutic recommendations based on CYP2D6-predicted categories of metaboliser phenotype. Novel algorithms to interpret CYP2D6 function from sequencing data that consider structural variants, and machine learning approaches to characterise the functional impact of novel variants, are being developed. However, CYP2D6 genotyping is yet to be implemented broadly into clinical practice, and so further effort and initiatives are required to overcome the implementation challenges and deliver the potential benefits to the bedside.

2,2,7-Trichloro-3,4-dihydro-naphthalen-1(2H)-one.

The title compound, C(10)H(7)Cl(3)O, obtained as a major byproduct from a classical Schmidt reaction. The cyclohexyl ring is distorted from a classical chair conformation, as observed for monocyclic analogues, presumably due to conjugation of the planar annulated benzo ring and the ketone group (r.m.s. deviation 0.024 Å). There are no significant intermolecular interactions.

Di-tert-butyl N-[2,6-bis-(methoxy-meth-oxy)phen-yl]imino-diacetate.

The title mol-ecule, C(20)H(31)NO(8), has pseudo-C2 symmetry about the C-N bond, with the bis-(tert-butoxy-carbon-yl)amino group twisted from the benzene ring plane by ca 60° and the bulky tert-butoxy-carbonyl (Boc) groups are orientated away from the substituted aniline group. As part of an anti-bacterial drug discovery programme furnishing analogues of platensimycin, we unexpectedly synthesized the bis-Boc-protected aniline.

8-Chloro-5-(4-phenethylpiperazin-1--yl)pyrido[2,3-b][1,5]benzoxazepine.

As part of an anti-psychotic drug discovery program, we report the crystal structure of the title compound, C(24)H(23)ClN(4)O. The mol-ecule has a tricyclic framework with a characteristic buckled V-shaped pyridobenzoxazepine unit, with the central seven-membered heterocycle in a boat configuration. The piperazine ring displays a chair conformation with the 2-phenyl-ethyl substituent assuming an equatorial orientation. There are two crystallographically independent, but virtually identical, mol-ecules in the asymmetric unit.

Synthesis and pharmacological evaluation of M4 muscarinic receptor positive allosteric modulators derived from VU10004.

The M4 mAChR is implicated in several CNS disorders and possesses an allosteric binding site for which ligands modulating the affinity and/or efficacy of ACh may be exploited for selective receptor targeting. We report the synthesis of a focused library of putative M4 PAMs derived from VU10004. These compounds investigate the pharmacological effects of target thieno[2,3-b]pyridines assembled from primary cycloalkanamines and cyclic secondary amines providing useful estimates of affinity (KB), cooperativity (αß), and direct agonist properties (τB).

Movement quality and links to measures of fitness in firefighters.

BACKGROUND/OBJECTIVE: Given the possible links between movement, fitness and injury, the goal of this study was to evaluate strength, endurance, and hip range of motion (ROM) (i.e. fitness); movement quality; and age in a population of firefighters. PARTICIPANTS: Male firefighters (n=282), all members of a major Canadian city fire department, volunteered to participate in this cross-sectional study. METHODS: Torso endurance, grip strength, pull-ups, hip ROM, movement quality (sum of 7 movement tasks graded on a 0-3 scale), age, body mass, height, body mass index, and hip and waist circumference were assessed in 282 Firefighters. Relationships between variables were evaluated and compared to relevant populations (i.e. police officers, other firefighters, athletes, students and healthy males). RESULTS: There was no relationship between age and fitness or movement quality. Compared to other populations, our firefighter population had poorer torso endurance but similar grip strength. Larger body mass, waist circumference and hip width were correlated (p< 0.01) with poorer Total Movement Score. Back extensor endurance (Biering-Sorensen test) was found to have the strongest relationship with Total Movement Score in comparison to all other tests. Notably, the extensor endurance and pull-ups were related to five of nine tasks each. The magnitude of hip ROM asymmetry related only to the stand-sit-stand movement test. In general, correlations between variables were relatively low, suggesting that most of these variables are independent and/or unrelated to one another. CONCLUSIONS: Movement quality is not strongly correlated with traditionally utilized markers of fitness, nor is movement linked to age. This would imply that training should include movement competency components together with traditional fitness objectives.

Probing structural requirements of positive allosteric modulators of the M4 muscarinic receptor.

The M4 mAChR is implicated in several CNS disorders and possesses an allosteric binding site for which ligands modulating the affinity and/or efficacy of ACh may be exploited for selective receptor targeting. We report the synthesis of a focused library of putative M4 PAMs derived from VU0152100 and VU10005. These compounds investigate the pharmacological effects of previously identified methoxy and fluoro substituents, providing useful estimates of affinity (KB), cooperativity (αß), and direct agonist properties (τB).

Homobivalent ligands of the atypical antipsychotic clozapine: design, synthesis, and pharmacological evaluation.

To date all typical and atypical antipsychotics target the dopamine D(2) receptor. Clozapine represents the best-characterized atypical antipsychotic, although it displays only moderate (submicromolar) affinity for the dopamine D(2) receptor. Herein, we present the design, synthesis, and pharmacological evaluation of three series of homobivalent ligands of clozapine, differing in the length and nature of the spacer and the point of attachment to the pharmacophore. Attachment of the spacer at the N4' position of clozapine yielded a series of homobivalent ligands that displayed spacer-length-dependent gains in affinity and activity for the dopamine D(2) receptor. The 16 and 18 atom spacer bivalent ligands were the highlight compounds, displaying marked low nanomolar receptor binding affinity (1.41 and 1.35 nM, respectively) and functional activity (23 and 44 nM), which correspond to significant gains in affinity (75- and 79-fold) and activity (9- and 5-fold) relative to the original pharmacophore, clozapine. As such these ligands represent useful tools with which to investigate dopamine receptor dimerization and the atypical nature of clozapine.

A three-dimensional model of the human immunodeficiency virus type 1 integration complex.

While the general features of HIV-1 integrase function are understood, there is still uncertainty about the composition of the integration complex and how integrase interacts with viral and host DNA. We propose an improved model of the integration complex based on current experimental evidence including a comparison with the homologous Tn5 transposase containing bound DNA and an analysis of DNA binding sites using Goodford's GRID. Our model comprises a pair of integrase dimers, two strands of DNA to represent the viral DNA ends and a strand of bent DNA representing the host chromosome. In our model, the terminal four base pairs of each of the viral DNA strands interact with the integrase dimer providing the active site, while bases one turn away interact with a flexible loop (residues 186-194) on the second integrase dimer. We propose that residues E152, Q148 and K156 are involved in the specific recognition of the conserved CA dinucleotide and that the active site mobile loop (residues 140-149) stabilises the integration complex by acting as a barrier to separate the two viral DNA ends. In addition, the residues responsible for DNA binding in our model show a high level of amino acid conservation.