KCNQ2/3 openers show differential selectivity and site of action across multiple KCNQ channels.

KCNQ2/3 voltage-gated potassium channels conduct low-threshold, slowly activating and non-inactivating currents to repolarize the neuronal resting membrane potential. The channels negatively regulate neuronal excitability and KCNQ2/3 openers are efficacious in hyperexcited states such as epilepsy and pain. We developed and utilized thallium influx assays to profile novel KCNQ2/3 channel openers with respect to selectivity across KCNQ subtypes and on requirement for tryptophan 236 of KCNQ2, a critical residue for activity of the KCNQ opener retigabine. Using distinct chemical series of openers, a quinazolinone series showed relatively poor selectivity across multiple KCNQ channels and lacked activity at the KCNQ2(W236L) mutant channel. In contrast, several novel benzimidazole openers showed selectivity for KCNQ2/3 and KCNQ2 and retain activity at KCNQ2(W236L). Profiling of several hundred KCNQ2/3 openers across multiple diverse chemical series revealed that openers show differential degrees of selectivity across subtypes, with selectivity most difficult to achieve against KCNQ2. In addition, we report the significant finding that KCNQ openers can pharmacologically differentiate between homomeric and heteromeric channels containing subtypes in common. Moreover, most openers assayed were dependent on the W236 for activity, whereas only a small number appear to use a distinct mechanism. Collectively, we provide novel insights into the molecular pharmacology of KCNQ channels by demonstrating differential selectivity and site of action for KCNQ2/3 openers. The high-throughput thallium influx assays should prove useful for rapid characterization of KCNQ openers and in guiding efforts to identify selective compounds for advancement towards the clinic.

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.

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.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

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.

G protein-dependent pharmacology of histamine H3 receptor ligands: evidence for heterogeneous active state receptor conformations.

Previously reported pharmacological studies using the imidazole-containing histamine H3 receptor ligands GT-2331 (Cipralisant) and proxyfan resulted in a range of classifications (antagonist, agonist, and protean) for these compounds. We examined the role that the signaling system, with particular emphasis on the type of G protein, had on the pharmacology observed for H3 ligands. Ligands were assessed using assays measuring neurotransmitter release, cAMP, and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. Whereas clobenpropit and ciproxifan were consistently antagonists, GT-2331, proxyfan, and imetit exhibited differential activity. Although GT-2331 and proxyfan exhibited little agonist activity in neurotransmitter release assays, both demonstrated full agonism relative to (R)-alpha-methylhistamine in cAMP assays. In [35S]GTPgammaS binding assays, GT-2331 and proxyfan demonstrated partial agonism. Imetit showed full agonism in most assays, but it was slightly less efficacious in a neurotransmitter release assay and in [35S]GTPgammaS binding at the human H3 receptor. To further examine these ligands, we coexpressed G alpha16 or chimeric G alpha q/i5 in human embryonic kidney cells expressing the human H3 receptor and assayed intracellular calcium and cAMP levels. GT-2331, proxyfan, and imetit demonstrated full agonism in all assays of cAMP activity. However, in cells expressing G alpha16, they exhibited minimal agonism in calcium mobilization assays, whereas imetit showed partial agonism. When G alpha q/i5 was used, the activity of both GT-2331 and proxyfan increased, whereas imetit became a full agonist. These results demonstrate that GT-2331 and proxyfan's differential pharmacology at the H3 receptor depends on the type of G protein used and provide indirect evidence for differential ligand-bound active states that mediate signaling by the H3 receptor.

Pharmacological and behavioral properties of A-349821, a selective and potent human histamine H3 receptor antagonist.

Histamine H3 receptors regulate the release of a variety of central neurotransmitters involved in cognitive processes. A-349821 ((4'-(3-((R,R)2,5-dimethyl-pyrrolidin-1-yl)-propoxy)-biphenyl-4-yl)-morpholin-4-yl-methanone) is a novel, non-imidazole H3 receptor ligand, displaying high affinity for recombinant rat and human H3 receptors, with pKi values of 9.4 and 8.8, respectively, and high selectivity for the H3 receptor versus H1, H2, and H4 histamine receptors. A-349821 is a potent H3 receptor antagonist in a variety of models using recombinant human and rat receptors, reversing agonist induced changes in cyclic AMP formation (pKb= 8.2 and pKb= 8.1, respectively), [35S]-GTPgammaS binding (pKb= 9.3 and pKb= 8.6, respectively) and calcium levels (human pKb= 8.3). In native systems, A-349821 competitively reversed agonist induced inhibition of electric field stimulated guinea-pig ileum (pA2= 9.5) and histamine-mediated inhibition of [3H]-histamine release from rat brain cortical synaptosomes (pKb= 9.2). Additionally, A-349821 inhibited constitutive GTPgammaS binding at both rat and human H3 receptors with respective pEC50 values of 9.1 and 8.6, demonstrating potent inverse agonist properties. In behavioral studies, A-349821 (0.4 mg/kg-4 mg/kg) potently blocked (R)-alpha-methylhistamine-induced dipsogenia in mice. The compound also enhanced cognitive activity in a five-trial inhibitory avoidance model in spontaneously hypertensive rat (SHR) pups at doses of 1-10mg/kg, with the 1mg/kg dose showing comparable efficacy to a fully efficacious dose of ciproxifan (3mg/kg). These doses of A-349821 were without effect on spontaneous locomotor activity. Thus, A-349821 is a novel, selective non-imidazole H3 antagonist/inverse agonist with balanced high potency across species and favorable cognition enhancing effects in rats.