Search

Recently reported compounds such as UR-COP78 (6) are among the most potent and selective ABCG2 modulators known so far but are prone to rapid enzymatic cleavage at the central benzanilide moiety. In search for more stable analogues, according to a bioisosteric approach, a series of N-(biphenyl-3-yl)quinoline carboxamides was prepared by solid phase and solution phase synthesis. The biphenyl moiety was constructed by Suzuki coupling. Inhibition of ABCB1 and ABCG2 was determined in a calcein-AM and a Hoechst 33342 microplate assay, respectively. Most synthesized compounds selectively inhibited the ABCG2 transporter at submicromolar concentrations with a maximal inhibitory effect (I max) over 90% (e.g., UR-COP228 (22a), IC50 591 nM, I max 109%; UR-COP258 (31), IC50 544 nM, I max 112%), though with lower potency and selectivity than 6. The biphenyl analogues are considerably more stable and demonstrate that the benzanilide core is not a crucial structural feature of quinoline carboxamide-type ABCG2 modulators.

Radioing in on NPY: Attachment of a [2,3-(3)H]propionyl group through an appropriate linker to the guanidine group of an (S)-argininamide-type neuropeptide Y (NPY) Y(2) receptor antagonist resulted in a subtype-selective radioligand.

Histamine is a biogenic amine that exerts its biological effects as a neurotransmitter and local mediator via four histamine receptor (HR) subtypes (H(x)Rs) - H(1)R, H(2)R, H(3)R, and H(4)R - belonging to the superfamily of G-protein-coupled receptors (GPCRs). All four H(x)Rs exhibit pronounced differences in agonist and/or antagonist pharmacology among various species orthologs. The species differences constitute a problem for animal experiments and drug development. This problem applies to GPCRs with diverse ligands. Here, we summarize our current knowledge on H(x)R orthologs as a case study for species-dependent activity of GPCR ligands. We show that species-specific pharmacology also provides unique opportunities to study important aspects of GPCR pharmacology in general, including ligand-binding sites, the roles of extracellular domains in ligand binding and receptor activation, agonist-independent (constitutive) receptor activity, thermodynamics of ligand/receptor interaction, receptor-activation mechanisms, and ligand-specific receptor conformations.

The human histamine receptors hH(1)R and hH(2)R constitute important drug targets, and hH(3)R and hH(4)R have substantial potential in this area. Considering the species-specificity of pharmacology of H(x)R orthologs, it is important to analyze hH(x)Rs. Here, we summarize current knowledge of hH(x)Rs endogenously expressed in human cells and hH(x)Rs recombinantly expressed in mammalian and insect cells. We present the advantages and disadvantages of the various systems. We also discuss problems associated with the use of hH(x)R antibodies, an issue of general relevance for G-protein-coupled receptors (GPCRs). There is much greater overlap in activity of 'selective' ligands for other hH(x)Rs than the cognate receptor subtype than generally appreciated. Studies with native and recombinant systems support the concept of ligand-specific receptor conformations, encompassing agonists and antagonists. It is emerging that for characterization of hH(x)R ligands, one cannot rely on a single test system and a single parameter. Rather, multiple systems and parameters have to be studied. Although such studies are time-consuming and expensive, ultimately, they will increase drug safety and efficacy.

Based on the dopamine D(4) receptor partial agonist FAUC 3019, a series of azulenylmethylpiperazines was synthesized and affinities for the monoaminergic GPCRs including dopamine, serotonin, histamine and α-adrenergic receptor subtypes were determined. Ligand efficacies of the most promising test compounds revealed the N,N-dimethylaminomethyl substituted azulene 11 to be the most potent D(4) partial agonist (EC(50)=0.41 nM). This candidate was investigated for its ability to promote penile erection. Applying an in vivo animal model, test compound 11 turned out to stimulate penile erection in male rats with superior potency in low concentrations when compared to apomorphine.

The diastereomeric mixture of d/l-2,7-diaminooctanedioyl-bis(YRLRY-NH2) (BVD-74D, 2) was described in the literature as a high affinity Y4 receptor agonist. Here we report on the synthesis and pharmacological characterization of the pure diastereomers (2R,7R)- and (2S,7S)-2 and a series of homo- and heterodimeric analogues in which octanedioic acid was used as an achiral linker. To investigate the role of the Arg residues, one or two arginines were replaced by Ala. Moreover, N(ω)-(6-aminohexylaminocarbonyl)Arg was introduced as an arginine replacement (17). (2R,7R)-2 was superior to (2S,7S)-2 in binding and functional cellular assays and equipotent with 17. [(3)H]Propionylation of one amino group in the linker of (2R,7R)-2 or at the primary amino group in 17 resulted in high affinity Y4R radioligands ([(3)H]-(2R,7R)-10, [(3)H]18) with subnanomolar Kd values.

Label-free cell-based assays have been attracting growing attention in drug research. Optical approaches based on evanescent electric fields (e.g. EPIC, RWG/DMR, SPR) and electrochemical impedance analysis (ECIS, xCELLigence) are by far the most widespread techniques for such purposes. We compared three label-free approaches (ECIS, RWG/DMR and SPR) with respect to the activation of the human histamine H1 receptor (H1R) expressed by U-373 MG glioblastoma and genetically engineered HEK 293T cells. HEK 293T cells were either expressing the hH1R alone or in combination with the adhesion protein hMSR1. The β2-adrenergic receptor (β2-AR) expressed by bovine aortic endothelial cells (BAEC) served as a second cell model. Reduced cell adhesion to the surface of the sensing devices affected both, the optical and the impedance-based readout, but became much more obvious in case of RWG- or SPR-based assays. By contrast, the co-expression of hH1R and hMSR1 in HEK 293T cells strongly enhanced the signal compared to hH1R expression alone. As the sensitivity of the optical readouts is confined to a distance of 100-200nm from the surface, depending on the wavelength of the incident light, this observation is in accordance with tighter adhesion of the co-transfectants, inducing a shorter distance between the cell membrane and the substrate. Combining ECIS and SPR, allowing for simultaneous registration of both signals for a single cell population, provided a direct correlation of both readouts, when H1R or β2-AR stimulation was investigated for the same cell populations. Cell adhesion was found to have a critical impact on the results of label-free cell monitoring, in particular when techniques based on evanescent electric fields are applied.

