The receptor concept in 3D: from hypothesis and metaphor to GPCR-ligand structures.

The first mentioning of the word "receptor" for the structure with which a bioactive compound should react for obtaining its specific influence on a physiological system goes back to the years around 1900. The receptor concept was adapted from the lock and key theory for the enzyme substrate and blockers interactions. Through the years the concept, in the beginning rather being a metaphor, not a model, was refined and became reality in recent years. Not only the structures of receptors were elucidated, also the receptor machineries were unraveled. Following a brief historical review we will describe how the recent breakthroughs in the experimental determination of G protein-coupled receptor (GPCR) crystal structures can be complemented by computational modeling, medicinal chemistry, biochemical, and molecular pharmacological studies to obtain new insights into the molecular determinants of GPCR-ligand binding and activation. We will furthermore discuss how this information can be used for structure-based discovery of novel GPCR ligands that bind specific (allosteric) binding sites with desired effects on GPCR functional activity.

Memoirs of the First 50 Years of the European Federation for Medicinal Chemistry (EFMC).

These memoirs span the first fifty years of the European Federation for Medicinal Chemistry (EFMC). They are the personal observations and remembrance of Prof. Henk Timmerman, who witnessed how the EFMC developed since its inception in December 1969, and are published at the occasion of the 50th anniversary of the EFMC. They include, with permission from the EFMC, material that was previously published in EFMC newsletters. These texts are for the first time united and completed, to tell the history of an organization that has accompanied and shaped the development of medicinal chemistry in Europe. They also highlight, through facts and anecdotes, the role of the men and women who are the scientific leaders and drivers of this extended scientific community.

Constitutively active mutants of the histamine H1 receptor suggest a conserved hydrophobic asparagine-cage that constrains the activation of class A G protein-coupled receptors.

The aim of this study was to create and characterize constitutively active mutant (CAM) histamine H(1) receptors (H(1)R) using random mutagenesis methods to further investigate the activation process of the rhodopsin-like family of G protein-coupled receptors (GPCRs). This approach identified position 6.40 in TM 6 as a "hot spot" because mutation of Ile6.40(420) either to Glu, Gly, Ala, Arg, Lys, or Ser resulted in highly active CAM H(1)Rs, for which almost no histamine-induced receptor activation response could be detected. The highly conserved hydrophobic amino acid at position 6.40 defines, in a computational model of the H(1)R, the asparagine cage motif that restrains the side chain of Asn7.49 of the NPxxY motif toward transmembrane domain (TM 6) in the inactive state of the receptor. Mutation of the asparagine cage into Ala or Gly, removing the interfering bulky constraints, increases the constitutive activity of the receptor. The fact that the Ile6.40(420)Arg/Lys/Glu mutant receptors are highly active CAM H(1)Rs leads us to suggest that a positively charged residue, presumably the highly conserved Arg3.50 from the DRY motif, interacts in a direct or an indirect (through other side chains or/and internal water molecules) manner with the acidic Asp2.50..Asn7.49 pair for receptor activation.

4-benzyl-1H-imidazoles with oxazoline termini as histamine H3 receptor agonists.

Research on the therapeutic applications of the histamine H3 receptor (H3R) has traditionally focused on antagonists/inverse agonists. In contrast, H3R agonists have received less attention despite their potential use in several disease areas. The lower availability of H3R agonists not only hampers their full therapeutic exploration, it also prevents an unequivocal understanding of the structural requirements for H3R activation. In the light of these important issues, we present our findings on 4-benzyl-1H-imidazole-based H3R agonists. Starting from two high throughput screen hits (10 and 11), the benzyl side chain was altered with lipophilic groups using combinatorial and classical chemical approaches (compounds 12-31). Alkyne- or oxazolino-substituents gave excellent affinities and agonist activities up to the single digit nM range. Our findings further substantiate the growing notion that basic ligand sidechains are not necessary for H 3R activation and reveal the oxazolino group as a hitherto unexplored functional group in H3R research.

The histamine H3 receptor: from gene cloning to H3 receptor drugs.

Since the cloning of the histamine H(3) receptor cDNA in 1999 by Lovenberg and co-workers, this histamine receptor has gained the interest of many pharmaceutical companies as a potential drug target for the treatment of various important disorders, including obesity, attention-deficit hyperactivity disorder, Alzheimer's disease, schizophrenia, as well as for myocardial ischaemia, migraine and inflammatory diseases. Here, we discuss relevant information on this target protein and describe the development of various H(3) receptor agonists and antagonists, and their effects in preclinical animal models.

