Synthesis and evaluation of N1-substituted-3-propyl-1,4-benzodiazepine-2-ones as cholecystokinin (CCK2) receptor ligands

A novel synthetic approach towards N1-alkylated 3-propyl-1,4-benzodiazepines was developed in five synthetic steps from 2-amino-4-chlorobenzophenone, in which the N-oxide 4 served as a key intermediate. The structure-activity relationship optimization of this 3-prophyl-1,4-benzodiazepine template was carried out on the N1-position by selective alkylation reactions and resulted in a ligand with an improved affinity on the cholecystokinin (CCK2) receptor. The N-allyl-3-propyl-benzodiazepine 6d displayed an affinity towards the CCK2 (CCK-B) receptor of 170 nM in a radiolabelled receptor-binding assay. The anxiolytic activity of this allyl-3-propyl-1,4-benzodiazepine 6d was subsequently determined in in-vivo psychotropic assays. This novel ligand had ED50 values of 4.7 and 5.2 mg kg-1 in the black and white box test and the x-maze, respectively, and no significant sedation/muscle relaxation was observed.

Synthesis and evaluation of 5-arylated 2(5H)-furanones and 2-arylated pyridazin-3(2H)-ones as anti-cancer agents

Bis-cyclic butenolides, 5-arylated 2(5H)-furanones 6a-c, 7a, b and the 3(2H)-pyridazones 9a-d were prepared by using the aldehyde form of muco halogen acids in electrophilic substitution reactions and in an aldol-like condensation reaction. The cytotoxicity of these simple and bis-cyclic butenolides have been evaluated in tissue culture studies on MAC 13 and MAC 16 murine colon cancer cell lines. The butyl furanone 3 displayed the highest cytotoxicity of 3 μM, as one selected example of a series of dichlorinated pseudoesters. The 5-arylated 2(5H)-furanones 6 and 7 did not show a structure-activity relationship (SAR) depending on the substitution pattern of the aromatic system. An IC50 (concentration inhibiting growth by 50%) was found within a range of 30-50 and 40-50 μM for the MAC 13 and MAC 16 cell lines, respectively. The pyridazine series 9 showed a maximum in-vitro activity for the p-methoxydrivative 9b, having an IC50 of 17 in MAC 13 and 11 μM in MAC 16 cell lines. Selected examples of each series and further novel 2(5H)-furanones such as the hydrazone 5 and the hydantoin 8 have been screened in-vivo in mice and the data are presented. For the pyridazines 9a-d, the in-vitro cytotoxicity correlated with an in-vivo inhibition of tumour growth. The ring expansion of the 5-membered 2(5H)-furanone ring system such as 6a into the 6-membered 3(2H)-pyridazone 9b led to an agent with improved antineoplastic properties. On the resistant MAC 16 cell line the pyridazone 9b displayed 52% tumour inhibition in mice at a dose of 50 mg kg-1 compared with 27% for the 5-FU standard.

Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

Elements of molecular neurobiology:instruction manual to 2r.e

Extensively updated, revised and illustrated this unique introductory text presents a molecular account of the structure, function and development of the brain and nervous systems. This book describes the latest research in neurobiology made possible by modern molecular biology techniques. The author synthesizes this new knowledge and demonstrates how an understanding at the molecular level can contribute towards a theory of the brain in health and disease.

Elements of molecular neurobiology

Extensively updated, revised and illustrated this unique introductory text presents a molecular account of the structure, function and development of the brain and nervous systems. This book describes the latest research in neurobiology made possible by modern molecular biology techniques. The author synthesizes this new knowledge and demonstrates how an understanding at the molecular level can contribute towards a theory of the brain in health and disease.

A quantitative study of the pathological changes in the cortical white matter in variant Creutzfeldt-Jakob disease (vCJD)

The objective of this study was to determine the degree of white matter pathology in the cerebral cortex in cases of variant Creutzfeldt-Jakob disease (vCJD) and to study the relationships between the white matter and grey matter pathologies. Hence, the pathological changes in cortical white matter were studied in individual gyri of the frontal, parietal, occipital, and temporal cortex in eleven cases of vCJD. Vacuolation (‘spongiform change’), deposition of the disease form of prion protein (PrPsc) in the form of discrete PrP deposits, and gliosis were observed in the white matter of virtually all cortical regions studied. Mean density of the vacuoles in the white matter was greater in the parietal lobe compared with the frontal, occipital, and temporal lobes but there were fewer glial cells in the occipital lobe compared with the other cortical regions. In the white matter of the frontal cortex, vacuole density was negatively correlated with the density of both glial cell nuclei and the PrP deposits. In addition, the densities of glial cells and PrP deposits were positively correlated in the frontal and parietal cortex. In the white matter of the frontal cortex and inferior temporal gyrus, there was a negative correlation between the densities of the vacuoles and the number of surviving neurons in laminae V/VI of the adjacent grey matter. In addition, in the frontal cortex, vacuole density in the white matter was negatively correlated with the density of the diffuse PrP deposits in laminae II/III and V/VI of the adjacent grey matter. The densities of PrP deposits in the white matter of the frontal cortex were positively correlated with the density of the diffuse PrP deposits in laminae II/III and V/V1 and with the number of surviving neurons in laminae V/V1. The data suggest that in the white matter in vCJD, gliosis is associated with the development of PrP deposits while the appearance of the vacuolation is a later development. In addition, neuronal loss and PrP deposition in the lower cortical laminae of the grey matter may be a consequence of axonal degeneration within the white matter.

