The identification, and optimisation of hERG selectivity, of a mixed NET/SERT re-uptake inhibitor for the treatment of pain.

Hit compound 1, a selective noradrenaline re-uptake transporter (NET) inhibitor was optimised to build in potency at the serotonin re-uptake transporter (SERT) whilst maintaining selectivity against the dopamine re-uptake transporter (DAT). During the optimisation of 1 it became clear that selectivity against the Kv11.1 potassium ion channel (hERG) was also a parameter for optimisation within the series. Discrete structural changes to the molecule as well as a lowering of global cLogP successfully increased the hERG selectivity to afford compound 11 m, which was efficacious in a mouse model of inflammatory pain, complete Freund's adjuvant (CFA) induced thermal hyperalgesia and a rat model of neuropathic pain, spinal nerve ligation (SNL) induced mechanical allodynia.

Fragment-based discovery of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Fragment-based NMR screening of a small literature focused library led to identification of a historical thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment optimized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based efficacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse. Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy for the series, however, it has a poor PK profile relative to 23A.

Pharmacological characterization of a fluorescent uptake assay for the noradrenaline transporter.

The noradrenaline transporter (NET) is a Na(+)/Cl(-) dependent monoamine transporter that mediates rapid clearance of noradrenaline from the synaptic cleft, thereby terminating neuronal signaling. NET is an important target for drug development and is known to be modulated by many psychoactive compounds, including psychostimulants and antidepressants. Here, the authors describe the development and pharmacological characterization of a nonhomogeneous fluorescent NET uptake assay using the compound 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP(+)). Data presented show that the pharmacology of both the classic radiolabeled (3)H-noradrenaline- and ASP(+)-based uptake assays are comparable, with an excellent correlation between potency obtained for known modulators of NET (r = 0.95, p < 0.0001). Furthermore, the fluorescent uptake assay is highly reproducible and has sufficiently large Z' values to be amenable for high-throughput screening (HTS). The advantage of this assay is compatibility with both 96- and 384-well formats and lack of radioactivity usage. Thus, the authors conclude that the assay is an inexpensive, viable approach for the identification and pharmacological profiling of small-molecule modulators of the monoamine transporter NET and may be amenable for HTS.

Short-term plasticity of cyclic adenosine 3',5'-monophosphate signaling in anterior pituitary corticotrope cells: the role of adenylyl cyclase isotypes.

Anterior pituitary corticotropes show a wide repertory of responses to hypothalamic neuropeptides and adrenal corticosteroids. The hypothesis that plasticity of the cAMP signaling system underlies this adaptive versatility was investigated. In dispersed rat anterior pituitary cells, depletion of intracellular Ca2+ stores with thapsigargin combined with ryanodine or caffeine enhanced the corticotropin releasing-factor (CRF)-evoked cAMP response by 4-fold, whereas reduction of Ca2+ entry alone had no effect. CRF-induced cAMP was amplified 15-fold by arginine-vasopressin (AVP) or phorbol-dibutyrate ester. In the presence of inhibitors of cyclic nucleotide phosphodiesterases and phorbol-dibutyrate ester, the depletion of Ca2+ stores had no further effect on CRF-induced cAMP accumulation. Adenohypophysial expression of mRNAs for the Ca2+-inhibited adenylyl cyclases (ACs) VI and IX, and the protein kinase C-stimulated ACs II and VII was demonstrated. ACIX was detected in corticotropes by immunocytochemistry, whereas ACII and ACVI were not present. The data show negative feedback regulation of CRF-induced cAMP levels by Ca2+ derived from ryanodine receptor-operated intracellular stores. Stimulation of protein kinase C by AVP enhances Ca2+-independent cAMP synthesis, thus changing the characteristics of intracellular Ca2+ feedback. It is proposed that the modulation of intracellular Ca2+ feedback in corticotropes by AVP is an important element of physiological control.

X-ray structures of the LXRalpha LBD in its homodimeric form and implications for heterodimer signaling.

Liver X receptors (LXRs) are nuclear receptors that are central regulators of cholesterol homeostasis, and synthetic LXR agonists have shown promise as promoters of reverse cholesterol transport and anti-inflammatory agents. Here, we present three X-ray structures of three different agonists bound to the ligand binding domain of LXRalpha. These compounds are GW3965, F(3)methylAA, and a benzisoxazole urea, and we show that these diverse chemical scaffolds address common structural themes, leading to high binding affinity for LXR. Our structures show the LXRalpha ligand binding domain in its homodimeric form, an arrangement previously thought to be stereochemically difficult. A comparison with existing structures of the LXRbeta homodimer and LXRalpha:RXR (retinoid X receptor) heterodimers explains differences in dimer affinity and leads us to propose a model for allosteric activation in nuclear receptor dimers, in which an unactivated RXR partner provides an inhibitory tail wrap to the cofactor binding pocket of LXR.

Small ligands modulating the activity of mammalian adenylyl cyclases: a novel mode of inhibition by calmidazolium.

Molecular cloning of membrane-spanning mammalian adenylyl cyclases (ACs) has led to the discovery of nine different isotypes, making ACs potentially useful therapeutic targets. This study investigated the mechanism by which fungicidal nitroimidazole compounds modulate AC activity. Current evidence indicates that biological control of AC activity occurs through the cytosolic domains. Hence, full-length ACII, ACIX, and recombinant fusion proteins composed of the cytoplasmic loops of human ACIX or the first and second cytoplasmic loops of rat ACV and ACII, respectively, were expressed in human embryonic kidney 293 cells. The AC activities of the respective proteins were characterized, and their modulation by nitroimidazoles was investigated. Calmidazolium inhibited the activities of both full-length ACs and soluble fusion proteins (IC(50), approximately 10 microM). Inhibition of ACIX by calmidazolium was mediated by direct interaction with the catalytic core in a noncompetitive fashion. ACIX was essentially insensitive to 2'-deoxyadenosine 3'-monophosphate, a known blocker of AC activity. The ACV-ACII fusion protein was inhibited by calmidazolium (IC(50), approximately 20 microM) as well as by 2'-deoxyadenosine 3'-AMP (IC(50), approximately 2 microM), in a manner indicating independent mechanisms of action. Taken together, the data demonstrate that ACIX is insensitive to adenosine analogs and that calmidazolium inhibits AC activity by a novel, noncompetitive mechanism.