The histamine H3 receptor antagonist clobenpropit enhances GABA release to protect against NMDA-induced excitotoxicity through the cAMP/protein kinase A pathway in cultured cortical neurons.

Using the histamine H3 receptor antagonist clobenpropit, the roles of histamine H3 receptors in NMDA-induced necrosis were investigated in rat cultured cortical neurons. Clobenpropit reversed the neurotoxicity in a concentration-dependent manner, and showed peak protection at a concentration of 10(-7) M. This protection was antagonized by the histamine H3 receptor agonist (R)-alpha-methylhistamine, but not by the histamine H1 receptor antagonist pyrilamine or the histamine H2 receptor antagonist cimetidine. In addition, the protection by clobenpropit was inhibited by the GABAA receptor antagonists picrotoxin and bicuculline. Further study demonstrated that the protection by clobenpropit was due to increased GABA release. The inducible GABA release was also inhibited by (R)-alpha-methylhistamine, but not by pyrilamine or cimetidine. Furthermore, both the adenylyl cyclase inhibitor SQ-22536 and the protein kinase A (PKA) inhibitor H-89 reversed the protection and the GABA release by clobenpropit. In addition, clobenpropit reversed the NMDA-induced increase in intracellular calcium level, which was antagonized by (R)-alpha-methylhistamine. These results indicate that clobenpropit enhanced GABA release to protect against NMDA-induced excitotoxicity, which was induced through the cAMP/PKA pathway, and reduction of intracellular calcium level may also be involved.

Joining Forces: The Chemical Biology-Medicinal Chemistry Continuum.

The scientific advances being made across all disciplines are creating ever-increasing opportunities to enhance our knowledge of biological systems and how they relate to human disease. One of the central driving forces in discovering new medicines is medicinal chemistry, where the design and synthesis of novel compounds has led to multiple drugs. Chemical biology, sitting at the interface of many disciplines, has now emerged as a major contributor to the understanding of biological systems and is becoming an integral part of drug discovery. Bringing chemistry and biology much closer and blurring the boundaries between disciplines is creating new opportunities to probe and understand biology; both disciplines play key roles and need to join forces and work together effectively to synergize their impact. The power of chemical biology will then reach its full potential and drive innovation, leading to the discovery of transformative medicines to treat patients. Advances in cancer biology and drug discovery highlight this potential.

A chemical switch for the modulation of the functional activity of higher homologues of histamine on the human histamine H3 receptor: effect of various substitutions at the primary amino function.

In an effort to establish the structural requirements for agonism, neutral antagonism, and inverse agonism at the human histamine H(3) receptor (H(3)R) we have prepared a series of higher homologues of histamine in which the terminal nitrogen of the side chain has been either mono- or disubstituted with several aliphatic, alicyclic, and aromatic moieties or incorporated in cyclic systems. The novel ligands have been pharmacologically investigated in vitro for their affinities on the human H(3)R and H(4)R subtypes by radioligand displacement experiments and for their intrinsic H(3)R activities via a CRE-mediated beta-galactosidase reporter gene assay. Subtle changes of the substitution pattern at the side chain nitrogen alter enormously the pharmacological activity of the ligands, resulting in a series of compounds with a wide spectrum of pharmacological activities. Among the several neutral H(3)R antagonists identified within this series, compounds 2b and 2h display an H(3)R affinity in the low nanomolar concentration range (pK(i) values of 8.1 and 8.4, respectively). A very potent and selective H(3)R agonist (1l, pEC(50) = 8.9, alpha = 0.94) and a very potent, though not highly selective, H(3)R inverse agonist (2k, pIC(50) = 8.9, alpha = -0.97) have been identified as well.

N-substituted piperidinyl alkyl imidazoles: discovery of methimepip as a potent and selective histamine H3 receptor agonist.

In this study, we continue our efforts toward the development of potent and highly selective histamine H(3) receptor agonists. We introduced various alkyl or aryl alkyl groups on the piperidine nitrogen of the known H(3)/H(4) agonist immepip and its analogues (1-3a). We observed that N-methyl-substituted immepip (methimepip) exhibits high affinity and agonist activity at the human histamine H(3) receptor (pK(i) = 9.0 and pEC(50) = 9.5) with a 2000-fold selectivity at the human H(3) receptor over the human H(4) receptor and more than a 10000-fold selectivity over the human histamine H(1) and H(2) receptors. Methimepip was also very effective as an H(3) receptor agonist at the guinea pig ileum (pD(2) = 8.26). Moreover, in vivo microdialysis (in rat brain) showed that methimepip reduces the basal level of brain histamine to about 25% after a 5 mg/kg intraperitoneal administration.

