Pharmazie
Refine
Year of publication
- 2007 (5) (remove)
Document Type
- Doctoral Thesis (3)
- Article (2)
Language
- English (5) (remove)
Has Fulltext
- yes (5)
Is part of the Bibliography
- no (5)
Keywords
- cyclooxygenase (1)
- hyperalgesia (1)
- mPGES-1 (1)
- pain (1)
- prostaglandin (1)
- spinal cord (1)
Institute
Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice
(2007)
Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice.
G protein-coupled receptors (GPCRs) constitute an important class of integral membrane proteins that are involved in several signaling pathways. About 50% of the currently available drugs are targeted against these receptors and high-resolution structures of these receptors will be of immense importance from the perspective of designing specific and potent drugs. However, structure determination of these receptors and of membrane proteins in general, has been a very challenging task till date. A major limitation in the structure determination of these proteins is that they are present in minute amounts in the native tissues and therefore, they must be produced heterologously. Additionally, crystallization of GPCRs is difficult owing to their flexible nature and limited hydrophilic surface area available for crystal contacts. The aim of my Ph.D. thesis work is two fold, first, to address the problem of GPCR crystallization by using a fusion protein complex approach and second, to tailor Rhodobacter sphaeroides as an expression system for the heterologous production of GPCRs. In the first approach, R. sphaeroides was used as an expression system to generate a fusion protein complex of the photosynthetic reaction center (RC) with a GPCR, expecting that such a complex would be easier to crystallize than the receptor alone. The notion behind this approach is that the RC will act as a scaffold in providing surface area to create crystal contacts and at the same time, it will also reduce the flexibility of the receptor, hopefully without perturbing the functionality of the receptor. Based on the computational modelling experiments, two ways to generate a fusion complex were assigned. Long linkers were inserted between the subunits of the RC and the GPCR. The linkers were designed with a possibility of straightforward alteration of their length as they contained a number of restriction enzyme sites. A series of these constructs were designed and expressed in R. sphaeroides deletion strain, which did not possess the chromosomal RC genes. Though most of these fusion constructs could be successfully expressed, as analyzed by western blot, majority of them were not functional in terms of ligand binding of the GPCR component of the fusion complex. Interestingly, one of these constructs, where the M subunit of RC was directly fused to the human angiotensin II type 1a receptor (AT1aR), exhibited significant functional expression. Based on saturation binding analysis using [125I] iodotyrosyl4Sar1Ile8-angiotensin II (an AT1aR subtype specific antagonist), an expression level of 40+5 pmol/mg of total membrane protein was calculated. This expression level corresponds to approximately 0.3 mg of functional receptor per liter culture and it is significantly higher than the AT1aR expression in native tissues. Additionally, the binding affinity of the recombinant receptor for its endogenous ligand angiotensin II was found to be 1±0.1 nM, which is similar to that observed for the AT1aR in native tissues. More interestingly, the RC part of the fusion complex was structurally assembled in other words, properly folded as judged by the presence of the characteristic peaks at 760 nm, 800 nm and 850 nm by absorption spectroscopy. However, a slight change in the intensity of the peak at 800 nm was observed while comparing the spectra of native RC with that in the fusion protein complex. This slight variation might be due to the change in the protein environment. The fusion protein complex RC-AT1aR was functionally solubilized and purified using a decahistidine tag fused at the c-terminus of the AT1aR. Subsequently, the monodispersity and integrity of the complex was confirmed by size exclusion chromatography, which revealed a homogeneous peak. Additionally, it was also possible to solubilize and purify this complex in the presence of a fluorescein tagged angiotensin II ligand which provides a nice tool to judge the functionality of the AT1aR and integrity of the complex at the same time. The purified RC-AT1aR fusion complex was then subjected to three-dimensional (3-D) crystallization trials and it was possible to obtain reproducible crystals of this complex. The crystals were fluorescent (as the complex was purified in presence of fluorescently labelled angiotensin II) and needle or tetragonal in shape, but produced a powdery diffraction pattern. Further attempts to improve the crystallization condition and to optimize the cryo-conditions are underway. In addition, attempts are also being made to obtain the crystals of this complex with the antagonist (e.g. losartan) bound to the receptor. In view of several limitations in the heterologous expression of GPCRs, as the second part of my Ph.D. thesis, I decided to explore the possibilities of