Synthesis and cytotoxity of oxysterols:studies on the A,B ring polyoxygenated sterols

Oxysterols (OS), the polyoxygenated sterols, represent a class of potent regulatory molecules for important biological actions. Cytotoxicity of OS is one of the most important aspects in studies of OS bioactivities. However, studies, the structure-activity relationship (SAR) study in particular, have been hampered by the limited availability of structurally diverse OS in numbers and amounts. The aim of this project was to develop robust synthetic methods for the preparation of polyhydroxyl sterols, thereof, evaluate their cytotoxicity and establish structure-activity relationship. First, we found hydrophobicity of the side chain is essential for 7-HC's cytotoxicity, and a limited number of hydroxyl groups and a desired configuration on the A, B ring are required for a potent cytotoxicity of an OS, after syntheses and tests of a number of 7-HC's analogues against cancer cell lines. Then polyoxygenation of cholesterol A, B rings was explored. A preparative method for the synthesis of four diastereomerically pure cholest-4-en-3,6-diols was developed. Epoxidation on these cholest-4-en-3,6-diols showed that an allyl group exerts an auxiliary role in producing products with desired configuration in syntheses of the eight diastereomerically pure 45-epoxycholestane-3,6-diols. Reduction of the eight 45-epoxycholestane-3,6-diols produced all eight isomers of the cytotoxic 5α-acholestane 3β,5,6β-triol (CT) for the first time. Epoxide ring opening with protic or Lewis acids on the eight 45-epoxycholestane-3,6-diols are carefully studied. The results demonstrated a combination of an acid and a solvent affected the outcomes of a reaction dramatically. Acyl group participation and migration play an important role with numbers of substrates under certain conditions. All the eight 4,5-trans cholestane- 3,4,5,6-tetrols were synthesised through manipulation of acyl participation. Furthermore these reaction conditions were tested when a number of cholestane-3,4, 5,6,7-pentols and other C3-C7 oxygenated sterols were synthesised for the first time. Introduction of an oxygenated functional group through cholest-2-ene derivatives was studied. The elimination of 3-(4-toluenesulfonate) esters showed the interaction between the existing hydroxyls or acyls with the reaction centre often resulted in different products. The allyl oxidation, epoxidation and Epoxide ring opening reactions are investigated with these cholest-2-enes.

Investigating the modulation of oral drug absorption using in vitro models

Dipeptides can be absorbed into cells via the dipeptide transporter (which also transported tripeptides and dipeptide derivatives). The optimum conditions for measuring the inhibition of Gly-Pro uptake in Caco-2 cells were identified. A number of structure-activity relationships were identified. These included the effects of increasing the amino-acid chain-length, and the presence of a thiol or hydroxyl group in the side-chain increased IC50 while the presence of a hydroxyl group did not. The benzyl esters had lower or equal IC50 values compared to the parent dipeptides while the methyl esters had higher values. These results indicated that while molecular properties did affect IC50, the size, charge and composition of three particular groups caused the most significant effects, supporting the structure-activity relationship identified. An assay was developed using calcein-AM to show the inhibition of p-glycoprotein activity. There was no significant change due to the presence of mannitol but there was in the presence of clyclosporin A (p<0.01). Incubating the cells with the test solution for 30 minutes before the addition of the ester resulted in a significant (p<0.001) difference. The assay was specific for p-glycoprotein, as the presence MRP inhibitors had no effect (p>0.05). The modified protocol allowed the identification of p-glycoprotein inhibitors quickly and simply using a cell suspension of unmodified cells. The clinically relevant buffering of grapefruit juice to pH 7 led to a four-fold increase in intracellular calcein and hence significant inhibition of p-glycoprotein. Buffered orange and lemon juices had no effect on the assay. Flavone derivatives had previously been found to be inhibitors of CYP3A4 yet neither naringin nor naringenin had any significant effect at concentrations found in grapefruit juice. Of the other (non-grapefruit) flavone derivatives tested, hesperidin, found in orange juice, had no significant effect, kaempferol and rutin also had no effect while genistein significantly inhibited p-glycoprotein (results that support previous studies). Hydroxycinnamic acids had no effect on p-glycoprotein. Studies on other compounds found that the balance between inhibiting p-glycoprotein and disrupting cell membranes depends on the compound containing an oxygen atom and the size of the negative charge on it, as well as three-dimensional arrangement of the atoms.