Non-imidazole histamine H3 ligands. Part III. New 4-n-propylpiperazines as non-imidazole histamine H3-antagonists.

In search for a new lead of non-imidazole histamine H3-receptor antagonists, a series of 1[(2-thiazolopyridine)-4-n-propyl]piperazines, the analogous 1-[(2-oxazolopyridine)-4-npropyl]piperazines, 1-[(2-benzothiazole)-4-n-propyl]piperazine and 1-[(2-benzooxazole)4-n-propyl]piperazine were prepared and in vitro tested as H3-receptor antagonists (the electrically evoked contraction of the guinea-pig jejunum). It appeared that by comparison of homologous pairs the thiazolo derivatives have slightly higher activity than their oxazolo analogues. The most potent compound of these series is the 1-(2-thiazolo[4,5-c]pyridine)-4-n-propylpiperazine (3c) with pA2 = 7.25 (its oxazole analogue (4g) showed pA2 = 6.9). The structure-activity relationships for compounds with various positions of the nitrogen in the benzene ring for the thiazoles compared with oxazoles are discussed.

Synthesis and pharmacological identification of neutral histamine H1-receptor antagonists.

In the present study we searched for neutral antagonists for the human histamine H(1)-receptor (H(1)R) by screening newly synthesized ligands that are structurally related to H(1)R agonists for their affinity using radioligand displacement studies and by assessing their functional activity via performing a NF-kappaB driven reporter-gene assay that allows for the detection of both agonistic and inverse agonistic responses. Starting from the endogenous agonist for the H(1)R, histamine, we synthesized and tested various analogues and ultimately identified several compounds with partial inverse agonistic properties and two neutral H(1)-receptor antagonists, namely 2-[2-(4,4-diphenylbutyl)-1H-imidazol-4-yl]ethylamine (histabudifen, 18d) (pK(i) = 5.8, alpha = 0.02) and 2-[2-(5,5-diphenylpentyl)-1H-imidazol-4-yl]ethylamine (histapendifen, 18e) (pK(i) = 5.9, alpha = -0.09).

Identification of 4-(1H-imidazol-4(5)-ylmethyl)pyridine (immethridine) as a novel, potent, and highly selective histamine H(3) receptor agonist.

In this study, the piperidine ring of immepip and its analogues was replaced by a rigid heterocyclic pyridine ring. Many compounds in the series exhibit high affinity and agonist activity at the human histamine H(3) receptor. Particularly, the 4-pyridinyl analogue of immepip (1c, immethridine) is identified as a novel potent and highly selective histamine H(3) receptor agonist (pK(i) = 9.07, pEC(50) = 9.74) with a 300-fold selectivity over the closely related H(4) receptor.

SYNOPSIS: SYNthesize and OPtimize System in Silico.

We present a de novo design program called SYNOPSIS, that includes a synthesis route for each generated molecule. SYNOPSIS designs novel molecules by starting from a database of available molecules and simulating organic synthesis steps. This way of generating molecules imposes synthetic accessibility on the molecules. In addition to a starting database, a fitness function is needed that calculates the value of a desired property for an arbitrary molecule. The values obtained from this function guide the design process in optimizing the molecules toward an optimal value of the calculated property. Two applications are described. The first uses an electric dipole moment calculation to generate molecules possessing a strong dipole moment. The second makes use of the three-dimensional structure of a viral enzyme in order to generate high affinity ligands. Twenty eight compounds designed with the program resulted in 18 synthesized and tested compounds, 10 of which showed HIV inhibitory activity in vitro.

Synthesis and structure-activity relationships of conformationally constrained histamine H(3) receptor agonists.

Immepip, a conformationally constrained analogue of the histamine congener imbutamine, shows high affinity and functional activity on the human H(3) receptor. Using histamine and its homologues as prototypes, other rigid analogues containing either a piperidine or pyrrolidine ring in the side chain were synthesized and tested for their activities at the human H(3) receptor and the closely related H(4) receptor. In the series of piperidine containing analogues, immepip was found to be the most potent H(3) receptor agonist, whereas its propylene analogue 13a was identified as a high-affinity neutral antagonist for the human H(3) receptor. Moreover, replacement of the piperidine ring of immepip by a pyrrolidine ring led to a pair of enantiomers that show a distinct stereoselectivity at the human H(3) and H(4) receptor.

Effect of the histamine H3-antagonist clobenpropit on spatial memory deficits induced by MK-801 as evaluated by radial maze in Sprague-Dawley rats.