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

Eosinophils play a crucial role in the pathogenesis of allergic diseases. Histamine activates eosinophils via the H(4)-receptor (H(4)R). However, pharmacological analysis of the H(4)R in eosinophils is still incomplete, and cell purity is a problem. The H(4)R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) has recently been reported to exhibit paradoxical stimulatory effects in some systems. Therefore, the first aim of our study was to pharmacologically re-examine H(x)R subtypes on human eosinophils using a highly purified preparation (97±2%). The second aim was to compare the effects of histamine with those induced by well-known activators of eosinophil functions, i.e. eotaxin-1 and formyl peptides. Histamine and the H(4)R-selective agonist 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine (UR-PI376) increased intracellular calcium concentration ([Ca(2+)](i)) and activated chemotaxis. JNJ7777120 per se exhibited no stimulatory effects but inhibited stimulation by histamine and UR-PI376. Blockade of the H(2)R by famotidine enhanced histamine-induced chemotaxis but not rises in [Ca(2+)](i). Compared to eotaxin and formyl peptides, the effect of histamine on eosinophil chemotaxis was only small. Formyl peptides but not histamine activated reactive oxygen species formation and release of eosinophil peroxidase. In conclusion, histamine is only an incomplete eosinophil activator with the H(2)R blunting the small chemotactic response to H(4)R activation. We also noted several differences in potencies of histamine, UR-PI376 and JNJ7777120 in calcium and chemotaxis assays and when compared to results in the literature. This indicates functional selectivity of H(4)R ligands, thus ligand-specific stabilization of distinct receptor conformations, inducing distinct biological responses.

Bivalent histamine H(2) receptor (H(2)R) agonists were synthesized by connecting pharmacophoric 3-(2-amino-4-methylthiazol-5-yl)-, 3-(2-aminothiazol-5-yl)-, 3-(imidazol-4-yl)-, or 3-(1,2,4-triazol-5-yl)propylguanidine moieties by N(G)-acylation with alkanedioic acids of various chain lengths. The compounds were investigated for H(2)R agonism in GTPase and [(35)S]GTPγS binding assays at guinea pig (gp) and human (h) H(2)R-Gsα(S) fusion proteins including various H(2)R mutants, at the isolated gp right atrium, and in GTPase assays for activity on recombinant H(1), H(3), and H(4) receptors. The bivalent ligands are H(2)R partial or full agonists, up to 2 orders of magnitude more potent than monovalent acylguanidines and, with octanedioyl or decanedioyl spacers, up to 4000 times more potent than histamine at the gpH(2)R. In contrast to their imidazole analogues, the aminothiazoles are highly selective for H(2)R vs other HR subtypes. Compounds with (theoretically) sufficient spacer length (20 CH(2) groups) to simultaneously occupy two orthosteric binding sites in H(2)R dimers are nearly inactive, whereas the highest potency resides in compounds with considerably shorter spacers. Thus, there is no evidence for interaction with H(2)R dimers. The high agonistic potency may result from interaction with an accessory binding site at the same receptor protomer.

The histamine H(4) receptor subtype (H(4)R) belongs to the class 1 of G protein-coupled receptors and is involved in inflammatory and immunological processes. The aim of this study was to elucidate the importance of extracellular regions for the large species differences between human (h) and canine (c) H(4)R. Therefore, chimeric receptors were generated by replacing corresponding domains of the hH(4)R with canine N-terminus (h(cNT)H(4)R) and three canine extracellular loops, respectively (h(cE1)H(4)R, h(cE2)H(4)R and h(cE3)H(4)R). Wild type and chimeric H(4) receptors were expressed in Sf9 insect cells and subsequently characterized in [(3)H]histamine-binding experiments and in steady-state GTPase activity assays, where standard H(4)R ligands histamine, 5-methylhistamine, thioperamide, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) and clozapine were examined. The exchange of N-terminus or first extracellular loop did not influence hH(4)R pharmacology. The effect of altered second extracellular loop (E2-loop) and third extracellular loop (E3-loop) was rather ligand specific than agonist/inverse agonist specific. At h(cE3)H(4)R, the potency of histamine and 5-methylhistamine significantly decreased. The efficacy of the inverse agonist thioperamide was strongly reduced at h(cE2)H(4)R and h(cE3)H(4)R. Surprisingly, JNJ7777120 as weak inverse agonist at hH(4)R exhibited partial agonistic activity at h(cE2)H(4)R and h(cE3)H(4)R. Molecular dynamic simulations suggest that the E2- and E3-loops are independently of each other involved in the partial/inverse agonism of JNJ7777120 and that E2- as well as E3-loop do not directly interact with JNJ7777120 in the binding pocket. In conclusion, our study indicates an involvement of the E2- and E3-loops in H(4)R activation process after binding of some but not all examined ligands.

