Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1-9) and a soluble AC (sAC, also referred to as AC10). This review ...will largely focus on mACs. mACs are activated by the G-protein G
and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene (
) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy.
In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 ...by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in 3Hhistamine binding and functional 35SGTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It is now well established that any given ligand for a G-protein-couple receptor (GPCR) does not simply possess a single defined
efficacy. Rather, a ligand possesses multiple efficacies, depending on ...the specific down-stream signal transduction pathway
analyzed. This diversity may be based on ligand-specific GPCR conformations and is often referred to as âfunctional selectivity.â
It has been known for a century that stereoisomers of catecholamines differ in their potency and, in some systems, also in
their efficacy. However, the molecular basis for efficacy differences of GPCR ligand stereoisomers has remained poorly defined.
In an elegant study published in this issue of Molecular Pharmacology , Woo et al. (p. 158) show that stereoisomers of the β 2 -adrenoceptor selective agonist fenoterol differentially activates G s - and G i -proteins in native rat cardiomyocytes. This study is so important because it is the first report to show that even the subtle
structural differences within a ligand stereoisomer pair are sufficient to discriminate between GPCR conformations with distinct
G-protein coupling properties. The study highlights of how important it is to examine the âmore activeâ (eutomer) and the
âless activeâ (distomer) stereoisomer to understand the mechanisms of action and the cellular effects of GPCR ligands. The
study by Woo et al. will ignite a renaissance of the analysis of ligand stereoisomers, using sensitive pharmacological and
biophysical assays. The available literature supports the notion that meticulous analysis of ligand stereoisomers is a goldmine
for understanding mechanisms of GPCR activation, analysis of signal transduction pathways, development of new therapies for
important diseases, and drug safety.
The histamine H(4) receptor (H(4)R) is expressed in several cell types of the immune system and is assumed to play an important pro-inflammatory role in various diseases, including bronchial asthma, ...atopic dermatitis, and pruritus. Accordingly, H(4)R antagonists have been suggested to provide valuable drugs for the treatment of these diseases. Over the past decade, the indole derivative 1-(5-chloro-1H-indol-2-yl)carbonyl-4-methylpiperazine (JNJ7777120) has become the "standard" H(4)R antagonist and has been extensively used to assess the pathophysiological role of the H(4)R. However, the situation has now become more complicated by recent data (p. 749 and Naunyn Schmiedebergs Arch Pharmacol doi: 10.1007/s00210-011-0612-3) showing that JNJ7777120 can also activate β-arrestin in a supposedly G(i)-protein-independent (pertussis toxin-insensitive) manner and that at certain H(4)R species orthologs, JNJ7777120 exhibits partial agonist efficacy with respect to G(i)-protein activation (steady-state high-affinity GTPase activity). These novel findings can be explained within the concept of functional selectivity or biased signaling, assuming unique ligand-specific receptor conformations with distinct signal transduction capabilities. Thus, great caution must be exerted when interpreting in vivo effects of JNJ7777120 as H(4)R antagonism. We discuss future directions to get out of the current dilemma in which there is no "standard" H(4)R antagonist available to the scientific community.
Recently, we identified high-affinity human histamine H3 (hH3R) and H4 receptor (hH4R) ligands among a series of NG-acylated imidazolylpropylguanidines, which were originally designed as histamine H2 ...receptor (H2R) agonists. Aiming at selectivity for hH4R, the acylguanidine group was replaced with related moieties. Within a series of cyanoguanidines, 2-cyano-1-4-(1H-imidazol-4-yl)butyl-3-(2-phenylthio)ethylguanidine (UR-PI376, 67) was identified as the most potent hH4R agonist (pEC50 = 7.47, α = 0.93) showing negligible hH1R and hH2R activities and significant selectivity over the hH3R (pK B = 6.00, α = −0.28), as determined in steady-state GTPase assays using membrane preparations of hH x R-expressing Sf9 cells. In contrast to previously described selective H4R agonists, this compound and other 3-substituted derivatives are devoid of agonistic activity at the other HR subtypes. Modeling of the binding mode of 67 suggests that the cyanoguanidine moiety forms charge-assisted hydrogen bonds not only with the conserved Asp-94 but also with the hH4R-specific Arg-341 residue. 2-Carbamoyl-1-2-(1H-imidazol-4-yl)ethyl-3-(3-phenylpropyl)guanidine (UR-PI97, 88) was unexpectedly identified as a highly potent and selective hH3R inverse agonist (pK B = 8.42, >300-fold selectivity over the other HR subtypes).
