International Union of Basic and Clinical Pharmacology. XCVIII. Histamine Receptors.

Histamine is a developmentally highly conserved autacoid found in most vertebrate tissues. Its physiological functions are mediated by four 7-transmembrane G protein-coupled receptors (H1R, H2R, H3R, H4R) that are all targets of pharmacological intervention. The receptors display molecular heterogeneity and constitutive activity. H1R antagonists are long known antiallergic and sedating drugs, whereas the H2R was identified in the 1970s and led to the development of H2R-antagonists that revolutionized stomach ulcer treatment. The crystal structure of ligand-bound H1R has rendered it possible to design new ligands with novel properties. The H3R is an autoreceptor and heteroreceptor providing negative feedback on histaminergic and inhibition on other neurons. A block of these actions promotes waking. The H4R occurs on immuncompetent cells and the development of anti-inflammatory drugs is anticipated.

Discovery of GLPG2737, a Potent Type 2 Corrector of CFTR for the Treatment of Cystic Fibrosis in Combination with a Potentiator and a Type 1 Co-corrector.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.

Targeting the Tyrosine Kinase 2 (TYK2) Pseudokinase Domain: Discovery of the Selective TYK2 Inhibitor ABBV-712.

Tyrosine kinase 2 (TYK2) is a nonreceptor tyrosine kinase that belongs to the JAK family also comprising JAK1, JAK2, and JAK3. TYK2 is an attractive target for various autoimmune diseases as it regulates signal transduction downstream of IL-23 and IL-12 receptors. Selective TYK2 inhibition offers a differentiated clinical profile compared to currently approved JAK inhibitors. However, selectivity for TYK2 versus other JAK family members has been difficult to achieve with small molecules that inhibit the catalytically active kinase domain. Successful targeting of the TYK2 pseudokinase domain as a strategy to achieve isoform selectivity was recently exemplified with deucravacitinib. Described herein is the optimization of selective TYK2 inhibitors targeting the pseudokinase domain, resulting in the discovery of the clinical candidate ABBV-712 (21).

Antagonism of supraspinal histamine H3 receptors modulates spinal neuronal activity in neuropathic rats.

There is growing evidence supporting a role for histamine H(3) receptors in the modulation of pathological pain. To further our understanding of this modulation, we examined the effects of a selective H(3) receptor antagonist, 6-((3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy)-N-methyl-3-pyridinecarboxamide (GSK189254), on spinal neuronal activity in neuropathic (L5 and L6 ligations) and sham rats. Systemic administration of GSK189254 (0.03-1 mg/kg i.v.) dose-dependently decreased both evoked (10-g von Frey hair for 15 s) and spontaneous firing of wide dynamic range (WDR) neurons in neuropathic, but not sham-operated, animals. The effects on spontaneous firing suggest that H(3) receptors may have a role in central sensitization and/or modulating non-evoked pain. Transection of the spinal cord (T9-T10) completely eliminated the effects (both evoked and spontaneous) of systemic GSK189254 (1 mg/kg, i.v.) on WDR neuronal firing in neuropathic rats, indicating that the descending modulatory system has an important role in the H(3)-related dampening of spinal neuronal activity. Subsequently, lesions of the locus coeruleus, or direct GSK189254 (3 and 10 nmol/0.5 µl) injections into this site, demonstrate that the locus coeruleus is a key component of the H(3) descending modulatory pathway. In summary, blockade of H(3) receptors reduces spontaneous firing as well as the responses of spinal nociceptive neurons to mechanical stimulation. This effect is in large part mediated via supraspinal sites, including the locus coeruleus, that send descending projections to modulate spinal neuronal activity.

Pharmacological properties and procognitive effects of ABT-288, a potent and selective histamine H3 receptor antagonist.

Blockade of the histamine H(3) receptor (H(3)R) enhances central neurotransmitter release, making it an attractive target for the treatment of cognitive disorders. Here, we present in vitro and in vivo pharmacological profiles for the H(3)R antagonist 2-[4'-((3aR,6aR)-5-methyl-hexahydro-pyrrolo[3,4-b]pyrrol-1-yl)-biphenyl-4-yl]-2H-pyridazin-3-one (ABT-288). ABT-288 is a competitive antagonist with high affinity and selectivity for human and rat H(3)Rs (K(i) = 1.9 and 8.2 nM, respectively) that enhances the release of acetylcholine and dopamine in rat prefrontal cortex. In rat behavioral tests, ABT-288 improved acquisition of a five-trial inhibitory avoidance test in rat pups (0.001-0.03 mg/kg), social recognition memory in adult rats (0.03-0.1 mg/kg), and spatial learning and reference memory in a rat water maze test (0.1-1.0 mg/kg). ABT-288 attenuated methamphetamine-induced hyperactivity in mice. In vivo rat brain H(3)R occupancy of ABT-288 was assessed in relation to rodent doses and exposure levels in behavioral tests. ABT-288 demonstrated a number of other favorable attributes, including good pharmacokinetics and oral bioavailability of 37 to 66%, with a wide central nervous system and cardiovascular safety margin. Thus, ABT-288 is a selective H(3)R antagonist with broad procognitive efficacy in rodents and excellent drug-like properties that support its advancement to the clinical area.

