SAR exploration of the non-imidazole histamine H3 receptor ligand ZEL-H16 reveals potent inverse agonism.

Histamine H3 receptor (H3 R) agonists without an imidazole moiety remain very scarce. Of these, ZEL-H16 (1) has been reported previously as a high-affinity non-imidazole H3 R (partial) agonist. Our structure-activity relationship analysis using derivatives of 1 identified both basic moieties as key interaction motifs and the distance of these from the central core as a determinant for H3 R affinity. However, in spite of the reported H3 R (partial) agonism, in our hands, 1 acts as an inverse agonist for Gαi signaling in a CRE-luciferase reporter gene assay and using an H3 R conformational sensor. Inverse agonism was also observed for all of the synthesized derivatives of 1. Docking studies and molecular dynamics simulations suggest ionic interactions/hydrogen bonds to H3 R residues D1143.32 and E2065.46 as essential interaction points.

1-[2-(1-Cyclobutylpiperidin-4-yloxy)-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-5-yl]propan-1-one: a Histamine H3 Receptor Inverse Agonist with Efficacy in Animal Models of Cognition.

A series of chemical optimizations, which was guided by in vitro affinity at histamine H3 receptor (H3 R), modulation of lipophilicity, ADME properties and preclinical efficacy resulted in the identification of 1-[2-(1-cyclobutylpiperidin-4-yloxy)-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-5-yl]propan-1-one (45 e) as a potent and selective (Ki =4.0 nM) H3 R inverse agonist. Dipsogenia induced by (R)-α-methylhistamine was dose dependently antagonized by 45 e, confirming its functional antagonism at H3 R. It is devoid of hERG and phospholipidosis issues. Compound 45 e has adequate oral exposures and favorable half-life in both rats and dogs. It has demonstrated high receptor occupancy (ED80 =0.22 mg/kg) and robust efficacy in object recognition task and, dose dependently increased acetylcholine levels in brain. The sub-therapeutic doses of 45 e in combination with donepezil significantly increased acetylcholine levels. The potent affinity, selectivity, in vivo efficacy and drug like properties together with safety, warrant for further development of this molecule for potential treatment of cognitive disorders associated with Alzheimer's disease.

Shining light on the histamine H2 receptor: Synthesis of carbamoylguanidine-type agonists as a pharmacological tool to study internalization.

So far, only little is known about the internalization process of the histamine H2 receptor (H2R). One promising approach to study such dynamic processes is the use of agonistic fluorescent ligands. Therefore, a series of carbamoylguanidine-type H2R agonists containing various fluorophores, heterocycles, and linkers (28-40) was synthesized. The ligands were pharmacologically characterized in several binding and functional assays. These studies revealed a significantly biased efficacy (Emax) for some of the compounds, e.g. 32: whereas 32 acted as strong partial (Emax: 0.77, mini-Gs recruitment) or full agonist (Emax: 1.04, [35S]GTPγS binding) with respect to G protein activation, it was only a weak partial agonist regarding ß-arrestin1/2 recruitment (Emax: 0.09-0.12) and failed to promote H2R internalization (confocal microscopy). On the other hand, H2R internalization was observed for compounds that exhibited moderate agonistic activity in the ß-arrestin1/2 pathways (Emax ≥ 0.22). The presented differently-biased fluorescent ligands are versatile molecular tools for future H2R studies on receptor trafficking and internalization e.g. using fluorescence microscopy.

Guided rational design with scaffold hopping leading to novel histamine H3 receptor ligands.