Strongly basic groups such as guanidine moieties are crucial structural elements but compromising drug-likeness of numerous biologically active compounds including ligands of G-protein coupled receptors (GPCRs). As part of a project focusing on the search for guanidine bioisosteres, argininamide-type neuropeptide Y (NPY) Y2 receptor (Y2R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, NG-acylated argininamides were preferably obtained by guanidinylation using tailor-made mono Boc-protected N-acyl-S-methylisothioureas. The compounds were investigated for Y2R antagonism (calcium assays), Y2R affinity and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the NG-substituted (S)-argininamides showed Y2R antagonistic activities and binding affinities comparable to the parent compound, whereas NG-acylated or -carbamoylated analogs containing a terminal amine were superior (Y2R: Ki and KB values in the low nanomolar range). This demonstrates that the basicity of the compounds, although being by 4-5 orders lower than that of guanidines, suffices to form key interactions with acidic amino acids of the Y2R. The acylguanidines bind with high affinity and selectivity to Y2R compared to Y1, Y4, and Y5 receptors. As derivatization of the amino group is tolerated, these compounds are considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y2R. The results support the concept of bioisosteric guanidine-acylguanidine exchange as a broadly applicable approach to retain pharmacological activity regardless of reduced basicity.

Aiming at structural optimization of potent and selective ABCG2 inhibitors, such as UR-ME22-1, from our laboratory, an efficient solid phase synthesis was developed to get convenient access to this class of compounds. 7-Carboxyisatoic anhydride was attached to Wang resin to give resin bound 2-aminoterephthalic acid. Acylation with quinoline-2- or -6-carbonyl chlorides, coupling with tetrahydroisoquinolinylethylphenylamine derivatives, cleavage of the carboxylic acids from solid support and treatment with trimethylsilydiazomethane gave the corresponding methyl esters. Among these esters highly potent and selective ABCG2 modulators were identified (inhibition of ABCB1 and ABCG2 determined in the calcein-AM and the Hoechst 33342 microplate assay, respectively). Interestingly, compounds bearing triethyleneglycol ether groups at the tetrahydroisoquinoline moiety (UR-COP77, UR-COP78) were comparable to UR-ME22-1 in potency but considerably more efficient (max inhibition 83% and 88% vs 60%, rel. to fumitremorgin c, 100%) These results support the hypothesis that solubility of the new ABCG2 modulators and of the reference compounds tariquidar and elacridar in aqueous media is the efficacy-limiting factor.

Fluorescently labelled NPY Y(1) receptor (Y(1)R) ligands were synthesized by connecting pyrylium and cyanine dyes with the argininamide-type Y(1)R antagonist core structure by linkers, covering a wide variety in length and chemical nature, attached to the guanidine group. The most promising fluorescent probes had Y(1)R affinities (radioligand binding) and antagonistic activities (calcium assay) in the one- to two-digit nanomolar range. These compounds turned out to be stable under assay conditions and to be appropriate for the detection of Y(1)Rs by confocal microscopy in live cells. To improve the signal-to-noise ratio by shifting the emission into the near infrared, a new benzothiazolium-type fluorescent cyanine dye (UR-DE99) was synthesized and attached to the parent antagonist via a carbamoyl linker yielding UR-MK131, a highly potent fluorescent Y(1)R probe, which was also successfully applied in flow cytometry.

The histamine H4 receptor (H4R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H4R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H4R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H4R in comparison to human H4R expressed in Sf9 insect cells. Only hH4R and cH4R exhibited a sufficiently high [3H]histamine affinity for radioligand binding studies. Generally, cH4R exhibited lower ligand-affinities than hH4R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH4R than at other H4R species orthologs. Unlike the other H4R species orthologs, hH4R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H4R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH4R, rH4R, and mH4R, whereas at hH4R, JNJ7777120 was a partial inverse agonist. H4R agonists from the class of NG-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H4R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H4R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H4R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of “the prototypical H4R antagonist” JNJ7777120 as H4R antagonism.

Starting from the working hypothesis that specific chemical labelling may be an attractive approach to detect and study G protein-coupled receptors (GPCRs) we synthesized catalytically active antagonists of the neuropeptide Y1 receptor (Y1R). An argininamide-type Y1R antagonist scaffold was combined with a DMAP moiety via spacers of different length and chemical nature. These hybrid compounds have Y1R affinities in the two-digit nanomolar range and are capable of catalysing acyl-transfer reaction to surrogates of bionucleophiles, as demonstrated in the absence of cells by using esters of fluorescent dyes as substrates in buffer. By contrast, selective staining of Y1Rs on living MCF-7 cells was not achieved due to significant non-catalysed (Y1R ligand independent) reaction with biomolecules and the limited density of Y1R on the cell surface. Although this may also depend on insufficient selectivity of the staining reagents, the results of this study suggest that the general applicability of catalytic staining to GPCRs has to be reconsidered, as this approach is hampered by a very low portion of receptor of interest compared to the total amount of membrane proteins.

Since its discovery 10 years ago the histamine H(4) receptor (H(4)R) has attracted attention as a potential drug target, for instance, for the treatment of inflammatory and allergic diseases. Potent and selective ligands including agonists are required as pharmacological tools to study the role of the H(4)R in vitro and in vivo. Many H(4)R agonists, which were identified among already known histamine receptor ligands, show only low or insufficient H(4)R selectivity. In addition, the investigation of numerous H(4)R agonists in animal models is hampered by species-dependent discrepancies regarding potencies and histamine receptor selectivities of the available compounds, especially when comparing human and rodent receptors. This article gives an overview about structures, potencies, and selectivities of various compounds showing H(4)R agonistic activity and summarizes the structure-activity relationships of selected compound classes.

