NMR imaging of the diffusion of water at 37 degrees C into poly(2-hydroxyethyl methacrylate) containing aspirin or vitamin B-12

The ingress of water into poly(2-hydroxyethyl methacrylate), PHEMA, loaded with either one of two model drugs, vitamin B-12 or aspirin, was studied at 37 degreesC using three-dimensional NMR imaging. PHEMA was loaded with 5 and 10 wt % of the drugs. From the imaging profiles, it was observed that incorporation of vitamin B-12 into PHEMA resulted in enhanced crack formation on sorption of water and the crack healing behind the diffusion front was slower than for PHEMA without added drug. This was accounted for by the anti-plasticization of PHEMA by vitamin B-12. Crack formation was inhibited in the P-HEMA-aspirin systems because of the plasticizing effect of the aspirin on the PHEMA matrix. All of the polymers were found to absorb water according to an underlying Fickian diffusion mechanism. For PHEMA loaded with 5 wt % of aspirin or vitamin B-12, the best values of the water diffusion coefficients were both found to be 1.3 +/- 0.1 x 10(-11) m(2) s(-1) at 37 degreesC, while the values for the polymer loaded with 10 wt % of the drugs were slightly higher, 1.5 +/- 0.1 x 10(-11) m(2) s(-1).

Countering cooperative effects in protease inhibitors using constrained b-strand-mimicking templates in focused combinatorial libraries

A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of dampening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally diverse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.

Mass uptake study of the diffusion of water and SBF into poly(2-hydroxyethyl methacrylate-co-tetrahydrofurfuryI methacrylate) containing aspirin or vitamin B-12

The ingress of water and Kokubo simulated body fluid (SBF) into poly (2-hydroxyethyl methacrylate) (PHEMA), and its co-polymers with tetrahydrofurduryl methacrylate (THFMA), loaded with either one of two model drugs, vitamin 1312 or aspirin, was studied by mass uptake over the temperature range 298-318 K. The polymers were studied as cylinders and were loaded with either 5 wt% or 10 wt% of the drugs. From DSC studies it was observed that vitamin B-12 behaved as a physical cross-linker restricting chain segmental mobility, and so had a small anti-plasticisation effect on PHEMA and the co-polymers rich in HEMA, but almost no effect on the T-g of co-polymers rich in THFMA. On the other hand, aspirin exhibited a plasticising effect on PHEMA and the copolymers. All of the polymers were found to absorb water and SBF according to a Fickian diffusion mechanism. The polymers were all found to swell to a greater extent in SBF than in water, which was attributed to the presence of Tris buffer in the SBF. The sorptions of the two penetrants were found to follow Fickian kinetics in all cases and the diffusion coefficients at 310 K for SBF were found to be smaller than those for water, except for the polymers containing aspirin where the diffusion coefficients were higher than for the other systems. For example, for sorption into PHEMA the diffusion coefficient for water was 1.41 X 10(-11) m(2)/s and for SBF was 0.79 x 10-11 m(2)/s, but in the presence of 5 wt% aspirin the corresponding values were 1.27 x 10(-1)1 m(2)/s and 1.25 x 10(-11) m(2)/s, respectively. The corresponding values for PHEMA loaded with 5 wt% B-12 were 1.25 x 10(-11) m(2)/s and 0.74 x 10(-11) m(2)/s, respectively.

Family resemblances? Ligand binding and activation of family A and B G-protein-coupled receptors

In April 2007, the Biochemical Society held a meeting to compare and contrast ligand binding and activation of Family A and B GPCRs (G-protein-coupled receptors). Being the largest class, Family A GPCRs usually receive the most attention, although a previous Biochemical Society meeting has focused on Family B GPCRs. The aim of the present meeting was to bring researchers of both families together in order to identify commonalities between the two. The present article introduces the proceedings of the meeting, briefly commenting on the focus of each of the following articles. ©The Authors.

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.