This study was performed to investigate whether or not the histamine H3-antagonist clobenpropit can ameliorate spatial memory deficits induced by MK-801 (0.3 microg per site) as evaluated by an eight-arm radial maze task of rats. A bilateral intrahippocampal (i.h.) injection of clobenpropit (5, 10 microg per site, dose-dependent) markedly improved the working and reference memory deficits induced by MK-801. Its ameliorating effect was potentiated by histidine, but completely antagonized by immepip (2.5 microg per site), a selective H3-agonist. alpha-Fluoromethylhistidine (FMH, 25 microg per site), a selective histidine decarboxylase inhibitor prevented the ameliorating effect of clobenpropit on the working memory deficits induced by MK-801. In addition, the H(1-antagonist pyrilamine, but not the H2-antagonist cimetidine, also inhibited the procognitive effects of clobenpropit. Both FMH and pyrilamine did not significantly modulate the effect of clobenpropit on reference memory. Therefore, the results of this study suggest that the procognitive effects of clobenpropit in MK-801-induced working memory deficits is mediated by increasing endogenous histamine release. In addition, the ameliorating effect of clobenpropit on reference memory might be due to the increased release of neurotransmitters other than histamine.

Improgan antinociception does not require neuronal histamine or histamine receptors.

Improgan, a chemical congener of the H(2) antagonist cimetidine, induces antinociception following intracerebroventricular (i.c.v.) administration in rodents, but the mechanism of action of this compound remains unknown. Because the chemical structure of improgan closely resembles those of histamine and certain histamine blockers, and because neuronal histamine is known to participate in pain-relieving responses, the antinociceptive actions of improgan were evaluated in mice containing null mutations in the genes for three histamine receptors (H(1), H(2), and H(3)) and also in the gene for histidine decarboxylase (the histamine biosynthetic enzyme). Similar to earlier findings in Swiss-Webster mice, improgan induced maximal, reversible, dose-related reductions in thermal nociceptive responses in ICR mice, but neither pre-improgan (baseline) nor post-improgan nociceptive latencies were changed in any of the mutant mice as compared with wild-type controls. Improgan also had weak inhibitory activity in vitro (pK(i)=4.7-4.9) on specific binding to three recently-discovered, recombinant isoforms of the rat H(3) receptor (H(3A), H(3B), and H(3C)). The present findings strongly support the hypothesis that neuronal histamine and its receptors fail to play a role in improgan-induced antinociception.

Effects of clobenpropit on pentylenetetrazole-kindled seizures in rats.

The purpose of this study was to investigate whether or not clobenpropit, a selective and potent histamine H(3) receptor antagonist, can protect from pentylenetetrazole (35 mg/kg)-kindled seizures in rats. I.c.v. injection with clobenpropit (10 and 20 microg) significantly delayed the seizure stage and prolonged the latency to the onset of myoclonic jerks and the latency to the clonic generalized seizure in a dose-dependent manner. The protection by clobenpropit (20 microg) was completely antagonized by both immepip (5 and 10 microg, i.c.v.), a selective potent histamine H(3) receptor agonist, and alpha-fluoromethylhistidine (alpha-FMH, 50 microg, i.c.v.), a selective histidine decarboxylase inhibitor. In addition, clobenpropit markedly potentiated the histidine (100 and 200 mg/kg)-induced inhibition of pentylenetetrazole-kindled seizures. Pyrilamine (2 and 5 microg, i.c.v.) reversed the inhibition of pentylenetetrazole-kindled seizures induced by clobenpropit, whereas cimetidine had no effect even at a high dose of 5 microg. These results indicate that clobenpropit protects against pentylenetetrazole-kindled seizures in rats, and that its action is mainly due to the activation of endogenous histamine by blocking autoinhibitory presynaptic histamine H(3) receptors.

Mutational analysis of the histamine H1-receptor binding pocket of histaprodifens.

Histaprodifens constitute a new class of histamine H(1)-receptor agonists. These ligands can be regarded as hybrid molecules, consisting of a histamine moiety linked at the two-position of the imidazole ring by a propyl chain to two phenyl rings, one of the characteristic features of several H(1)-receptor antagonists. To delineate the binding site of various histaprodifen-like ligands, we generated mutant histamine H(1) receptors, in which various amino acids, involved in the binding of either histamine or H(1)-receptor antagonists, were replaced by alanine. Wild-type and mutant H(1) receptors were transiently expressed in African green monkey kidney cells (COS-7) and evaluated for their interaction with histamine and various histaprodifens by [(3)H]mepyramine radioligand-binding studies and by nuclear factor kappaB (NF-kappaB) reporter-gene assays. Our data show that, within the histamine H(1)-receptor binding pocket, histaprodifens interact with both agonist and antagonist binding sites, resulting in high affinity histamine H(1)-receptor agonists.