Identification of pyridazin-3-one derivatives as potent, selective histamine H₃ receptor inverse agonists with robust wake activity

H(3)R structure-activity relationships on a novel class of pyridazin-3-one H(3)R antagonists/inverse agonists are disclosed. Modifications of the pyridazinone core, central phenyl ring and linker led to the identification of molecules with excellent target potency, selectivity and pharmacokinetic properties. Compounds 13 and 21 displayed potent functional H(3)R antagonism in vivo in the rat dipsogenia model and demonstrated robust wake activity in the rat EEG/EMG model.     

Synthesis and evaluation of pyridone-phenoxypropyl-R-2-methylpyrrolidine analogues as histamine H3 receptor antagonists

6-{4-[3-(R)-2-Methylpyrrolidin-1-yl)propoxy]-phenyl}-2H-pyridazin-3-one 6 (Irdabisant; CEP-26401) was recently reported as a potent H(3)R antagonist with excellent drug-like properties and in vivo activity that advanced into clinical evaluation. A series of pyridone analogs of 6 was synthesized and evaluated as H(3)R antagonists. Structure-activity relationships revealed that the 5-pyridone regiomer was optimal for H(3)R affinity. N-Methyl 9b showed excellent H(3)R affinity, acceptable pharmacokinetics and pharmaceutical properties. In vivo evaluation of 9b showed potent activity in the rat dipsogenia model and robust wake-promoting activity in the rat EEG model.     

Increased severity of reperfusion arrhythmias in mouse hearts lacking histamine H3-receptors

We had previously reported that activation of histamine H(3)-receptors (H(3)R) on cardiac adrenergic nerve terminals decreases norepinephrine (NE) overflow from ischemic hearts and alleviates reperfusion arrhythmias. Thus, we used transgenic mice lacking H(3)R (H(3)R(-/-)) to investigate whether ischemic arrhythmias might be more severe in H(3)R(-/-) hearts than in hearts with intact H(3)R (H(3)R(+/+)). We report a greater incidence and longer duration of ventricular fibrillation (VF) in H(3)R(-/-) hearts subjected to ischemia. VF duration was linearly correlated with NE overflow, suggesting a possible cause-effect relationship between magnitude of NE release and severity of reperfusion arrhythmias. Thus, our findings strengthen a protective antiarrhythmic role of H(3)R in myocardial ischemia. Since malignant tachyarrhythmias cause sudden death in ischemic heart disease, attenuation of NE release by selective H(3)R agonists may represent a new approach in the prevention and treatment of ischemic arrhythmias.     

Mepyramine-JNJ7777120-hybrid compounds show high affinity to hH(1)R, but low affinity to hH(4)R

In literature, a synergism between histamine H(1) and H(4) receptor is discussed. Furthermore, it was shown, that the combined application of mepyramine, a H(1) antagonist and JNJ7777120, a H(4) receptor ligand leads to a synergistic effect in the acute murine asthma model. Thus, the aim of this study was to develop new hybrid ligands, containing one H(1) and one H(4) pharmacophor, connected by an appropriate spacer, in order to address both, H(1)R and H(4)R. Within this study, we synthesized nine hybrid compounds, which were pharmacologically characterized at hH(1)R and hH(4)R. The new compounds revealed (high) affinity to hH(1)R, but showed only low affinity to hH(4)R. Additionally, we performed molecular dynamic studies for some selected compounds at hH(1)R, in order to obtain information about the binding mode of these compounds on molecular level.     

Amine-constrained pyridazinone histamine H₃ receptor antagonists

Pyridazinone 1 was recently reported as a potent H(3)R antagonist with good drug-like properties and in vivo activity. A series of constrained amine analogs of 1 was synthesized to identify compounds with improved pharmacokinetic profiles. From these efforts, a new class of (S)-2-pyrrolidin-1-ylmethyl-1-pyrrolidinyl amides was identified.     

Comparing methods to quantify experimental transmission of infectious agents

Transmission of an infectious agent can be quantified from experimental data using the transient-state (TS) algorithm. The TS algorithm is based on the stochastic SIR model and provides a time-dependent probability distribution over the number of infected individuals during an epidemic, with no need for the experiment to end in final-size (e.g., where no more infections can occur). Because of numerical limitations, the application of the TS algorithm is limited to populations with only a few individuals. We investigated the error of using the easily applicable, time-independent final-size (FS) algorithm knowing that the FS situation was not reached. We conclude that the methods based on the FS algorithm: (i) underestimate R(0), (ii) are liberal when testing H(0):R(0)1 against H(1):R(0)<1, (iii) are conservative when testing H(0):R(0)1 against H(1):R(0)>1, and (iv) are conservative when testing H(0):R(control)=R(treatment) against H(1):R(control)>R(treatment). Furthermore, a new method is presented to find a difference in transmission between two treatment groups (MaxDiff test). The MaxDiff test is compared to tests based on FS and TS algorithms. The TS test and the MaxDiff test were most powerful (approximately equally powerful) in finding a difference, whereas the FS test was less powerful (especially, when both R(control) and R(treatment) are >1).     

