Kenneth S. Koblan

Design, Synthesis, and Evaluation of a Novel 4-Aminomethyl-4-fluoropiperidine as a T-Type Ca2+ Channel Antagonist

The novel T-type antagonist ( S)- 5 has been prepared and evaluated in in vitro and in vivo assays for T-type calcium ion channel activity. Structural modification of the piperidine leads 1 and 2 afforded the fluorinated piperidine ( S)- 5, a potent and selective antagonist that displayed in vivo CNS efficacy without adverse cardiovascular effects.

Discovery of 1,4-Substituted Piperidines as Potent and Selective Inhibitors of T-Type Calcium Channels

The discovery of a novel series of potent and selective T-type calcium channel antagonists is reported. Initial optimization of high-throughput screening leads afforded a 1,4-substituted piperidine amide 6 with good potency and limited selectivity over hERG and L-type channels and other off-target activities. Further SAR on reducing the basicity of the piperidine and introducing polarity led to the discovery of 3-axial fluoropiperidine 30 with a significantly improved selectivity profile. Compound 30 showed good oral bioavailability and brain penetration across species. In a rat genetic model of absence epilepsy, compound 30 demonstrated a robust reduction in the number and duration of seizures at 33 nM plasma concentration, with no cardiovascular effects at up to 5.6 microM. Compound 30 also showed good efficacy in rodent models of essential tremor and Parkinsons disease. Compound 30 thus demonstrates a wide margin between CNS and peripheral effects and is a useful tool for probing the effects of T-type calcium channel inhibition.

In Vitro Characterization of T-Type Calcium Channel Antagonist TTA-A2 and In Vivo Effects on Arousal in Mice

T-type calcium channels have been implicated in many behaviorally important neurophysiological processes, and altered channel activity has been linked to the pathophysiology of neurological disorders such as insomnia, epilepsy, Parkinsons disease, depression, schizophrenia, and pain. We have previously identified a number of potent and selective T-type channel antagonists (Barrow et al., 2007; Shipe et al., 2008; Yang et al., 2008). Here we describe the properties of the antagonist TTA-A2 [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl}ethyl)acetamide], assessed in patch-clamp experiments. TTA-A2 blocks T-type channels (Cav3.1, 3.2, 3.3) voltage dependently and with high potency (IC50 ∼100 nM). Stimulation at 3 Hz revealed additional use dependence of inhibition. A hyperpolarized shift of the channel availability curve and delayed channel recovery from inactivation suggest that the compound preferentially interacts with and stabilizes inactivated channels. The compound showed a ∼300-fold selectivity for Cav3 channels over high-voltage activated calcium channels. Inhibitory effects on native T-type currents were confirmed in brain slice recordings from the dorsal lateral geniculate nucleus and the subthalamic nucleus. Furthermore, we demonstrate that in vivo T-type channel inhibition by TTA-A2 suppresses active wake and promotes slow-wave sleep in wild-type mice but not in mice lacking both Cav3.1 and Cav3.3, suggesting the selective effect of TTA-A2 on recurrent thalamocortical network activity. The discovery of the potent and selective T-type channel antagonist TTA-A2 has enabled us to study the in vivo effects of pharmacological T-channel inhibition on arousal in mice, and it will help to explore the validity of these channels as potential drug targets for sleep-related and other neurological diseases.

Antagonism of T-type calcium channels inhibits high-fat diet–induced weight gain in mice

The epidemics of obesity and metabolic disorders have well-recognized health and economic burdens. Pharmacologic treatments for these diseases remain unsatisfactory with respect to both efficacy and side-effect profiles. Here, we have identified a potential central role for T-type calcium channels in regulating body weight maintenance and sleep. Previously, it was shown that mice lacking CaV3.1 T-type calcium channels have altered sleep/wake activity. We found that these mice were also resistant to high-fat diet-induced weight gain, without changes in food intake or sensitivity to high-fat diet-induced disruptions of diurnal rhythm. Administration of a potent and selective antagonist of T-type calcium channels, TTA-A2, to normal-weight animals prior to the inactive phase acutely increased sleep, decreased body core temperature, and prevented high-fat diet-induced weight gain. Administration of TTA-A2 to obese rodents reduced body weight and fat mass while concurrently increasing lean muscle mass. These effects likely result from better alignment of diurnal feeding patterns with daily changes in circadian physiology and potentially an increased metabolic rate during the active phase. Together, these studies reveal what we believe to be a previously unknown role for T-type calcium channels in the regulation of sleep and weight maintenance and suggest the potential for a novel therapeutic approach to treating obesity.

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.

EJ-Ras Inhibits Phospholipase Cγ1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase

Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21ras (EJ-Ras) produces morphogenic changes characterized by decreased attachment to the substratum, with retraction and rounding of the cell body. Transformed fibroblasts lose their stressed conformation and adopt a relaxed morphology. The specific molecular mechanisms responsible for these changes remain uncharacterized. We found that EJ-Ras transformation of NIH 3T3 cells decreased the cellular content of polymerized actin, particularly at the expense of actin stress fibers, but induced the accumulation of actin filaments in peripheral ruffling membranes. Polymerization of actin could be induced in EJ-Ras-transformed cells by exposure to platelet-derived growth factor (PDGF)-BB to an extent similar to that observed in wild-type NIH 3T3 cells. In EJ-Ras cells, actin polymerization was independent of phospholipase C gamma 1 (PLC gamma 1) activity, because inositol tris-phosphate (IP3) production observed in control NIH 3T3 cells in response to PDGF-BB was absent. Although PDGF-BB did stimulate tyrosine phosphorylation of PLC gamma 1, the phospholipase was strongly inhibited by an inhibitory factor present in the cytoplasm of EJ-Ras-transformed cells. In addition, cytoplasmic extracts of EJ-Ras, but not of control cells, inhibited phosphatidylinositol 4,5-diphosphate (PIP2) hydrolysis catalyzed by a recombinant PLC gamma 1 in vitro. Although PIP2 hydrolysis could not contribute to the reorganization of the actin cytoskeleton induced by PDGF-BB in EJ-Ras-transformed cells, phosphatidylinositol 3-kinase (PI3-K) was necessary for actin polymerization. Wortmannin, a specific PI3-K inhibitor, not only blocked actin polymerization in both control and EJ-Ras-transformed cells but actually led to rapid actin depolymerization when these cells were exposed to PDGF-BB. Thus, in EJ-Ras-transformed cells, cell morphogenic changes in response to PDGF-BB rely importantly on PI3-K and can occur in the complete absence of IP3 production despite tyrosine phosphorylation of PLC gamma 1.

Automation of In Vitro Dose-Inhibition Assays Utilizing the Tecan Genesis and an Integrated Software Package to Support the Drug Discovery Process

We have automated in vitro dose-inhibition assays to evaluate newly synthesized chemical entities and to rapidly produce and disseminate the results with minimal personnel. A variety of assay metho...

Biochemical and biological analyses of farnesyl-protein transferase inhibitors.

The methods outlined in Subheading 3. provide a logical sequence of assays with which to evaluate the biochemical and biological properties of potential FPTase inhibitors. The clinical predictability of these assays must await the evaluation of one or more of these compounds in humans.