Discovery of histamine H3 antagonists for the treatment of cognitive disorders and Alzheimer's disease.

H(3) antagonists increase the release of brain histamine, acetylcholine, noradrenaline, and dopamine, neurotransmitters that are known to modulate cognitive processes. The ability to release brain histamine supports the effect on attention and vigilance, but histamine also modulates other cognitive domains such as short-term and long-term memory. A number of H(3) antagonists, including 1-{3-[3-(4-chlorophenyl)propoxy]propyl}piperidine hydrochloride (BF2.649), (1R,3R)-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-ylmethyl)phenyl]cyclobutane-1-carboxamide (PF-03654746), 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254), MK-0249 (structure not yet disclosed), JNJ-17216498 (structure not yet disclosed), and ABT-288 (structure not yet disclosed), have advanced to the clinical area for the potential treatment of human cognitive disorders. H(3) antagonists exhibited wake-promoting effects in humans and efficacy in narcoleptic patients, indicating target engagement, but some of them were not efficacious in patients suffering from attention-deficit hyperactivity disorder and schizophrenic patients. Preclinical studies have also shown that H(3) antagonists activate intracellular signaling pathways that may improve cognitive efficacy and disease-modifying effects in Alzheimer's disease. Ongoing clinical studies will be able to determine the utility of H(3) antagonists for the treatment of cognitive disorders in humans.

Discovery of GLPG2451, a Novel Once Daily Potentiator for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is a life-threatening recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR). With the discovery of Ivacaftor and Lumacaftor, it has been shown that administration of one or more small molecules can partially restore the CFTR function. Correctors are small molecules that enhance the amount of CFTR on the cell surface, while potentiators improve the gating function of the CFTR channel. Herein, we describe the discovery and optimization of a novel potentiator series. Scaffold hopping, focusing on retaining the different intramolecular contacts, was crucial in the whole discovery process to identify a novel series devoid of genotoxic liabilities. From this series, the clinical candidate GLPG2451 was selected based on its pharmacokinetic properties, allowing QD dosing and based on its low CYP induction potential.

In vitro studies on a class of quinoline containing histamine H3 antagonists.

A series of quinoline containing histamine H(3) antagonists is reported herein. These analogs were synthesized via the Friedlander quinoline synthesis between an aminoaldehyde intermediate and a methyl ketone allowing for a wide diversity of substituents at the 2-position of the quinoline ring.

Rigidified 2-aminopyrimidines as histamine H4 receptor antagonists: effects of substitution about the rigidifying ring.

Three novel series of histamine H(4) receptor (H(4)R) antagonists containing the 2-aminopyrimidine motif are reported. The best of these compounds display good in vitro potency in both functional and binding assays. In addition, representative compounds are able to completely block itch responses when dosed ip in a mouse model of H(4)-agonist induced scratching, thus demonstrating their activities as H(4)R antagonists.

Identification of GLPG/ABBV-2737, a Novel Class of Corrector, Which Exerts Functional Synergy With Other CFTR Modulators.

The deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) causes a severe defect in folding and trafficking of the chloride channel resulting in its absence at the plasma membrane of epithelial cells leading to cystic fibrosis. Progress in the understanding of the disease increased over the past decades and led to the awareness that combinations of mechanistically different CFTR modulators are required to obtain meaningful clinical benefit. Today, there remains an unmet need for identification and development of more effective CFTR modulator combinations to improve existing therapies for patients carrying the F508del mutation. Here, we describe the identification of a novel F508del corrector using functional assays. We provide experimental evidence that the clinical candidate GLPG/ABBV-2737 represents a novel class of corrector exerting activity both on its own and in combination with VX809 or GLPG/ABBV-2222.

A robust and high-capacity [(35)S]GTPgammaS binding assay for determining antagonist and inverse agonist pharmacological parameters of histamine H(3) receptor ligands.

Guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays were established and utilized as a reliable and high-capacity functional assay for determining antagonist and inverse agonist pharmacological parameters of novel histamine H(3) ligands, at the recombinant human H(3) receptor. [(35)S]GTPgammaS binding assays were performed with membranes prepared from human embryonic kidney 293 cells stably expressing the full-length (445 amino acids) human H(3) receptor isoform, at approximately 1 pmol/mg of protein. Utilizing robotic liquid handling, assay filtration, and scintillation counting in a 96-well format, concentration-response curves were determined for up to 40 compounds per assay. The imidazole-containing H(3) receptor antagonist ciproxifan and the non-imidazole antagonist ABT-239 inhibited (R)-alpha-methylhistamine (RAMH)-stimulated [(35)S]GTPgammaS binding in a competitive manner, and negative logarithm of the dissociation equilibrium constant (pK(b)) values determined for nearly 200 structurally diverse H(3) antagonists were very similar to the respective negative logarithm of the equilibrium inhibition constant values from N-alpha-[(3)H]methylhistamine competition binding assays. H(3) antagonists also concentration-dependently decreased basal [(35)S]GTPgammaS binding, thereby displaying inverse agonism at the constitutively active H(3) receptor. At maximally effective concentrations, non-imidazole H(3) antagonists inhibited basal [(35)S]GTPgammaS binding by approximately 20%. For over 100 of these antagonists, negative logarithm of the 50% effective concentration values for inverse agonism were very similar to the respective pK(b) values. Both H(3) receptor agonist-dependent and -independent (constitutive) [(35)S]GTPgammaS binding were sensitive to changes in assay concentrations of sodium, magnesium, and the guanine nucleotide GDP; however, the potency of ABT-239 for inhibition of RAMH-stimulated [(35)S]GTPgammaS binding was not significantly affected. These robust and reliable [(35)S]GTPgammaS binding assays have become one of the important tools in our pharmacological analysis and development of novel histamine H(3) receptor antagonists/inverse agonists.

In vitro SAR of pyrrolidine-containing histamine H3 receptor antagonists: trends across multiple chemical series.

Structure-activity relationships (SAR) were analyzed within a library of diverse yet simple compounds prepared as histamine H3 antagonists. The libraries were constructed with a variety of low molecular weight pyrrolidines, selected from (R)-2-methylpyrrolidine, (S)-2-methylpyrrolidine, and pyrrolidine.

An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H3 isoform confers pharmacological differences and constitutive activity.

In this article, we pharmacologically characterized two naturally occurring human histamine H3 receptor (hH3R) isoforms, hH3R(445) and hH3R(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. In this report, we show that the hH3R(365) is differentially expressed compared with the hH3R(445) and has a higher affinity and potency for H3R agonists and conversely a lower potency and affinity for H3R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH3R(365) compared with the hH3R(445) in both guanosine-5'-O-(3-[35S]thio) triphosphate binding and cAMP assays, likely explaining the observed differences in hH3R pharmacology of the two isoforms. Because H3R ligands are beneficial in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder and currently entered clinical trails, these differences in H3R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H3R ligands.

Synthesis, potency, and in vivo profiles of quinoline containing histamine H3 receptor inverse agonists.

A new structural series of histamine H3 receptor antagonist was developed. The new compounds are based on a quinoline core, appended with a required basic aminoethyl moiety, and with potency- and property-modulating heterocyclic substituents. The analogs have nanomolar and subnanomolar potency for the rat and human H3R in various in vitro assays, including radioligand competition binding as well as functional tests of H3 receptor-mediated calcium mobilization and GTPgammaS binding. The compounds possessed favorable drug-like properties, such as good PK, CNS penetration, and moderate protein binding across species. Several compounds were found to be efficacious in animal behavioral models of cognition and attention. Further studies on the pharmaceutic properties of this series of quinolines discovered a potential problem with photochemical instability, an issue which contributed to the discontinuation of this series from further development.

Novel heterocyclic-substituted benzofuran histamine H3 receptor antagonists: in vitro properties, drug-likeness, and behavioral activity.