Guanidine and acylguanidine groups are crucial structural features of numerous biologically active compounds. Depending on the biological target, acylguanidines may be considered as considerably less basic bioisosteres of guanidines with improved pharmacokinetics and pharmacodynamics, as recently reported for N'-monoalkylated N-acylguanidines as ligands of G-protein-coupled receptors (GPCRs). The molecular basis for enhanced ligand-receptor interactions of acylguanidines is far from being understood. So far, only a few and contradictory results about their conformational preferences have been reported. In this study, the conformations, conformational preferences, and conformational exchange of four unprotonated and seven protonated monoalkylated acylguanidines with up to six anions and with bisphosphonate tweezers are investigated by NMR. Furthermore, the effects of the acceptor properties in acylguanidine salts, of microsolvation by dimethylsulfoxide, and of varying acyl and alkyl substituents are studied. Throughout the whole study, exclusively two out of eight possible acylguanidine conformations were detected, independent of the compound, the anion, or the solvent used. For the first time, it is shown that the strength and number of intermolecular interactions with anions, solvent molecules, or biomimetic receptors decide the conformational preferences and exchange rates. One recently presented and two new crystal structures resemble the conformational preferences observed in solution. Thus, consistent conformational trends are found throughout the structurally diverse compound pool, including two potent GPCR ligands, different anions, and receptors. The presented results may contribute to a better understanding of the mechanism of action at the molecular level and to the prediction and rational design of these biologically active compounds.

More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y 1 -receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic “and-gate” that grants cell access only to those particles that carry a receptor ligand “and” where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type.

A set of chiral imidazolylpropylguanidines and 2-aminothiazolylpropylguanidines bearing N(G)-3-phenyl- or N(G)-3-cyclohexylbutanoyl residues was synthesized and investigated for histamine H(2) receptor (H(2)R) agonism (guinea pig (gp) right atrium, GTPase assay on recombinant gp and human (h)H(2)R) and for hH(2)R selectivity compared to hH(1)R, hH(3)R and hH(4)R. In contrast to previous studies on arpromidine derivatives, the present investigation of acylguanidine-type compounds revealed only very low eudismic ratios (1.1-3.2), indicating the stereochemistry of the acyl moiety to play only a minor role in this series of H(2)R agonists.

The bioisosteric replacement of the guanidino group in arpromidine-like histamine H(2) receptor (H(2)R) agonists by an acylguanidine moiety is a useful approach to obtain potent H(2)R agonists with improved oral bioavailability and blood-brain barrier penetration. Unfortunately, the selectivity of such N(G)-acylated imidazolylpropylguanidines for the H(2)R is poor, in particular versus histamine H(3) (H(3)R) and H(4) receptors (H(4)R). This drawback appears to depend on the "privileged" imidazolylpropylguanidine structure. The 2-amino-4-methylthiazol-5-yl moiety is a bioisostere of the imidazole ring in the moderately potent H(2)R-selective histamine analogue amthamine. This approach was successfully applied to acylguanidine-type H(2)R agonists. The aminothiazoles are nearly equipotent to the corresponding imidazoles as H(2)R agonists. Compared with histamine, the potency is increased up to 40-fold on the guinea pig right atrium, and up to 125- and 280-fold in GTPase assays with human and guinea pig H(2)R-G(salphaS) fusion proteins expressed in Sf9 insect cells, respectively. Docking studies on H(2)R models support the hypothesis that 2-aminothiazolyl and imidazolyl derivatives interact with H(2)Rs as bioisosteres. In contrast to the imidazoles, the aminothiazoles are devoid of agonistic or relevant antagonistic effects on H(1), H(3), and H(4) receptors. Moreover, unlike amthamine, the 4-methyl group does not significantly contribute to the H(2)R agonism of N(G)-acylated 2-amino-4-methylthiazol-5-ylpropylguanidines.

This study reports the synthesis and pharmacological characterization of tritium-labeled N(1)-[3-(1H-imidazol-4-yl)propyl]-N(2)-propionylguanidine ([(3)H]UR-PI294), a novel and readily accessible radioligand for the human histamine H(3) receptor (hH(3)R) and H(4) receptor (hH(4)R). The radioligand displays high affinity for both histamine receptor subtypes (K(D) (hH(3)R)=1.1 nM, K(D) (hH(4)R)=5.1 nM) and is shown to be a valuable pharmacological tool for the determination of hH(3)R and hH(4)R affinities.

N1-Aryl(heteroaryl)alkyl-N2-[3-(1H-imidazol-4-yl)propyl]guanidines are potent histamine H2-receptor (H2R) agonists, but their applicability is compromised by the lack of oral bioavailability and CNS penetration. To improve pharmacokinetics, we introduced carbonyl instead of methylene adjacent to the guanidine moiety, decreasing the basicity of the novel H2R agonists by 4-5 orders of magnitude. Some acylguanidines with one phenyl ring were even more potent than their diaryl analogues. As demonstrated by HPLC-MS, the acylguanidines (bioisosteres of the alkylguanidines) were absorbed from the gut of mice and detected in brain. In GTPase assays using recombinant receptors, acylguanidines were more potent at the guinea pig than at the human H2R. At the hH1R and hH3R, the compounds were weak to moderate antagonists or partial agonists. Moreover, potent partial hH4R agonists were identified. Receptor subtype selectivity depends on the imidazolylpropylguanidine moiety (privileged structure), opening an avenue to distinct pharmacological tools including potent H4R agonists.