During the past decades, histamine H3 receptors have received widespread attention in pharmaceutical research due to their involvement in pathophysiology of several diseases such as neurodegenerative disorders. In this context, blocking of these receptors is of paramount importance in progression of such diseases. In the current investigation, novel histamine H3 receptor ligands were designed by exploiting scaffold-hopping drug-design strategy. We inspected the designed molecules in terms of ADME properties, drug-likeness, as well as toxicity profiles. Additionally molecular docking and dynamics simulation studies were performed to predict binding mode and binding free energy calculations, respectively. Among the designed structures, we selected compound d2 and its demethylated derivative as examples for synthesis and affinity measurement. In vitro binding assays of the synthesized molecules demonstrated that d2 has lower binding affinity (Ki = 2.61 µM) in radioligand displacement assay to hH3R than that of demethylated form (Ki = 12.53 µM). The newly designed compounds avoid of any toxicity predictors resulted from extended in silico and experimental studies, can offer another scaffold for histamine H3R antagonists for further structure-activity relationship studies.

Metabolic benefits of novel histamine H3 receptor ligands in the model of excessive eating: The importance of intrinsic activity and pharmacokinetic properties.

AIMS: One of the therapeutic approaches in the treatment of obesity is the use of histamine H3 receptor ligands. Histamine plays a significant role in eating behavior because it causes a loss of appetite and is considered to be a satiety signal released during food intake. MATERIAL AND METHODS: Histamine ligands were selected based on the preliminary studies which included determination of intrinsic activity and selected pharmacokinetic parameters. Female Wistar rats were fed palatable feed for 28 days and simultaneously the tested compounds were administered intraperitoneally at a dose of 10 mg/kg b.w./day. Rats' weight was evaluated daily and calories intake was evaluated once per week. At the end of experiment insulin and glucose tolerance tests was performed. Plasma levels of cholesterol, triglycerides, leptin, insulin, glucose, C-peptide and CRP were also determined. In order to rule out false-positive results the influence of tested compounds on spontaneous activity of rats was monitored. RESULTS: Animals fed palatable feed and treated with KSK-61 or KSK-63 compounds showed the slowest weight gain which was comparable to the one observed in control animals. Both compounds with the highest pharmacological activity have also similar pharmacokinetic properties with quite long half-life and high volume of distribution indicating that they can freely cross most biological barriers. Some compounds, especially KSK-63, compensated for metabolic disorders. CONCLUSION: The presented study proves that search among the active histamine H3 receptor ligands for the new therapeutic agents to treat obesity is justified. Compounds KSK-61 and KSK-63 can be considered as the leading structures.

Molecular Modeling of Histamine Receptors-Recent Advances in Drug Discovery.

The recent developments of fast reliable docking, virtual screening and other algorithms gave rise to discovery of many novel ligands of histamine receptors that could be used for treatment of allergic inflammatory disorders, central nervous system pathologies, pain, cancer and obesity. Furthermore, the pharmacological profiles of ligands clearly indicate that these receptors may be considered as targets not only for selective but also for multi-target drugs that could be used for treatment of complex disorders such as Alzheimer's disease. Therefore, analysis of protein-ligand recognition in the binding site of histamine receptors and also other molecular targets has become a valuable tool in drug design toolkit. This review covers the period 2014-2020 in the field of theoretical investigations of histamine receptors mostly based on molecular modeling as well as the experimental characterization of novel ligands of these receptors.

Activation of carbonic anhydrase isoforms involved in modulation of emotional memory and cognitive disorders with histamine agonists, antagonists and derivatives.

Carbonic anhydrases (CAs, EC 4.2.1.1) activators were shown to be involved in memory enhancement and learning in animal models of cognition. Here we investigated the CA activating effects of a large series of histamine based compounds, including histamine receptors (H1R - H4R) agonists, antagonists and other derivatives of this autacoid. CA activators may be thus useful for improving cognition as well as in diverse therapeutic areas (phobias, obsessive-compulsive disorder, generalised anxiety, post-traumatic stress disorders), for which activation of this enzyme was recently shown to be involved.

Discovery of a G Protein-Biased Radioligand for the Histamine H2 Receptor with Reversible Binding Properties.