Three novel heterocyclic benzofurans A-688057 (1), A-687136 (2), and A-698418 (3) were profiled for their in vitro and in vivo properties as a new series of histamine H(3) receptor antagonists. The compounds were all found to have nanomolar potency in vitro at histamine H(3) receptors, and when profiled in vivo for CNS activity, all were found active in an animal behavioral model of attention. The compound with the most benign profile versus CNS side effects was selected for greater scrutiny of its in vitro properties and overall drug-likeness. This compound, A-688057, in addition to its potent and robust efficacy in two rodent behavioral models at blood levels ranging 0.2-19 nM, possessed other favorable features, including high selectivity for H(3) receptors (H(3), K(i)=1.5 nM) versus off-target receptors and channels (including the hERG K(+) channel, K(i)>9000 nM), low molecular weight (295), high solubility, moderate lipophilicity (logD(pH7.4)=2.05), and good CNS penetration (blood/brain 3.4x). In vitro toxicological tests indicated low potential for phospholipidosis, genotoxicity, and CYP(450) inhibition. Even though pharmacokinetic testing uncovered only moderate to poor oral bioavailability in rat (26%), dog (30%), and monkey (8%), and only moderate blood half-lives after i.v. administration (t(1/2) in rat of 2.9h, 1.7h in dog, 1.8h in monkey), suggesting poor human pharmacokinetics, the data overall indicated that A-688057 has an excellent profile for use as a pharmacological tool compound.

Crystal structures of human adenosine kinase inhibitor complexes reveal two distinct binding modes.

Adenosine kinase (AK) is an enzyme responsible for converting endogenous adenosine (ADO) to adenosine monophosphate (AMP) in an adenosine triphosphate- (ATP-) dependent manner. The structure of AK consists of two domains, the first a large alpha/beta Rossmann-like nucleotide binding domain that forms the ATP binding site, and a smaller mixed alpha/beta domain, which, in combination with the larger domain, forms the ADO binding site and the site of phosphoryl transfer. AK inhibitors have been under investigation as antinociceptive, antiinflammatory, and anticonvulsant as well as antiinfective agents. In this work, we report the structures of AK in complex with two classes of inhibitors: the first, ADO-like, and the second, a novel alkynylpyrimidine series. The two classes of structures, which contain structurally similar substituents, reveal distinct binding modes in which the AK structure accommodates the inhibitor classes by a 30 degrees rotation of the small domain relative to the large domain. This change in binding mode stabilizes an open and a closed intermediate structural state and provide structural insight into the transition required for catalysis. This results in a significant rearrangement of both the protein active site and the orientation of the alkynylpyrimidine ligand when compared to the observed orientation of nucleosidic inhibitors or substrates.

Discovery of 3-methyl-N-(1-oxy-3',4',5',6'-tetrahydro-2'H-[2,4'-bipyridine]-1'-ylmethyl)benzamide (ABT-670), an orally bioavailable dopamine D4 agonist for the treatment of erectile dysfunction.

The goal of this study was to identify a structurally distinct D(4)-selective agonist with superior oral bioavailability to our first-generation clinical candidate 1a (ABT-724) for the potential treatment of erectile dysfunction. Arylpiperazines such as (heteroarylmethyl)piperazine 1a, benzamide 2, and acetamides such as 3a,b exhibit poor oral bioavailability. Structure-activity relationship (SAR) studies with the arylpiperidine template provided potent partial agonists such as 4d and 5k that demonstrated no improvement in oral bioavailability. Further optimization with the (N-oxy-2-pyridinyl)piperidine template led to the discovery of compound 6b (ABT-670), which exhibited excellent oral bioavailability in rat, dog, and monkey (68%, 85%, and 91%, respectively) with comparable efficacy, safety, and tolerability to 1a. The N-oxy-2-pyridinyl moiety not only provided the structural motif required for agonist function but also reduced metabolism rates. The SAR study leading to the discovery of 6b is described herein.

Lack of efficacy of melanin-concentrating hormone-1 receptor antagonists in models of depression and anxiety.

The aim of this study was to validate melanin-concentrating hormone (MCH)-1 receptor antagonism as a potential treatment of mood disorders. We attempted to replicate the effects previously reported with SNAP-7941 and expanded the investigation to three other orally bioavailable MCH-1 receptor antagonists with good brain penetration. SNAP-7941 (3-30 mg/kg, i.p.) and T-226296 (5-60 mg/kg, p.o.) (+/- racemate), were evaluated in the rat forced swim and mouse tail suspension tests. (+)SNAP-7941 (3-10 mg/kg, p.o.) was also tested in a modified 5-min rat forced swim protocol as previously reported. A-665798 (3-30 mg/kg, p.o.) and A-777903 (3-30 mg/kg, p.o.) were tested in mouse tail suspension and rat Vogel tests. None of the compounds showed meaningful efficacy in the paradigms tested. The lack of efficacy with four structurally different MCH-1 receptor antagonists does not support a role for therapeutic treatment of depression/anxiety via this mechanism of action.

Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 Antagonists.

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.