Pyrrolidin-3-yl-N-methylbenzamides as potent histamine 3 receptor antagonists

On the basis of the previously reported benzimidazole 1,3'-bipyrrolidine benzamides (1), a series of related pyrrolidin-3-yl-N-methylbenzamides were synthesized and evaluated as H(3) receptor antagonists. In particular, compound 32 exhibits potent H(3) receptor binding affinity, improved pharmaceutical properties and a favorable in vivo profile.     

Epoxidation and reduction of cholesterol, 1,4,6-cholestatrien-3-one and 4,6-cholestadien-3beta-ol

Many naturally occurring polyhydroxylated sterols and oxysterols exhibit potent biologic activities. This paper describes reagent and position selectivity of epoxidation and reduction of cholesterol derivatives. Cholesterol was reacted with m-chloroperoxybenzoic acid (m-CPBA) to form 5alpha,6alpha-epoxycholestan-3beta-ol, but in reaction with 30% H(2)O(2), it did not reacted. 1,4,6-cholestatrien-3-one was obtained from cholesterol and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in dioxane. 1,4,6-cholestatrien-3-one was reacted with 30% H(2)O(2) and 5% NaOH in methanol to give 1alpha,2alpha-epoxy-4,6-cholestadien-3-one, which was stereoselectively reduced with NaBH(4) to form 1alpha,2alpha-epoxy-4,6-cholestadien-3beta-ol and reduced with Li metal in absolute ethanol to give 2-ethoxy-1,4,6-cholestatrien-3-one. And 1,4,6-cholestatrien-3-one was epoxidized with m-CPBA in dichloromethane to afford 6alpha,7alpha-epoxy-1,4-cholestadien-3-one, which was reacted with NaBH(4) to synthesize 6alpha-hydroxy-4-cholesten-3-one and reduced Li metal in absolute ethanol to form 2-ethoxy-1,4,6-cholestatrien-3-one, respectively. 1,4,6-cholestatrien-3-one was reduced with NaBH(4) in absolute ethanol to form 4,6-cholestadien-3beta-ol, which was reacted with 30% H(2)O(2) to leave original compound, but was reacted with m-CPBA to give 4beta,5beta-epoxy-6-cholesten-3beta-ol as the major product and 4beta,5beta-epoxy-6alpha,7alpha-epoxycholestan-3beta-ol as the minor product.     

Activation/deactivation of acetylcholinesterase by H2O2: more evidence for oxidative stress in vitiligo

Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.     

Evidence for tolerance following repeated dosing in rats with ciproxifan, but not with A-304121

Blockade of presynaptic histamine H(3) receptors with potent and selective ligands improves cognitive function in rodents and there is significant interest in developing such drugs for long-term symptomatic treatment of CNS disorders such as attention deficit hyperactivity disorder (ADHD). Unfortunately, little is known about the effects of repeated exposure to H(3) receptor antagonists/inverse agonists. We therefore investigated the effects of acute and repeated daily administration of two potent, brain penetrating H(3) receptor antagonists/inverse agonists, ciproxifan and A-304121, on rat body weight, food and water intake, core temperature and locomotor activity, as well as H(3) receptor density and gene expression levels. Methylphenidate, used clinically for the treatment of ADHD, was included as an additional comparator. Ciproxifan, an imidazole-based compound, decreased food intake over the first 10 days and locomotor activity acutely, but these effects were lost after further repeated administration. The ex vivo binding studies revealed increased H(3) receptor density in rats following repeated administration of ciproxifan for 10 or 15 days; however, H(3) receptor gene expression was not changed. In contrast, rats treated with the non-imidazole, A-304121, did not differ from controls on any measure during the observation period, while rats treated with methylphenidate exhibited hyperthermia and hyperactivity. The implications for potential long-term treatment with H(3) receptor antagonists in CNS disorders such as ADHD are discussed.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Evidence that the tertiary structure of 20(S)-ginsenoside Rg(3) with tight hydrophobic packing near the chiral center is important for Na(+) channel regulation

Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg(3) exists as two stereoisomers of carbon-20: 20-S-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(S)-Rg(3)) and 20-R-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(R)-Rg(3)). Recently, we reported that 20(S)-Rg(3) regulates voltage-dependent Ca(2+) channel activity and several types of ligand-gated ion channels, whereas 20(R)-Rg(3) does not have this activity. In this study, we investigated the structure-activity relationship of these two stereoisomers by NMR spectroscopy and by measurement of the current in Xenopus oocytes expressing the mouse cardiac voltage-dependent Na(+) channel (Na(v)1.5). We found that 20(S)-Rg(3) but not 20(R)-Rg(3) inhibited Na(+) channel current in a dose- and voltage-dependent manner. The difference between Rg(3) epimers in voltage-dependent ion channel regulation indicates that the structure of 20(S)-Rg(3) may be geometrically better aligned than that of 20(R)-Rg(3) for interaction with receptor regions in Na(+) channels. The (1)H and (13)C NMR chemical shifts, including all hydroxyl protons of 20(S)-Rg(3) and 20(R)-Rg(3), were completely assigned, and their tertiary structures were determined. 20(S)-Rg(3) has more tight hydrophobic packing near the chiral center than 20(R)-Rg(3). Tertiary structures and activities of 20(S)-Rg(3) and 20(R)-Rg(3) indicate that 20(S)-Rg(3) may have stronger interactions with the receptor region in ion channels than 20(R)-Rg(3). This may result in different stereoselectivity of Rg(3) stereoisomers in the regulation of voltage-dependent Na(+) channel activity. This is the first structural approach to ginsenoside action on ion channel.     

The role of protein binding of trivalent arsenicals in arsenic carcinogenesis and toxicity

Three of the most plausible biological theories of arsenic carcinogenesis are protein binding, oxidative stress and altered DNA methylation. This review presents the role of trivalent arsenicals binding to proteins in arsenic carcinogenesis. Using vacuum filtration based receptor dissociation binding techniques, the lifetimes of unidentate (<1s), bidentate (1-2min) and tridentate (1-2h) arsenite containing peptide binding complexes were estimated. According to our experimental data some of the protein targets to which arsenite may bind in vivo include tubulin, poly(ADP-ribose)polymerase (PARP-1), thioredoxin reductase, estrogen receptor-alpha, arsenic(+3)methyltransferase and Keap-1. Arsenite binding to tubulin can lead to several of the genetic effects observed after arsenic exposures (aneuploidy, polyploidy and mitotic arrests). Among many other possible arsenite binding sites are rat hemoglobin, the DNA repair enzyme xeroderma pigmentosum protein A (XPA), and other C2H2, C3H and C4 zinc finger proteins including members of the steroid receptor superfamily (e.g. glucocorticoid receptor). Macromolecules to which arsenite does not bind to include calf thymus DNA, mixed Type II-A histones and bovine H3/H4 histone. Although all six tested arsenicals released iron from ferritin, radioactive arsenite did not bind to the protein horse ferritin.     

Signaling Pathway of Histamine H1 Receptor-Mediated Histamine H1 Receptor Gene Upregulation Induced by Histamine in U-373 MG Cells

Histamine H₁ receptor (H1R) is one of the targets of histamine in the nervous system and the peripheral tissues. Protein kinase Cδ (PKCδ) signaling is involved in histamine-induced upregulation of H1R gene expression in HeLa cells. Histamine also upregulates H1R gene expression in U-373 MG cells. However, the molecular signaling of this upregulation is still unclear. Here, we investigated the molecular mechanism of histamine-induced H1R gene upregulation in U-373 MG cells. Histamine-induced H1R gene upregulation was inhibited by H1R antagonist d-chlorpheniramine, but not by ranitidine, ciproxifan, or JNJ77777120, and H2R, H3R, or H4R antagonists, respectively. Ro-31-8220 and Go6976 also suppressed this upregulation, however, the PKCδ selective inhibitor rottlerin and the PKCβ selective inhibitor {"type":"entrez-nucleotide","attrs":{"text":"Ly333531","term_id":"1257370768"}}Ly333531 did not. Time-course studies showed distinct kinetics of H1R gene upregulation in U-373 MG cells from that in HeLa cells. A promoter assay revealed that the promoter region responsible for H1R gene upregulation in U-373 MG cells was different from that of HeLa cells. These data suggest that the H1R-activated H1R gene expression signaling pathway in U-373 MG cells is different from that in HeLa cells, possibly by using different promoters. The involvement of PKCα also suggests that compounds that target PKCδ could work as peripheral type H1R-selective inhibitors without a sedative effect.     

Regulation of tyrosinase by tetrahydropteridines and H2O2

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.     

Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H₄ and the ion channel serotonin 5-HT₃A

A fragment library was screened against the G protein-coupled histamine H(4) receptor (H(4)R) and the ligand-gated ion channel serotonin 5-HT(3A) (5-HT(3A)R). Interestingly, significant overlap was found between H(4)R and 5-HT(3A)R hit sets. The data indicates that dual active H(4)R and 5 HT(3A)R fragments have a higher complexity than the selective compounds which has important implications for chemical genomics approaches. The results of our fragment-based library screening study illustrate similarities in ligand recognition between H(4)R and 5-HT(3A)R and have important consequences for selectivity profiling in ongoing drug discovery efforts on H(4)R and 5-HT(3A)R. The affinity profiles of our fragment screening studies furthermore match the chemical properties of the H(4)R and 5-HT(3A)R binding sites and can be used to define molecular interaction fingerprints to guide the in silico prediction of protein-ligand interactions and structure.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).