The histamine H
4
receptor (H
4
R) 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 H
4
R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H
4
R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H
4
R in comparison to human H
4
R expressed in Sf9 insect cells. Only hH
4
R and cH
4
R exhibited a sufficiently high
3
Hhistamine affinity for radioligand binding studies. Generally, cH
4
R exhibited lower ligand-affinities than hH
4
R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH
4
R than at other H
4
R species orthologs. Unlike the other H
4
R species orthologs, hH
4
R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H
4
R antagonist (1-(5-chloro-1H-indol-2-yl)carbonyl-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH
4
R, rH
4
R, and mH
4
R, whereas at hH
4
R, JNJ7777120 was a partial inverse agonist. H
4
R agonists from the class of
N
G
-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H
4
R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H
4
R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H
4
R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of “the prototypical H
4
R antagonist” JNJ7777120 as H
4
R antagonism.
Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic ...efficacy and fewer adverse effects. Dual coupling of the β
2
-adrenoceptor (β
2
AR) triggers a signal transduction via G
s
α and G
i
α proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using β
2
AR-G
s
α and β
2
AR-G
i
α fusion proteins, (
R
,
S’
)- and (
S
,
S’
)-isomers of 4′-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for G
s
. G protein-independent signaling via β-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of β
2
AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of G
s
-biased β
2
AR agonists.
Edema factor (EF) is a calmodulin (CaM)-activated adenylyl cyclase (AC) toxin from Bacillus anthracis that contributes to anthrax pathogenesis. Anthrax is an important medical problem, but treatment ...of B. anthracis infections is still unsatisfying. Thus, selective EF inhibitors could be valuable drugs in the treatment of anthrax infection, most importantly shock. The catalytic site of EF, the EF/CaM interaction site and allosteric sites constitute potential drug targets. To this end, most efforts have been directed towards targeting the catalytic site. A major challenge in the field is to obtain compounds with high selectivity for AC toxins relative to mammalian membranous ACs (mACs). 3'-(N-methyl)anthraniloyl-2'-deoxyadenosine-5'-triphosphate is the most potent EF inhibitor known so far (Ki, 10nM), but selectivity relative to mACs needs to be improved (currently ~5-50-fold, depending on the specific mAC isoform considered). AC toxin inhibitors can be identified in virtual screening studies based on available EF crystal structures and examined in cellular test systems or at the level of purified toxin using classic radioisotopic or non-radioactive fluorescence assays. Binding of certain MANT-nucleotides to AC toxins elicits large direct fluorescence- or fluorescence resonance energy transfer signals upon interaction with CaM, and these signals can be used to identify toxin inhibitors in competition binding studies. Collectively, potent EF inhibitors are available, but before they can be used clinically, selectivity against mACs must be improved. However, several methodological approaches, complementing each other, are now available to direct the development of potent, selective, orally applicable and clinically useful EF inhibitors.
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.
Mammalian membranous and soluble adenylyl cyclases (mAC, sAC) and soluble guanylyl cyclases (sGC) generate cAMP and cGMP from ATP and GTP, respectively, as substrates. mACs (nine human isoenzymes), ...sAC, and sGC differ in their overall structures owing to specific membrane-spanning and regulatory domains but consist of two similarly folded catalytic domains C1 and C2 with high structure-based homology between the cyclase species. Comparison of available crystal structures - VC1:IIC2 (a construct of domains C1a from dog mAC5 and C2a from rat mAC2), human sAC and sGC, mostly in complex with substrates, substrate analogs, inhibitors, metal ions, and/or modulators - reveals that especially the nucleotide binding sites are closely related. An evolutionarily well-conserved catalytic mechanism is based on common binding modes, interactions, and structural transformations, including the participation of two metal ions in catalysis. Nucleobase selectivity relies on only few mutations. Since in all cases the nucleoside moiety is embedded in a relatively spacious cavity, mACs, sAC, and sGC are rather promiscuous and bind nearly all purine and pyrimidine nucleotides, including CTP and UTP, and many of their derivatives as inhibitors with often high affinity. By contrast, substrate specificity of mammalian adenylyl and guanylyl cyclases is high due to selective dynamic rearrangements during turnover.
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