developing a novel expression system based on R. sphaeroides for production of recombinant GPCRs. The notion behind using this host is that lack of inclusion bodies and high concentration of membranes in R. sphaeroides would result in efficient functional overexpression of recombinant membrane proteins. For this purpose, a R. sphaeroides strain, modified by the deletion of the genes encoding the RC and the light harvesting proteins LH1 and LH2, was used. The genes for RC and LHs constitute about 85-90% of total membrane proteins in a R. sphaeroides cell. These membranes are normally housed in special membrane vesicles called intracytoplasmic membranes (ICMs) that can fill almost the entire cell volume under certain growth conditions. Synthesis of a heterologous protein under the control of the moderately strong photosynthetic superoperonic promoter should be coordinated with the synthesis of new membranes to harbour these proteins, thus acting as a natural induction system. Moreover, as most of the native membrane proteins are absent in this deletion strain, heterologously produced protein should not experience a shortage of molecular chaperones for proper folding and insertion. Additionally, the absence of inclusion bodies in this host should enhance the functional and homogenous population of the recombinant proteins. Three human GPCRs, namely the adenosine A2a receptor (A2a), the angiotensin II type 1a receptor (AT1aR) and the bradykinin subtype 2 receptor (B2R) were tested for expression and functionality in this system. Two different constructs were used to determine the optimal position and ribosome-binding site (RBS) in the superoperon for the highest expression level. Of these three receptors, the AT1aR and B2R were successfully produced, while the A2aR failed to express, producing green carotenoid free R. sphaeroides mutants, for unknown reasons. For the recombinant B2R, [3H] bradykinin binding analysis revealed a low functional expression level of 0.7-0.8 pmol/mg of total membrane protein. This expression level corresponds to 0.01 mg functional receptor per liter of culture and is not sufficient for large-scale expression of this receptor. However, for the recombinant AT1aR, [125I] iodotyrosyl4Sar1Ile8- angiotensin II binding analysis revealed an expression level of 12±1 pmol/mg of total membrane protein. This expression level corresponds to approximately 0.1 mg functional receptor per liter culture and this is significantly higher than the AT1aR expression in native tissues. This expression system is still in the nascent stages of development and there are several parameters, which are still to be assessed for the optimal use of this system for the production of GPCRs and other membrane proteins. In conclusion, my Ph.D. work presents a novel fusion protein complex based approach for obtaining crystallizable GPCRs and a novel expression system for producing heterologous GPCRs. It was possible, for the first time, to produce a functional RC-GPCR complex that could easily be crystallized, though further finetuning of the system is required. R. sphaeroides based novel expression system was successfully used to produce functional human GPCRs under the control of a moderately strong photosynthetic superoperonic promoter. This expression system represents a naturally induced system where the expression of a heterologous protein is coordinated with the synthesis of new membranes to harbour the recombinant protein. The fusion protein complex approach and the expression system presented here can hopefully be used as a general method to facilitate the expression and crystallization of other membrane proteins.
The experience of pain is mediated by a specialized sensory system, the nociceptive system. There is considerable evidence that the cGMP/cGMP kinase I (cGKI) signaling pathway modulates the nociceptive processing within the spinal cord. However, downstream targets of cGKI in this context have not been identified to date. In this study we investigated whether cysteine-rich protein 2 (CRP2) is a downstream effector of cGKI in the spinal cord and is involved in nociceptive processing. Immunohistochemistry of the mouse spinal cord revealed that CRP2 is expressed in superficial laminae of the dorsal horn. CRP2 is colocalized with cGKI and with markers of primary afferent C fibers. Importantly, the majority of CRP2 mRNA-positive dorsal root ganglion (DRG) neurons express cGKI and CRP2 is phosphorylated in a cGMP-dependent manner. To elucidate the functional role of CRP2 in nociception, we investigated the nociceptive behavior of CRP2-deficient (CRP2-/-) mice. Touch perception and acute thermal nociception were unaltered in CRP2-/- mice. However, CRP2-/- mice showed an increased nociceptive behavior in models of persistent pain as compared to wild type mice. Intrathecal administration of cGKI activating cGMP analogs increased the nociceptive behavior in wild type but not in CRP2-/- mice, indicating that the presence of CRP2 was essential for cGMP/cGKI-mediated nociception. These data indicate that CRP2 is a new downstream effector of cGKI-mediated spinal nociceptive processing and point to an inhibitory role of CRP2 in the generation of inflammatory pain.