Currently employed histamine H2 receptor (H2R) radioligands possess several drawbacks, for example, high non-specificity, insurmountable binding, or short half-life. We report the synthesis and the chemical and pharmacological characterization of the highly stable carbamoylguanidine-type radioligand [3H]UR-KAT479 ([3H]23), a subtype selective histamine H2 receptor G protein-biased agonist. [3H]23 was characterized by saturation, kinetic, and competition binding assays at the human, guinea pig, and mouse H2 receptors (co-)expressed in HEK293(T) cells. [3H]23 reversibly bound to the respective H2Rs with moderate to high affinity (human/guinea pig/mouse Kd: 24/28/94 nM). In order to investigate the applicability of carbamoylguanidine-type ligands in animal studies elucidating the role of the H2R in the brain, we performed a preliminary partitioning experiment in the whole human/mouse blood, which indicated a low binding of [3H]23 to red blood cells. These properties turn [3H]23 into a powerful tool for the determination of binding affinities and demonstrate the promising pharmacokinetic profile of carbamoylguanidine-type ligands.

Development of New Synthetic Reactions Using Hetero-Heavy Atoms and Their Application to Synthesis of Biofunctional Molecules.

Novel reactions using hetero-heavy atoms (P, S, Si, Se, and Sn) were developed and applied to the synthesis of biofunctional molecules and some medicine-candidates, in which the following items are covered. 1) Development of introduction of C1-unit using cyanophosphates (CPs). 2) Carbene-generation under neutral condition from CPs and its application to organic synthesis. 3) [3,3]Sigmatropic rearrangement-ring expansion reactions of medium-sized cyclic thionocarbonates containing a sulfur atom and their application to natural product synthesis. 4) Stereoselective synthesis of novel ß-imidazole C-nucleosides via diazafulvene intermediates and their application to investigating ribozyme reaction mechanism. 5) Developments of novel histamine H3- and H4-receptor ligands using new synthetic methods involving Se or Sn atoms.

Covalent Inhibition of the Histamine H3 Receptor.

Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H3 receptor (H3R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H3R ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H3R was validated with washout experiments and leads to inverse agonism on H3R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H3R conformation and to study the consequences of prolonged inhibition of the H3R.

4-(3-Aminoazetidin-1-yl)pyrimidin-2-amines as High-Affinity Non-imidazole Histamine H3 Receptor Agonists with in Vivo Central Nervous System Activity.

Despite the high diversity of histamine H3 receptor (H3R) antagonist/inverse agonist structures, partial or full H3R agonists have typically been imidazole derivatives. An in-house screening campaign intriguingly afforded the non-imidazole 4-(3-azetidin-1-yl)pyrimidin-2-amine 11b as a partial H3R agonist. Here, the design, synthesis, and structure-activity relationships of 11b analogues are described. This series yields several non-imidazole full agonists with potencies varying with the alkyl substitution pattern on the basic amine following the in vitro evaluation of H3R agonism using a cyclic adenosine monophosphate response element-luciferase reporter gene assay. The key compound VUF16839 (14d) combines nanomolar on-target activity (pKi = 8.5, pEC50 = 9.5) with weak activity on cytochrome P450 enzymes and good metabolic stability. The proposed H3R binding mode of 14d indicates key interactions similar to those attained by histamine. In vivo evaluation of 14d in a social recognition test in mice revealed an amnesic effect at 5 mg/kg intraperitoneally. The excellent in vitro and in vivo pharmacological profiles and the non-imidazole structure of 14d make it a promising tool compound in H3R research.

Synthesis and biological evaluation of heteroalicyclic cyanoguanidines at histamine receptors.