Automated synthesis and evaluation of potential new anti-microbial agents

A series of N1-benzylideneheteroarylcarboxamidrazones was prepared in an automated fashion, and tested against Mycobacterium fortuitum in a rapid screen for antimycobacterial activity. Many of the compounds from this series were also tested against Mycobacterium tuberculosis, and the usefulness as M.fortuitum as a rapid, initial screen for anti-tubercular activity evaluated. Various deletions were made to the N1-benzylideneheteroarylcarboxamidrazone structure in order to establish the minimum structural requirements for activity. The N1-benzylideneheteroarylcarbox-amidrazones were then subjected to molecular modelling studies and their activities against M.fortuitum and M.tuberculosis were analysed using quantitative structure-analysis relationship (QSAR) techniques in the computational package TSAR (Oxford Molecular Ltd.). A set of equations predictive of antimycobacterial activity was hereby obtained. The series of N1-benzylidenehetero-arylcarboxamidrazones was also tested against a multidrug-resistant strain of Staphylococcus aureus (MRSA), followed by a panel of Gram-positive and Gram-negative bacteria, if activity was observed for MRSA. A set of antimycobacterial N1-benzylideneheteroarylcarboxamidrazones was hereby discovered, the best of which had MICs against m. fortuitum in the range 4-8μgml-1 and displayed 94% inhibition of M.tuberculosis at a concentration of 6.25μgml-1. The antimycobacterial activity of these compounds appeared to be specific, since the same compounds were shown to be inactive against other classes of organisms. Compounds which were found to be sufficiently active in any screen were also tested for their toxicity against human mononuclear leucocytes. Polyethylene glycol (PEG) was used as a soluble polymeric support for the synthesis of some fatty acid derivatives, containing an isoxazoline group, which may inhibit mycolic acid synthesis in mycobacteria. Both the PEG-bound products and the cleaved, isolated products themselves were tested against M.fortuitum and some low levels of antimycobacterial activity were observed, which may serve as lead compounds for further studies.

Studies on the mode of cytotoxicity of imidazotetraziones

The irnidazotetrazinones are a novel group of anti tumour agents which have demonstrated good activity against a range of murine tumours and human xenografts. They possess a structure activity relationship similar to the anti tumour triazenes, with the chloroethyl (mitozolomide) and methyl (temozolomide) analogues being active antitumour agents, whilst the ethyl (CCRG 82019) and higher homologues are inactive. This thesiS attempts to elucidate the biological mechanisms responsible for the strict structure-activity relationship observed amongst the imidazotetrazinones. Mitozolomide is the only agent chemically capable of cross-linking DNA , which has been suggested to be responsible fo r the cytotoxicity of this group of agents. Only mitozolomide and ternozolornide Exhibit a marked ditferential toxicity towards the 0 -alkylguanine-DNA alkyltransferase deficient GM892A (Mer-) cell line rather than the proficient Raji cell line (Mer+). The rate of uptake of imidazotetrazinones into cells is similar for all three agents in both cell lines, and does not explain the differing sensitivities to these agents. The effect of drug treatment on the incorporation of precursors into macromolecules, and their pool sizes, was examined. Temozolomide administration was found to alter de novo protein synthesis in both GM892A and Raji cells. Flow cytometric analysis revealed that temozolomide and CCRG 82019 block cells in late S/G2/M phase of the cell cycle , similar to that observed with mitozolomide. The extent of reaction of all three drugs with isolated macromolecules and cellular macromolecules was determined, and differences found, with cellular repair processes influencing the number of alkyl lesions remaining bound to macromolecules. The specific bases formed in calf thymus DNA after treatment with either temozolornide and CCRG 82019 was measured, and it was found that the types and relative amounts of lesions formed, differed, as well as the total level of alkylation. Whereas DNA extracted from imidazotetrazinone treated cells is not affected in its ability to support RNA polymerase activity, an effect is observed on the ability to extract DNA polymerase from drug treated cells. This may suggest that the alkylated DNA must be in intact chromatin for the lesion to manifest its effects. Temozolomide and methyl methanesulphonate do got appear to act with a synergistic mode of action. The 0 -position of guanine is suspected to be a critical site for the action of these types of drugs.

