Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period.

The circadian clock links our daily cycles of sleep and activity to the external environment. Deregulation of the clock is implicated in a number of human disorders, including depression, seasonal affective disorder, and metabolic disorders. Casein kinase 1 epsilon (CK1epsilon) and casein kinase 1 delta (CK1delta) are closely related Ser-Thr protein kinases that serve as key clock regulators as demonstrated by mammalian mutations in each that dramatically alter the circadian period. Therefore, inhibitors of CK1delta/epsilon may have utility in treating circadian disorders. Although we previously demonstrated that a pan-CK1delta/epsilon inhibitor, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), causes a significant phase delay in animal models of circadian rhythm, it remains unclear whether one of the kinases has a predominant role in regulating the circadian clock. To test this, we have characterized 3-(3-chloro-phenoxymethyl)-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PF-4800567), a novel and potent inhibitor of CK1epsilon (IC(50) = 32 nM) with greater than 20-fold selectivity over CK1delta. PF-4800567 completely blocks CK1epsilon-mediated PER3 nuclear localization and PER2 degradation. In cycling Rat1 fibroblasts and a mouse model of circadian rhythm, however, PF-4800567 has only a minimal effect on the circadian clock at concentrations substantially over its CK1epsilon IC(50). This is in contrast to the pan-CK1delta/epsilon inhibitor PF-670462 that robustly alters the circadian clock under similar conditions. These data indicate that CK1epsilon is not the predominant mediator of circadian timing relative to CK1delta. PF-4800567 should prove useful in probing unique roles between these two kinases in multiple signaling pathways.

Synthesis and SAR of 2-aryloxy-4-alkoxy-pyridines as potent orally active corticotropin-releasing factor 1 receptor antagonists.

A series of 2-aryloxy-4-alkoxy-pyridines ( 1) was identified as novel, selective, and orally active antagonists of the corticotropin-releasing factor 1 (CRF 1) receptor. Among these, compound 2 (CP-316311) is a potent and selective CRF 1 receptor antagonist with an IC 50 value of 6.8 nM in receptor binding and demonstrates oral efficacy in central nervous system (CNS) in vivo models. The regiochemistry of compounds in this series was determined by an X-ray structural analysis. A method to control regioselectivity via pyridine- N-oxides was developed. The synthesis of compounds in series 1 (Figure ) and [ (3)H]- 2 as well as the structure-activity relationship (SAR) are discussed. The in vitro, ex vivo, and in vivo properties of representative compounds are described herein. Compound 2 was advanced to phase II depression trials to test the hypothesis that CRF 1 antagonists could be used clinically as antidepressant drugs.

2-aryloxy-4-alkylaminopyridines: discovery of novel corticotropin-releasing factor 1 antagonists.

An orally active clinical candidate of corticotropin-releasing factor 1 (CRF 1) antagonist 1 showed a significant positive food effect in dog and human after oral administration. Efforts to address the food effect issue led us to explore and discover compounds in series 2 as orally active CRF 1 receptor antagonists, in which some compounds showed improved physicochemical properties while retaining desired pharmacological properties. Compound 3a (CP-376395) was selected for further development, due not only to its reduced food effects but also its greater efficacy in CNS models. Compound 3a was advanced to the clinic. The synthesis of representative potential candidates and their in vitro, ex vivo, and in vivo data are described.

Thiazole-diamides as potent gamma-secretase inhibitors.

The thiazole-diamide series (1) has been identified as highly potent gamma-secretase inhibitors. Several representative compounds showed IC(50) values of <0.3 nM. The synthesis and SAR, as well as a radiolabeled synthesis of [(3)H]-2a, are described.

Concentration-dependent modulation of amyloid-beta in vivo and in vitro using the gamma-secretase inhibitor, LY-450139.

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.