Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists

Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood–brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited ¹²⁵I-NPS binding in the CNS when administered to rats.     

Pyridyl amides as potent inhibitors of T-type calcium channels

A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-type antagonist 37 that displayed in vivo efficacy in rodent models of epilepsy and sleep.     

Development of a fluorescence polarization binding assay for asialoglycoprotein receptor

Asialoglycoprotein receptor (ASGP-R) has been actively investigated for targeted delivery of therapeutic agents into hepatocytes because this receptor is selectively and highly expressed in liver and has a high internalization rate. Synthetic cluster glycopeptides (e.g., triGalNAc) bind with high affinity to ASGP-R and, when conjugated to a therapeutic agent, can drive receptor-mediated uptake in liver. We developed a novel fluorescent polarization (FP) ASGP-R binding assay to determine the binding affinities of ASGP-R-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from rat liver as a source of ASGP-R and Cy5 fluorophore-labeled triGalNAc synthetic ligand as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent in determining binding affinities to ASGP-R. At the optimized conditions, a Z' factor of 0.73 was achieved in a 96-well format.     

Distinct domains of the voltage-gated K+ channel Kvbeta 1.3 beta -subunit affect voltage-dependent gating

The Kv1.3 subunit confers a voltage-dependent, partialinactivation (time constant = 5.76 ± 0.14 ms at +50 mV), anenhanced slow inactivation, a hyperpolarizing shift in the activationmidpoint, and an increase in the deactivation time constant of theKv1.5 delayed rectifier. Removal of the first 10 amino acids fromKv1.3 eliminated the effects on fast and slow inactivation but notthe voltage shift in activation. Addition of the first 87 amino acids of Kv1.3 to the amino terminus of Kv1.5 reconstituted fast and slowinactivation without altering the midpoint of activation. Although aninternal pore mutation that alters quinidine block (V512A) did notaffect Kv1.3-mediated inactivation, a mutation of the external mouthof the pore (R485Y) increased the extent of fast inactivation whilepreventing the enhancement of slow inactivation. These data suggestthat 1) Kv1.3-mediated effects involve at least two distinct domains of this -subunit,2) inactivation involves openchannel block that is allosterically linked to the external pore, and3) the Kv1.3-induced shift in theactivation midpoint is functionally distinct from inactivation.

     

Tricyclic imidazole antagonists of the Neuropeptide S Receptor

A new structural class of potent antagonists of the Neuropeptide S Receptor (NPSR) is reported. High-throughput screening identified a tricyclic imidazole antagonist of NPSR, and medicinal chemistry optimization of this structure was undertaken to improve potency against the receptor as well as CNS penetration. Detailed herein are synthetic and medicinal chemistry studies that led to the identification of antagonists 15 and NPSR-PI1, which demonstrate potent in vitro NPSR antagonism and central exposure in vivo.     

Molecular characterization of lantibiotic-synthesizing enzyme EpiD reveals a function for bacterial Dfp proteins in coenzyme A biosynthesis

The lantibiotic-synthesizing flavoprotein EpiD catalyzes the oxidative decarboxylation of peptidylcysteines to peptidyl-aminoenethiols. The sequence motif responsible for flavin coenzyme binding and enzyme activity is conserved in different proteins from all kingdoms of life. Dfp proteins of eubacteria and archaebacteria and salt tolerance proteins of yeasts and plants belong to this new family of flavoproteins. The enzymatic function of all these proteins was not known, but our experiments suggested that they catalyze a similar reaction like EpiD and/or may have similar substrates and are homododecameric flavoproteins. We demonstrate that the N-terminal domain of the Escherichia coli Dfp protein catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine. This reaction is essential for coenzyme A biosynthesis.     

Discovery and expanded SAR of 4,4-disubstituted quinazolin-2-ones as potent T-type calcium channel antagonists

The discovery and synthesis of 4,4-disubstituted quinazolinones as T-type calcium channel antagonists is reported. Based on lead compounds 2 and 3, a focused SAR campaign driven by the optimization of potency, metabolic stability, and pharmacokinetic profile identified 45 as a potent T-type Ca²⁺ channel antagonist with minimized PXR activation. In vivo, 45 suppressed seizure frequency in a rat model of absence epilepsy and showed significant alterations of sleep architecture after oral dosing to rats as measured by EEG.     

Role of voltage-gated calcium channels in potassium-stimulated aldosterone secretion from rat adrenal zona glomerulosa cells

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K(+) activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K(+)-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni(2+), and selected L-type antagonist dihydropyridines, inhibited K(+)-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat alpha1H T-type Ca(2+) currents. In contrast, below 10 microM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K(+)-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K(+)-stimulated aldosterone secretion.     

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.     

Positive Allosteric Interaction of Structurally Diverse T-Type Calcium Channel Antagonists

Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.