The goal of this thesis was to gain further insight into the binding behavior of ligands in the heptahelical domain (HD) of group I metabotropic glutamate receptors (mGluRs). This was realized by the establishment of strategies for the detection and optimization of molecules acting as non-competitive antagonists of group I mGluRs (mGluR1/5). These strategies should guarantee high diversity in the retrieved chemotypes of the detected compounds not resembling original reference molecules (“scaffold-hopping”). The detection of new scaffolds, in turn, was divided into two approaches: First the development of pharmacological assays to screen compounds at a certain target for bioactivity (here: affinity towards the allosteric recognition site of mGluR1 and mGluR5), and second the evaluation of computer assisted methods for the identification of virtual hits to be screened afterwards on the pharmacological assays established before. Promising molecules should be optimized with respect to activity/affinity and selectivity, their binding mode investigated and, finally, compared to existing lead compounds. Initially, membrane based binding assays for the HD of mGlu1 and mGlu5 receptors with enhanced throughput (shifting from 24-well plates to 96-well plates) were set up. For the mGluR1 assay the potent antagonist EMQMCM exhibited high affinity towards the binding site (Ki ~3nM), which is in accordance with published data from Mabire et al. (functional IC50 3nM). For mGluR5 the reference antagonist MPEP binds with high affinity to the receptor (binding IC50 13.8nM), which confirmed earlier findings from Anderson et al. (binding IC50 15nM). In another series of experiments the properties of rat cerebellar (mGluR1) and corticalmembranes (mGluR5) as well as of radiotracers were investigated by means of binding saturation studies and kinetic experiments. Furthermore, the influence of the solvent DMSO, necessary for compound screening of lipophilic substances, on positive and negative controls was evaluated. As the precise architecture of the HD of mGluR1 is still not known our efforts in identifying new ligands for this receptor focused on the ligand-based approach. All computer assisted methods that were applied to virtually screen large compound collections and to retrieve potential hits (“activity-enriched subsets”) acting at the heptahelical domain of mGluR1 relied on the existence of a valid dataset of reference molecules. This was realized by an initial compilation of a mGluR reference data collection comprising in total 357 entries predominantly negative but also some positive allosteric modulators for mGluR1 and mGluR5. In the next step a pharmacophore model for non-competitive mGluR1 antagonists was constructed. It was based upon six selective, potent and structurally diverse ligands. Prospective virtual screening was performed using the CATS atom-pair descriptor. The Asinex Gold-Collection was screened for each seed compound and some of the most similar compounds (according to the CATS descriptor) were ordered and tested forbinding affinity and functional activity at mGluR1. A high hit rate of approximately 26% (IC50 < 15 micro M) was yielded confirming the applicability of this method. One compound exerted functional activity below one micro molar (IC50-value of C-07:362nM ± 0.03). Moreover, non-linear principal component analysis was employed. Again the Asinex vendor database served as test database and was filtered by the pharmacophore model for mGluR1 established before. Test molecules that were adjacently located with mGluR1 antagonist references were selected. 15 compounds were tested on mGluR1 in binding and functional assays and three of them exhibited functional activity (IC50) below 15 micro M. The most potent molecule P-06 revealed an IC50-value of 1.11 micro M (± 0.41). The COBRA database comprising 5,376 structurally diverse bioactive molecules affecting various targets was encoded with the CATS descriptor and used for training two selforganizing maps (SOM). The encoded mGluR reference data collection was projected onto this map according to the SOM algorithm. This projection allowed to clearly distinguish between antagonists of mGluR1 and mGluR5 subtype. 28 compounds were ordered and tested on activity and affinity for mGluR1. They exhibited functional activity down to the sub-micro molar range (IC50-value of S-08: 744nM ± 0.29) yielding a final hit rate of 46% (<15 micro M). Then, the Asinex collection was screened using the SOM approach. For a predicted target panel including the muscarinic mACh (M1) receptor, the histamine H1-receptor and the dopamine D2/D3 receptors, the tested mGluR ligands exhibited the calculated binding pattern. This virtual screening concept might provide a basis for early recognition of potential sideeffects in lead discovery. We superimposed a set of 39 quinoline derivatives as non-competitive mGluR1 antagonists that were recently published by Mabire and co-workers. A CoMFA model (QSAR) was established and the influence of several side chains on functional activity was investigated. The coumarine derivative C-07 was obtained as a result of similarity searching. Starting from this compound a series of chemical derivatives was synthesized. This led to the discovery of potent (B-28, IC50: 58nM ± 0.008; Ki: 293nM ± 0.022) and selective (rmGluR5 IC50: 28.6 micro M) mGluR1 antagonists. From a homology model of mGluR1 we derived a potential binding mode for coumarines within the allosteric transmembrane region. Potential interacting patterns with amino acids were proposed considering the difference of the binding pockets between rat and human receptors. The proposed binding modes for quinolines (here:EMQMCM) and coumarines (here:B-04) were compared and discussed considering in particular the influence on activity of several side chains of quinolines obtained from the QSAR studies. The present studies demonstrated the applicability of ligand-based virtual screening for non-competitive antagonists of a G-protein coupled receptor, resulting in novel, potent and selective agents.