Insulin release from cultured B-cells

Established RlNm5F and lN111 R1 and newly available HlT-T15 and UMR 407/3 B-cell lines have been successfully maintained in vitro. With the exclusion of UMR 407/3 cells, all lines were continuously propagable. Doubling times and plating efficiencies for HlT-T15, RlNm5F, lN111 R1 and UMR 407/3 cells were 20 hours and 85%, 31 hours and 76%, 24 hours and 80% and 38 hours and 94% respectively. All the cell lines were anchorage dependent, but only UMR 407/3 cells grew to confluence. Only HlT-T15 and UMR 407/3 cells produced a true insulin response to glucose but glucose markedly increased the rate of D-[U14C]glucose oxidation by all the cell lines. Glucose induced insulin release from HlT-T15 cells was biphasic with an exaggerated first phase. Insulin release from HlT-T15, RlNm5F and IN111 R1 cells was stimulated by amino acids and sulphonylureas. Glucagon stimulated insulin release from HlT-T15 and RlNm5F cells while somatostatin and pancreatic polypeptide inhibited release. These observations suggest that net insulin release from the whole islet may be the result of significant paracrine interaction. HlT-T15 and RlNm5F cell insulin release was stimulated by forskolin and inhibited by imidazole. Ca2+ channel blockade and calmodulin inhibition suppressed insulin release from HlT-T15, RlNm5F and IN111 R1 cells. In addition phorbol esters stimulated insulin release from RlNm5F cells. These data implicate cAMP, Ca2+ and protein kinase-C in the regulation of insulin release from cultured B-cells. Acetylcholine increased insulin release from HlT-T15 and RlNm5F cells. Inhibition of the response by atropine confirmed the involvement of muscarinic receptors. HlT-T15 cell insulin release was also inhibited by adrenaline. These observations suggest a possible role for the autonomic nervous system in the modulation of insulin release. Preliminary studies with a human insulinoma maintained in monolayer culture have demonstrated a limited life span of some seven weeks, a continuous low level of insulin release but no insulin response to glucose challenge.

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

Secretin family (Class B) G protein-coupled receptors - from molecular to clinical perspectives

Family B G protein-coupled receptors represent an important but under-researched group of receptors. This edition of the British Journal of Pharmacology considers the roles and pharmacology of a number of these receptors. Whilst common themes emerge, it is clear that more work is needed to understand the details of each receptor in order to properly exploit them therapeutically.

Calcitonin and calcitonin receptor-like receptors:common themes with family B GPCRs?

The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR.

Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg(2.39), His(2.43) and Glu(3.46), which makes a polar lock with T(6.37). These alignments and models provide useful tools for understanding class B GPCR function.

Contribuições para o processo de verificação de satisfatibilidade módulo teoria em Event-B

Event-B is a formal method for modeling and verification of discrete transition systems. Event-B development yields proof obligations that must be verified (i.e. proved valid) in order to keep the produced models consistent. Satisfiability Modulo Theory solvers are automated theorem provers used to verify the satisfiability of logic formulas considering a background theory (or combination of theories). SMT solvers not only handle large firstorder formulas, but can also generate models and proofs, as well as identify unsatisfiable subsets of hypotheses (unsat-cores). Tool support for Event-B is provided by the Rodin platform: an extensible Eclipse based IDE that combines modeling and proving features. A SMT plug-in for Rodin has been developed intending to integrate alternative, efficient verification techniques to the platform. We implemented a series of complements to the SMT solver plug-in for Rodin, namely improvements to the user interface for when proof obligations are reported as invalid by the plug-in. Additionally, we modified some of the plug-in features, such as support for proof generation and unsat-core extraction, to comply with the SMT-LIB standard for SMT solvers. We undertook tests using applicable proof obligations to demonstrate the new features. The contributions described can potentially affect productivity in a positive manner.

Dr. David Einhorn's ausgewählte Predigten und Reden

herausgegeben von Dr. K. Kohler