Recent studies on histamine receptor (HR) subtypes identified imidazolyl butyl cyanoguanidines, like UR-PI376, as highly potent agonists at the human histamine H4 receptor (hH4 R). While imidazole-containing compounds display drawbacks in pharmacokinetics, we studied the possibility of replacing the heteroaromatic cycle by nonaromatic six-membered heterocycles (piperidine, morpholine, thiomorpholine, and N-methylpiperazine) as potential bioisosteres. Beyond that, this approach should give more information about the indispensability of the aromatic ring as a basic head group. Besides these changes, a variation of the spacer length (C3 -C5 ) connecting the heterocycle and the cyanoguanidine moiety has been made to possibly trigger the selectivity towards the respective HRs. Investigations in radioligand-binding assays exhibited only very weak activity at the hH1 R and hH3 R, while nearly all compounds were inactive at the hH2 R and hH4 R. In the case of piperidine-containing compounds, moderate affinities at the hH3 R over the single-digit micromolar range were detected.

The Discovery of LML134, a Histamine H3 Receptor Inverse Agonist for the Clinical Treatment of Excessive Sleep Disorders.

Histamine H3 receptor (H3R) inverse agonists that have been in clinical trials for the treatment of excessive sleep disorders, have been plagued with insomnia as a mechanism-based side effect. We focused on the identification of compounds that achieve high receptor occupancy within a short time, followed by rapid disengagement from the receptor, a target profile that could provide therapeutic benefits without the undesired side effect of insomnia. This article describes the optimization work that led to the discovery of 1-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl 4-cyclobutylpiperazine-1-carboxylate (18 b, LML134).

A Photoswitchable Agonist for the Histamine H3 Receptor, a Prototypic Family A G-Protein-Coupled Receptor.

Spatiotemporal control over biochemical signaling processes involving G protein-coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H3 receptor (hH3 R). Upon illumination, key compound 65 decreases its affinity for the hH3 R by 8.5-fold and its potency in hH3 R-mediated Gi protein activation by over 20-fold, with the trans and cis isomer both acting as full agonist. In real-time two-electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light-induced modulation of hH3 R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.

Discovery and Development of N-[4-(1-Cyclobutylpiperidin-4-yloxy)phenyl]-2-(morpholin-4-yl)acetamide Dihydrochloride (SUVN-G3031): A Novel, Potent, Selective, and Orally Active Histamine H3 Receptor Inverse Agonist with Robust Wake-Promoting Activity.

A series of chemical optimizations guided by in vitro affinity at a histamine H3 receptor (H3R), physicochemical properties, and pharmacokinetics in rats resulted in identification of N-[4-(1-cyclobutyl-piperidin-4-yloxy)phenyl]-2-(morpholin-4-yl)acetamide dihydrochloride (17v, SUVN-G3031) as a clinical candidate. Compound 17v is a potent (hH3R Ki = 8.73 nM) inverse agonist at H3R with selectivity over other 70 targets, Compound 17v has adequate oral exposures and favorable elimination half-lives both in rats and dogs. It demonstrated high receptor occupancy and marked wake-promoting effects with decreased rapid-eye-movement sleep in orexin-B saporin lesioned rats supporting its potential therapeutic utility in treating human sleep disorders. It had no effect on the locomotor activity at doses several fold higher than its efficacious dose. It is devoid of hERG and phospholipidosis issues. Phase-1 evaluation for safety, tolerability, and pharmacokinetics, and long-term safety studies in animals have been successfully completed without any concern for further development.

QSAR Modeling of Histamine H3R Antagonists/inverse Agonists as Future Drugs for Neurodegenerative Diseases.

BACKGROUND: Histamine H3 receptor (H3R) is associated with several neuropsychological diseases, and thus it is an important target involved in several CNS disorders, such as narcolepsy, attention deficit hyperactivity disorder and schizophrenia. Since QSAR modeling is a feasible approach to explain the role of the molecular substituents in the biological activity, it can help in improving the design of better H3R ligands for these conditions. METHODS: This article reviews papers previously published in literature to show the current status of the contribution from QSAR modeling to reach H3R antagonists/inverse agonists. RESULTS: Classical and 3D-QSAR models were retrieved, showing that the steric and hydrophobic properties of the H3R ligands are most important to reach good affinity. CONCLUSION: Although QSAR methods are valuable to design better H3R antagonists/inverse agonists, pharmacokinetics should also be considered in future models to ensure good CNS penetration.