Residues in the second extracellular loop (e2) play a role in ligand binding in certain aminergic G protein coupled receptors (GPCRs). N-[3-(1H-Imidazol-4-yl)propyl)]guanidines and N (G)-acylated derivatives are more efficacious and potent agonists at fusion proteins of the guinea pig histamine H(2) receptor and the short splice variant of G(salpha), G(salphaS) (gpH(2)R-G(salphaS)) than at the human isoform (hH(2)R-G(salphaS)). To elucidate the structural basis for this species-selectivity, we generated a mutant hH(2)R-G(salphaS) fusion protein with the four e2 residues differing in both species isoforms mutated into the gpH(2)R sequence, and a reverse mutant of the gpH(2)R-G(salphaS) with the corresponding mutations into the human species. In a steady-state GTPase activity assay, efficacies and potencies of guanidine-type agonists were similar at mutant and wild-type receptors indicating that e2 does not contribute to the species-selectivity. In several class 1 GPCRs, amino acids in the vicinity of a highly conserved cysteine in e2 participate in ligand binding. A three-dimensional homology model of the hH(2)R predicted Lys-173 and Lys-175, adjacent to Cys-174 in e2, to be in close proximity to the binding pocket of guanidine-type agonists. To elucidate the putative role of both residues for interactions with the agonists, two hH(2)R-G(salphaS) fusion proteins, with single-point mutations of Lys-173--Ala-173 and Lys-175--Ala-175 respectively, were generated. With these mutants, the efficacies and potencies of small and bulky H(2)R agonists did not significantly change. However, increases in GTPase activity upon agonist stimulation were reduced, suggesting an impact of both residues on the efficiency of receptor coupling to G(salphaS). In conclusion, none of the point mutations generated within this study substantially altered the efficacies and potencies of guanidine-type agonists relative to the wild-type receptors, suggesting that these residues do not directly face the H(2)R guanidine-binding pocket. Thus, agonist binding to residues in e2 is relevant for some but not all aminergic GPCRs.

PURPOSE: Previously, we studied the effect of co-administration of paclitaxel with the second generation ABCB1 (p-gp) modulator valspodar on the intracerebral growth of human U118-MG glioblastoma in nude mice. Valspodar significantly increased the brain levels of paclitaxel by inhibition of p-gp expressed at the blood brain barrier. Thus, the tumour burden was reduced by 90%, which was considered as a proof of concept. However, the paclitaxel dose had to be reduced because of toxic side effects resulting from increased drug levels due to p-gp modulation in peripheral tissues. Therefore, in the present study we examined the co-application of paclitaxel with the third generation ABCB1 modulators elacridar and tariquidar, which were supposed to preferentially modulate p-gp in brain capillaries. METHODS: The inhibitory activity of the modulators was measured by a flow cytometric and a chemosensitivity assay in vitro. To determine the distribution of paclitaxel in vivo, nude mice received 50 mg/kg of valspodar, elacridar or tariquidar p.o. (control: vehicle) 4 h before i.v. injection of 8 mg/kg of paclitaxel. Brain, liver, kidney and plasma were collected and analyzed by RP-HPLC. RESULTS: Our in vitro experiments demonstrate that the new modulators are about 80 times more effective in comparison to valspodar. Co-administration of paclitaxel with elacridar and tariquidar led to a long lasting fivefold increase in the concentration of the cytostatic in the brain. Although the increase (2.5- to 7-fold) tended to be lower compared to that induced by co-administered valspodar (six- to eightfold), the brain/plasma ratios achieved with the new modulators were 2-15 times higher. CONCLUSIONS: Elacridar and tariquidar seem to modulate p-glycoprotein preferentially at the blood-brain barrier. Our results suggest that the systemic toxicity of cytostatics combined with elacridar or tariquidar should be lower than in combination with valspodar.

The decomposition of some NG-(ω-aminoalkanoyl)argininamides, which are key intermediates for the preparation of radiolabeled and fluorescent neuropeptide Y receptor ligands, prompted us to synthesize a small series of simple 1-(ω-aminoalkanoyl)guanidines, and to investigate these model compounds for stability in alkaline buffers. The degradation of acylguanidines was monitored by time resolved UV spectroscopy. The most labile compound, 1-(5-aminopentanoyl)guanidine, decomposed with a half life of 19 s to yield piperidin-2-one (pH 10.4 at 25 °C). In contrast the half life of 1-(6-aminohexanoyl)guanidine is 7.7 h, which is comparable to the hydrolysis of acetylguanidine (t1/2 = 9.6 h) in alkaline solution.

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.

Less lipophilic and better water soluble tariquidar analogues were synthesised from one central anthranilic acid derived building block by CuI-catalysed N/O-arylation reactions. The compounds were tested for their inhibitory activity against the ABCB1 transporter in a flow cytometric calcein-AM efflux assay. A correlation between their calculated log P values and their activities was observed, with the more lipophilic analogues being as potent as the reference substance tariquidar.

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.

Previous studies revealed pharmacological differences between human and guinea pig histamine H(2) receptors (H(2)Rs) with respect to the interaction with guanidine-type agonists. Because H(2)R species variants are structurally very similar, comparative studies are suited to relate different properties of H(2)R species isoforms to few molecular determinants. Therefore, we systematically compared H(2)Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH(2)R, gpH(2)R, rH(2)R, and cH(2)R, respectively, and the short splice variant of G(salpha), G(salphaS), were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH(2)R-G(salphaS) but neither gpH(2)R-G(salphaS) nor rH(2)R-G(salphaS) showed the hallmarks of increased constitutive activity compared with hH(2)R-G(salphaS), i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH(2)R-G(salphaS), increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H(2)Rs without or together with mammalian G(salphaS) or H(2)R-G(salpha) fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH(2)R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH(2)R-G(salphaS), gpH(2)R-G(salphaS), and rH(2)R-G(salphaS) in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH(2)R-G(salphaS). In conclusion, the cH(2)R exhibits increased constitutive activity compared with hH(2)R, gpH(2)R, and rH(2)R, and there is evidence for ligand-specific conformations in H(2)R species isoforms.

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.

Imidazolylpropylguanidines derived from impromidine and arpromidine are more potent and efficacious agonists at the guinea pig histamine H2 receptor (gpH2R) than at the human H2R (hH2R) in the GTPase assay. Additionally, such guanidines are histamine H1 receptor (H1R) antagonists with preference for the human relative to the guinea pig receptor. The purpose of this study was to examine structure-activity relationships of guanidines at human and guinea pig H1R and H2R species isoforms expressed in Sf9 insect cells. Three impromidine analogues and six arpromidine analogues exhibited agonistic activity at H2R and antagonistic activity at H1R as assessed in the steady-state GTPase assay. Species selectivity of derivatives was similar as compared with the parent compounds. None of the structural modifications examined (different aromatic ring systems and different ring substituents) was superior in terms of H2R potency and efficacy relative to impromidine and arpromidine, respectively. These data point to substantial structural constraints at the agonist binding site of H2R. Guanidines exhibited distinct structure-activity relationships for H1R antagonism in a radioligand competition binding assay and the GTPase assay and for H1R inverse agonism. Our data indicate that it is difficult to obtain guanidine-type agonists with high potency and high efficacy for hH2R, but those compounds may be useful tools for exploring the antagonist binding site and constitutive activity of H1R.

The negatively charged polysaccharide Hyaluronic acid (HA) has diverse physiological and pathophysiological functions depending on its chain size. Space‑filling, anti‑inflammatory and antiangiogenic effects are triggered by high molecular weight HA (HMW HA) (>20 kDa). Hydrolyzation of HMW HA by Hyal‑1 results in low molecular weight HA (LMW HA) (

A series of chromones, bearing substituted amino groups or N-substituted carboxamide moieties in position 2, was synthesized and characterized in cellular assays for modulation of the ABC transporters ABCC1 (MDCKII-MRP1 cells), ABCB1 (Kb-V1 cells) and ABCG2 (MCF-7/Topo cells). The most potent ABCC1 modulators identified among these flavonoid-type compounds were comparable to the reference compound reversan regarding potency, but superior in terms of selectivity concerning ABCB1 and ABCG2 (2-[4-(Benzo[c][1,2,5]oxadiazol-5-ylmethyl)piperazin-1-yl]-5,7-dimethoxy-4H-chromen-4-one (51): ABCC1, IC50 11.3 μM; inactive at ABCB1 and ABCG2). Compound 51 was as effective as reversan in reverting ABCC1-mediated resistance to cytostatics in MDCKII-MRP1 cells and proved to be stable in mouse plasma and cell culture medium. Modulators, such as compound 51, are of potential value as pharmacological tools for the investigation of the (patho)physiological role of ABCC1.

The histamine (HA) receptor subtype 1 (H1R) and H4R are expressed on immune cells and contribute to an inflammatory reaction. Both receptor subtypes individually enhance the intracellular concentrations of calcium and regulate the accumulation of cAMP, increase MAPK activity, and regulate expression of e.g., inflammatory genes. In a previous study we characterized and compared signaling pathways of the murine orthologs of the H1R and the H4R recombinantly expressed at comparable levels in HEK 293 cells. In the present study, we aimed at analyzing possible interactions of the signaling pathways emerging at the mH1R and the mH4R. Therefore, we co-expressed both receptor subtypes at comparable levels in HEK 293 cells and investigated HA-induced signaling parameters such as the concentrations of intracellular calcium and cAMP, phosphorylation of the MAPKs p38, ERK 1, and ERK 2, and of the transcription factor CREB, and expression of the immediate early gene EGR-1. We demonstrate that the intracellular concentrations of calcium and cAMP as well as the EGR-1 expression are regulated exclusively via the mH1R. In contrast, both receptor subtypes H1R and H4R synergize in HA-induced MAPK activation. This synergism most probably relies on signaling pathways independent of the second messenger calcium and cAMP. In summary, we provide evidence that the mH1R inhibits or dampens the function of the co-expressed mH4R regarding specific parameters, while other signaling events are regulated cooperatively by the mH1R and the mH4R.

The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

Previously, we identified ascorbic acid 6-O-hexadecanoate as an up to 1500 times more potent inhibitor of bacterial and bovine hyaluronidases than the parent compound, vitamin C, and determined a crystal structure of hyaluronidase from Streptococcus pneumoniae in complex with the inhibitor. As the alkanoyl chain interacts with a hydrophobic patch of the enzyme we synthesized other 6-O-acylated vitamin C derivatives bearing various lipophilic residues and investigated the inhibition of Streptococcus agalactiae strain 4755 hyaluronidase (SagHyal(4755)) and of bovine testicular hyaluronidases (BTH) in a turbidimetric assay. All compounds showed selectivity for the bacterial enzyme. Whereas vitamin C 6-O-hexanoate only weakly inhibited SagHyal(4755), the inhibition of both enzymes increased with the length of the aliphatic chain. In the case of the 6-O-octadecanoate, IC(50) values of 0.9 and 39microM for SagHyal(4755) and BTH, respectively, were determined. Partial replacement of the aliphatic chain with a phenyl, p-phenylene or p-biphenylyl group resulted in inhibitors with activity in the lower micromolar range, too. The title compounds are among the most potent inhibitors of both enzymes known to date.

The quantification of pharmacological parameters at G protein-coupled receptors (GPCRs) is indispensable in drug research but costly and time-consuming when conventional methods are sequentially applied. With neuropeptide Y (NPY) Y(1), Y(2), and Y(5) receptors as model systems, a homogenous flow cytometric method for the simultaneous determination of the affinity, selectivity, and activity of GPCR ligands was developed. Mixtures of cells expressing the receptors of interest and cyanine-labeled NPY as a universal fluorescent Y(1), Y(2), and Y(5) receptor agonist were used. Calcium mobilization was measured in different channels with the aid of fluo-4 and fura red. A combination of dye-loaded HEL-Y(1) and CHO-Y(2)-Galpha(qi5) cells with unloaded HEC-1B-Y(5) cells allowed the simultaneous determination of Y(1), Y(2), and Y(5) receptor selectivity preceded by the Y(1) and Y(2) receptor-mediated response with one and the same sample. The data are in good agreement with those determined by radioligand binding and spectrofluorimetry. The convenient, robust, and inexpensive multiparametric procedure offers a broad range of applications in the pharmacological characterization of GPCR ligands.

In an effort to develop a non-radioactive alternative to the [3H]tiotidine and [125I]iodoaminopotentidine binding assays for the histamine H2-receptor (H2R), primary amines related to aminopotentidine were prepared and coupled with the succinimidyl esters of the bulky fluorescent dyes S0536 and BODIPY 650/665-X. The primary amines exhibited different degrees of antagonistic potency at the human and guinea pig H2R. Surprisingly, one compound (5) coupled to the cyanine dye S0536 acted as potent partial agonist/antagonist at the H2R (KB approximately 50 nM; EC50 approximately 100-150 nM). Compounds coupled to the BODIPY dye exhibited moderately high H2R-affinity, too. Thus, the H2R accommodates bulky fluorophores, probably through interaction with extracellular receptor domains. The compounds presented herein provide a starting point for the optimization of fluorescent H2R ligands with respect to affinity and fluorescence as valuable tools to analyze the molecular mechanisms of H2R activation.

Purpose: The aim of this study was to investigate the role of platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) in the proliferation of human glioblastoma cells as a prerequisite for a new therapeutic approach to the treatment of malignant brain tumors with selective tyrosine kinase inhibitors such as imatinib. Methods and results: In the human glioblastoma cell lines U-87 MG, U-118 MG and U-373 MG different PDGF and PDGFR mRNAs were detected by RT-PCR, and the expression of the receptor proteins was demonstrated by immunostaining and flow cytometry. Moreover, functional activity of PDGFRs was demonstrated in PDGFRβ expressing glioblastoma cell variants by measuring the mobilization of intracellular Ca2+ upon PDGF-BB stimulation. However, addition of PDGF-BB to the serum-free culture medium had no stimulatory effect on cell proliferation. Furthermore, cell growth in serum-supplemented and serum-free medium was not affected by imatinib, leflunomide and AG-1296 at therapeutically relevant concentrations. Conclusion: Our results suggest that clinical antitumor effects of imatinib on glioblastoma, if any, are not mediated by the PDGFR.

The inhibition of kinesin Eg5 by small molecules such as monastrol is currently evaluated as an approach to develop a novel class of antiproliferative drugs for the treatment of malignant tumours. Therefore, we studied the effects of the new monastrol analogues enastron, dimethylenastron and vasastrol VS-83 on the proliferation of human glioblastoma cells in the kinetic crystal violet assay. Compared to monastrol, the new cell cycle specific compounds showed an at least one order of magnitude higher anti proliferative activity against U-87 MG, U-118 MG, and U-373 MG glioblastoma cells. The compounds were neither inactivated by hydrolysis nor by binding to serum proteins. Moreover, we demonstrated the characteristic monoaster formation after incubation of cells with the new compounds by confocal laser scanning microscopy. We also showed that the arrangement of beta-actin and tubulin, vital components of the cyto-skeleton of mitotic and quiescent cells, were not affected by the new compounds. Due to the necessity of overcoming the blood-brain barrier in the treatment of brain tumours, we investigated if the new monastrol analogues are modulators or substrates of the p-glycoprotein (p-gp) 170 by a flow cytometric calcein-AM efflux assay. The tested compounds showed no modulating effects on the p-gp function. With respect to the treatment of primary and secondary CNS tumours, the results of our experiments suggest that the new monastrol analogues represent an interesting class of potential anticancer drugs, predicted to be less neurotoxic in comparison to classical tubulin inhibitors.

The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3-diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2-(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 microM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound's inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 microM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties.

Potent and specific inhibitors of hyaluronidases, a group of enzymes preferentially catalysing the hydrolysis of hyaluronic acid, are not known so far. Such compounds could be useful as pharmacological tools for studying the physiological and pathophysiological role of both hyaluronan and hyaluronidases. The effects of sulphated and non-sulphated structurally different oligosaccharides on bovine testicular hyaluronidase, hyaluronidase from bee venom and hyaluronate lyase from Streptococcus agalactiae (hylB (4755)) were studied with the Morgan-Elson reaction. Several active compounds were identified within a series of sulphated beta-(1,4)-galacto-oligosaccharides. The determined IC (50) values of these sulphated oligosaccharides ranged from 4 microM to 630 microM for all hyaluronan-degrading enzymes. Sulphated oligosaccharides like verbascose, planteose and neomycin showed comparable inhibition on all hyaluronidases, thereby possessing 100 - 500 times the activity of the widely accepted hyaluronidase inhibitor apigenin.

Hyaluronidases depolymerize hyaluronic acid, an important component of the extracellular matrix. Bacterial hyaluronan lyases (EC 4.2.2.1) contribute to the spreading of microorganisms in tissues. Potent and selective inhibitors are not known up to now, but would be interesting pharmacological tools and potential drugs for the treatment of bacterial infections. Starting from two crystal structures of streptococcal hyaluronan lyases, a homology model of the S. agalactiae strain 4755 enzyme hylB4755 was generated and applied to structure-based inhibitor design with the de novo design program LUDI. The databases LeadQuest® Vol. 1&2, Accelrys and ChemACX containing 228000 compounds were screened resulting in 1275 hits. Based on high LUDI scores, synthetic feasibility and commercial availability, 19 of these hits were investigated for inhibition of hylB4755. 13 compounds were active in the milli- and submillimolar range. 1,3-diacetylbenzimidazole-2-thione with IC50 values of 5 M and 160 M at physiological and optimum pH, respectively, is one of the two most potent inhibitors of hyaluronan lyases known to date. Depending on the LUDI sphere radius, it was docked in two different poses with similar scores. To analyze whether the hits represent (physico)chemical properties typical for drugability and optimal pharmacokinetics, the distributions of six descriptors within all hits from LeadQuest® and ChemACX and within the original databases were compared. Generally, the hit distributions represent the type of the original ones, but are narrower with slightly different medians. More than 80% of the hits have molecular weights between 150 and 350, XLOGP values between 1 and 4, one H-donor, 1-3 H-acceptors, 0-3 rotatable bonds and 1-3 rings. In conclusion, the application of LUDI to both databases has led to the accumulation of relatively rigid, drug-like molecules.

Although a few experimental approaches to isolated limb perfusion (ILP) are described in the literature, none of these animal models mimics the clinical perfusion techniques adequately to improve the technique of ILP on the basis of valid preclinical data. Therefore, we developed an ILP setup in rats allowing online monitoring of essential perfusion parameters such as temperature (in perfusate, various tissues, and rectum), pH (perfusate), perfusion pressure, and O(2) concentration (in perfusate, tissue), by a tailor-made data acquisition system. This setup permits close supervision of vital parameters during ILP. Various interdependencies, concerning the flow rate and the pressure of perfusate as well as tissue oxygenation were registered. For the measurement of pO(2) values in the perfusate and in different regions of the perfused hind limb, a novel type of microoptode based on quenching of a fluorescent dye was devised. Stable normothermic (37 degrees C) perfusion conditions were maintained at a constant perfusion pressure in the range of 40-60 mm Hg by administration of the spasmo lytic moxaverine (0.5 mg/mL of perfusate as initial dose) at a perfusate flow rate of 0.5 mL/min for 60 min. At the end of an ILP, there were no signs of tissue damage, neither concerning laboratory data (K(+), myoglobin, creatine kinase, lactic dehydrogenase) nor histopathological criteria. The reported ILP model is not only well suited to investigate the effects of hyperthermia but also to assess the efficacy of new antineoplastic approaches, when nude rats, bearing human tumours in the hind limbs, are used.

Recent research on histamine H2 receptor agonists was focused on quantitative structure-activity relationships and receptor models explaining the activity of imidazolylpropylguanidines. Their selectivity for guinea pig vs. human isoforms was investigated using H2 receptor-Gsalpha fusion proteins and attributed to amino acid differences in transmembrane domains 1 and 7. New antagonists result from approaches to improve pharmacokinetic properties and to design hybrid drugs which additionally have gastroprotective or anti H. pylori activity.

Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix. The mammalian hyaluronidases are considered to be involved in many (patho)physiological processes like fertilization, tumor growth, and metastasis. Bacterial hyaluronidases, also termed hyaluronate lyases, contribute to the spreading of microorganisms in tissues. Such roles for hyaluronidases suggest that inhibitors could be useful pharmacological tools. Potent and selective inhibitors are not known to date, although L-ascorbic acid has been reported to be a weak inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL). The x-ray structure of SpnHL complexed with L-ascorbic acid has been elucidated suggesting that additional hydrophobic interactions might increase inhibitory activity. Here we show that L-ascorbic acid 6-hexadecanoate (Vcpal) is a potent inhibitor of both streptococcal and bovine testicular hyaluronidase (BTH). Vcpal showed strong inhibition of Streptococcus agalactiae hyaluronate lyase with an IC(50) of 4 microM and weaker inhibition of SpnHL and BTH with IC(50) values of 100 and 56 microM, respectively. To date, Vcpal has proved to be one of the most potent inhibitors of hyaluronidase. We also determined the x-ray structure of the SpnHL-Vcpal complex and confirmed the hypothesis that additional hydrophobic interactions with Phe-343, His-399, and Thr-400 in the active site led to increased inhibition. A homology structural model of BTH was also generated to suggest binding modes of Vcpal to this hyaluronidase. The long alkyl chain seemed to interact with an extended, hydrophobic channel formed by mostly conserved amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 in BTH.

A tissue-protective effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from animal models of inflammatory bowel disease (IBD). Despite the fact that the clinical usefulness of the anti-inflammatory effects of this cytokine is presently investigated in patients with IBD, there are no data available regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express the interleukin-11 receptor alpha-chain (IL-11Ralpha) and an additional signal-transducing subunit (gp130). In this study, we identified the target cells for IL-11 within the human colon with a new IL-11Ralpha monoclonal antibody and investigated the functional expression of the receptor and downstream effects of IL-11-induced signaling. Immunohistochemistry revealed expression of the IL-11Ralpha selectively on colonic epithelial cells. HT-29 and colonic epithelial cells (CEC) constitutively expressed IL-11Ralpha mRNA and protein. Co-expression of the signal-transducing subunit gp130 was also demonstrated. IL-11 induced signaling through triggering activation of the Jak-STAT pathway without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. However, IL-11 stimulation resulted in a dose-dependent tyrosine phosphorylation of Akt, a decreased activation of caspase-9, and a reduced induction of apoptosis in cultured CEC. In HLA-B27 transgenic rats treated with IL-11, a reduction of apoptotic cell numbers was found. This study demonstrates functional expression of the IL-11Ralpha restricted on CEC within the human colonic mucosa. IL-11 induced signaling through triggering activation of the Jak-STAT pathway, without inducing anti-inflammatory or proliferative effects. The beneficial effects of IL-11 therapy are likely to be mediated by CEC via activation of the Akt-survival pathway, mediating antiapoptotic effects to support mucosal integrity.