The development of novel drugs targeting GPCRs is of particular interest since modulation of subfamilies of this receptor class highly influences neurotransmission in the central nervous system. This study has focused on the development of ligands for the dopamine D3 receptor. The receptor belongs to the dopamine D2-like family among the biogenic amine binding GPCRs. The dopamine D3 receptor is involved in neurological and neuropsychiatric disorders such as Parkinson’s disease, schizophrenia and drug addiction. Due to its close structural similarity to the dopamine D2 receptor subtype, it is still a challenge to identify and further optimize new leads. Therefore an in vitro screening assay, which also allows elucidating comprehensive structure-affinity relationships, is required. In this investigation the implementation and evaluation of radioligand binding assays for human dopamine D2S and dopamine D3 receptors and for the related aminergic human histamine H1 receptor stably expressed in Chinese hamster ovary (CHO) cells has been performed. Saturation binding experiments with [³H]spiperone at dopamine D2S and D3 receptors and with [³H]mepyramine at histamine H1 receptors were carried out. The determined equilibrium dissociation constant of radioligands (Kd) and the total number of specific binding sites (Bmax) of the receptor membrane preparations were in good agreement with reference data. Inhibition constants (Ki) of reference ligands obtained in radioligand competition binding experiments at dopamine hD2S, hD3 and histamine H1 receptors validated the reliability and reproducibility of the assay. In order to discriminate agonists from antagonists, a GTP shift assay has been investigated for dopamine D2S and D3 receptors. In competition binding studies at dopamine D2S receptors the high- and low affinity state in the absence of the GTP analogue Gpp(NH)p has been recognized for the agonists pramipexole and the seleno analogue 54. In the presence of Gpp(NH)p a decrease in affinity, referred to as “GTP shift”, has been revealed for agonists at dopamine D2S and D3 receptors. An effect of Gpp(NH)p on dopamine D2S receptor binding has not been observed for the antagonists ST 198 and BP 897, while a reverse “GTP shift” has been noticed at the dopamine D3 receptor. For the development of novel ligands with high affinity and selectivity for dopamine D3 receptors, investigation in refined structure-affinity relationships (SAR) of analogues of the lead BP 897 has been performed. Replacement of the naphthalen-2-carboxamide of BP 897 by aryl amide residues (1 - 4) had a clear influence on affinity binding and selectivity for dopamine D3 receptors. Introduction of the benzo[b]thiophen-2-carboxamide (1) has markedly improved binding with subnanomolar affinity and enhanced selectivity for dopamine D3 receptors. Exchanging the aryl substituted basic alkanamine residue of 1 by a 1,2,3,4-tetrahydroisoquinoline moiety (6) emphasized the benefit of the 4-(2-methoxyphenyl) piperazine residue of BP 897 regarding dopamine D2 and D3 receptor affinities. The change of particular elements of BP 897 and the rearrangement of the amide functionality resulted in inverse amide compounds with new chemical properties. Moderate affinity binding data, as obtained for the isoindol-1-carbonyl compound 11, suggest that inverse amides provide a worthwhile new lead structure with a novel structural scaffold. A hybrid approach combining privileged scaffolds of histamine H1 receptor antagonists and fragments of dopamine D3 receptor-preferring ligands, related to BP 897and analogues has been investigated. Various benzhydrylpiperazine derivatives and related structures have shown moderate to high affinities for dopamine D3 receptors with the impressive enhancement of the cinnamide substituted bamipine-related hybrid 39, exhibiting the highest affinity and selectivity for dopamine D3 receptors. Improved affinity profiles of structural modified histamine H1 receptor antagonists for dopamine D2 and D3 receptors and a refined SAR has been achieved. A SAR of derivatives of the dopamine agonist pramipexole and the related etrabamine has been studied. The propargyl substituted etrabamine derivative 61 demonstrated highest affinity and selectivity. The ligand attracts attention since neuroprotective properties have been reported for the propargyl functionality. Further development resulted in the most promising compound 64, a cinnamide derivative with 4-fluoro substitution on the phenyl ring. Subnanomolar affinity and remarkable selectivity for dopamine D3 receptors has aroused particular interest in this ligand due to its development potential as a radioligand for PET studies. Radioligand binding studies in combination with virtual screening and different classification techniques of chemoinformatic methods resulted in further elucidation of SAR. New leads with novel chemical scaffolds have been found in the bicycle[2.2.1]heptane derivative 95 and the benzhydrylidene substituted pyrrolidindione 112 and can be further optimized by chemical modifications. The outcome of the studies provides the development of various novel high affine and dopamine D3 receptor selective ligands. Modifications of lead structures or application of chemoinformatic tools in combination with radioligand competition binding assays have resulted in new leads with different chemical scaffolds. Furthermore, a comprehensive insight into structure-affinity relationships of ligands at dopamine D3 receptors has been revealed. This refined SAR is valuable to develop more affine and selective drug candidates with a designed pharmacological receptor profile.