Flavones and related compounds as inhibitors of protein tyrosine kinases

Aberrant tyrosine protein kinase activity has been implicated in the formation and maintenance of malignancy and so presents a potential target for cancer chemotherapy. Quercetin, a naturally occuring flavonoid, inhibits the tyrosine protein kinase encoded by the Rous sarcoma virus but also exhibits many other effects. Analogues of this compound were synthesised by the acylation of suitable 2-hydroxyacetophenones with appropriately substituted aromatic (or alicyclic) acid chlorides, followed by base catalysed rearrangement to the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones. Acid catalysed ring closure furnished flavones. The majority of the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones were shown by NMR to exist in the enol form. This was supported by the crystal structure of 1-(2-hydroxy-4-methoxyphenyl)-3-phenylpropan-1,3-dione. In contrast, 1.(4,6-dimethoxy-2-hydroxyphenyl)-3-phenylpropan-1,3-dione did not exhibit keto-enol tautomerism in the NMR spectrum and was shown in its crystal structure to assume a twisted conformation. Assessment of the biological activity of the analogues of quercetin was carried out using whole cells and the kinase domain of the tyrosine protein kinase encoded by the Abelson murine leukaemia virus, ptab150 kinase. Single cell suspension cultures and clonogenic potential of murine fibroblasts transformed by the Abelson Murine leukaemia virus (ANN-1 cells) did not indicate the existence of any structure activity relationship required for cytotoxicity or cytostasis. No selective toxicity was apparent when the `normal' parent cell line, (3T3), was used to assess the cytotoxic potential of quercetin. The ICS50 for these compounds were generally in the region of 1-100M. The potential for these compounds to inhibit ptab150 kinase was determined. A definite substitution requirement emerged from these experiments indicating a necessity for substituents in the A ring or in the 3-position of the flavone nucleus. Kinetic data showed these inhibitors to be competitive for ATP.

The use of site-directed mutagenesis to study GPCRs

G protein coupled receptors (GPCRs) are highly flexible and dynamic proteins, which are able to interact with diverse ligands, effectors, and regulatory proteins. Site-directed mutagenesis (SDM) is a powerful tool for providing insight into how these proteins actually work, both in its own right and when used in conjunction with information provided by other techniques such as crystallography or molecular modelling. Mutagenesis has been used to identify and characterise a myriad of functionally important residues, motifs and domains within the GPCR architecture, and to identify aspects of similarity and differences between the major families of GPCRs. This chapter presents the necessary information for undertaking informative SDM of these proteins. Whilst this is relevant to protein structure/function studies in -general, specific pitfalls and protocols suited to investigating GPCRs in particular will be highlighted.

Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors:identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.

Identifying a novel functional domain within the p115 tethering factor required for Golgi ribbon assembly and membrane trafficking

The tethering factor p115 has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that RNAi-mediated depletion of p115 in C. elegans causes accumulation of the yolk protein (YP170) in body cavity and the retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes.Structure-function analyses of p115 have identified two homology (H1-2) regions within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a novel C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants lacking the fourth CC domain (CC4) act in a dominant negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi-mediated "replacement" strategy we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115.p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function and suggest that both the CC1 and the CC4 SNARE-binding motifs may participate in p115-mediated membrane tethering.

The antimicrobial action of pseudin-2

The amphibian antimicrobial peptide pseudin-2 is a peptide derived from the skin of the South-American frog Pseudis paradoxa (Olson et al., 2001). This peptide possesses tremendous potential as a therapeutic lead since it has been shown to possess both antimicrobial as well insulin-releasing properties (Olson et al., 2001; Abdel-Wahab et al., 2008). This study aimed to develop pseudin-2’s potential by understanding and improving its properties as an antimicrobial agent. The structure-function relationships of pseudin-2 were explored using a combination of in-vitro and in-silico techniques, with an aim to predict how the structure of the peptide may be altered in order to improve its efficacy. A library of pseudin-2 mutants was generated by randomizing codons at positions 10, 14 and 18 of a synthetic gene, using NNK saturation mutagenesis. Analysis of these novel peptides broadly confirmed, in line with literature precedent, that anti-microbial activity increases with increased positive charge. Specifically, 2 positively-charged residues at positions 10 and 14 and a hydrophobic at position 18 are preferred. However, substitution at position 14 with some polar, non-charged residues also created peptides with antimicrobial activity. Interestingly, the pseudin-2 analogue [10-E, 14-Q, 18-L] which is identical to pseudin-2, except that the residues at positions 10 and 14 are switched, showed no anti-microbial activity at all. Molecular dynamics simulations of pseudin-2 showed that the peptide possesses two equilibrium structures in a membrane environment: a linear and a kinked a-helix which both embed into the membrane at an angle. Biophysical characterization using circular dichroism spectroscopy confirmed that the peptide is helical within the membrane environment whilst linear dichroism established that the peptide has no defined orientation within the membrane. Collectively, these data indicate that Pseudin-2 exerts its antimicrobial activity via the carpet model.