Betahistine dihydrochloride transdermal delivery via optimized thermosensitive gels: percutaneous absorption evaluation using rat growth as a biomarker.

The aim of this study was to develop and optimize a betahistine dihydrochloride (BH) thermoreversible bioadhesive gel intended for transdermal delivery. The gels were obtained via cold method. A full factorial design was employed to investigate the joint effect of Poloxamer 407 concentration (18 and 20%), adhesive polymer type (Polyvinyl pyrolidone, Hydroxypropyl methylcellulose, and Carbopol 934), and adhesive polymer concentration (0.5 and 1.5%) on gelling temperature, viscosity at 37 °C, and adhesion strength. Data collected were analyzed using multiple linear regression. A desirability index approach with relative importance weight was used to choose the most desirable formulation. F4 (20% Poloxamer+1.5% Carbopol) was selected for further characterization. F4 released 96.97% drug in 12 h across hairless rat skin. F4 gelation temperature and time were 36 ± 0.35 °C, and 6 ± 0.7 min, respectively. F4 adhesive force was 8835.68 dyne/cm2. F4 was tested for its appetite suppressing effect in a rat model and it was evaluated histopathologically. Rats' chow intake and weight gain was significantly decreased with no signs of inflammation or lipolysis when the optimized BH gel formulation, F4, was compared with untreated animals and animals treated with BH free gel. The results suggest that BH is percutaneously absorbed from the gel base and that the BH gel is tolerable. The desirability index approach with relative importance weight of responses was effective in determination of the optimum formulation. BH is systemically effective and well-tolerated when applied topically in hydrogel-based systems. The Carbopol-Poloxamer gel is a promising modality for transdermal delivery of BH.

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists.

Emerging from an HTS campaign, novel steroid-based histamine H3 receptor antagonists were identified and characterized. Structural moieties of the hit compounds were combined to improve binding affinities which resulted in compound 4 as lead molecule. During the lead optimization due to the versatile modifications of diamino steroid derivatives, several in vitro potent compounds with subnanomolar binding affinities to histamine H3 receptors were found. The unfavorable binding to rat muscarinic receptors was successfully reduced by tuning the basicity. Compound 20 showed significant in vivo activity in the rat dipsogenia model and could serve as a pharmacological tool in the future.

Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method.

Virtual screening offers an efficient alternative to high-throughput screening in the identification of pharmacological tools and lead compounds. Virtual screening is typically based on the matching of target structures or ligand pharmacophores to commercial or in-house compound catalogues. This study provides the first proof-of-concept for our recently reported method where pharmacophores are instead constructed based on the inference of residue-ligand fragments from crystal structures. We demonstrate its unique utility for G protein-coupled receptors, which represent the largest families of human membrane proteins and drug targets. We identified five neutral antagonists and one inverse agonist for the histamine H3 receptor with potencies of 0.7-8.5 µM in a recombinant receptor cell-based inositol phosphate accumulation assay and validated their activity using a radioligand competition binding assay. H3 receptor antagonism is of large therapeutic value and our ligands could serve as starting points for further lead optimisation. The six ligands exhibit four chemical scaffolds, whereof three have high novelty in comparison to the known H3 receptor ligands in the ChEMBL database. The complete pharmacophore fragment library is freely available through the GPCR database, GPCRdb, allowing the successful application herein to be repeated for most of the 285 class A GPCR targets. The method could also easily be adapted to other protein families.

Isoform-Specific Biased Agonism of Histamine H3 Receptor Agonists.

The human histamine H3 receptor (hH3R) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the H3R; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hH3R splice variants (hH3R445 and hH3R365) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3ß (GSK3ß) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former's designation as a "protean" agonist. The 80 amino acid IL3 deleted isoform hH3R365 is more permissive in its signaling than hH3R445: 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hH3R365 was completely unable to stimulate GSK3ß phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G protein-coupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants.