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Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) ε or δ can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1ε and CK1δ using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1δ is the principal regulator of the clock period: pharmacological inhibition of CK1δ, but not CK1ε, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1δ inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1δ inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2 −/− mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2 −/− mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1δ offers an avenue for therapeutic modulation of perturbed circadian behavior.

The recent increase in the number of X-ray crystal structures of G-protein coupled receptors (GPCRs) has been enabling for structure-based drug design (SBDD) efforts. These structures have revealed that GPCRs are highly dynamic macromolecules whose function is dependent on their intrinsic flexibility. Unfortunately, the use of static structures to understand ligand binding can potentially be misleading, especially in systems with an inherently high degree of conformational flexibility. Here, we show that docking a set of dopamine D3 receptor compounds into the existing eticlopride-bound dopamine D3 receptor (D3R) X-ray crystal structure resulted in poses that were not consistent with results obtained from site-directed mutagenesis experiments. We overcame the limitations of static docking by using large-scale high-throughput molecular dynamics (MD) simulations and Markov state models (MSMs) to determine an alternative pose consistent with the mutation data. The new pose maintains critical interactions observed in the D3R/eticlopride X-ray crystal structure and suggests that a cryptic pocket forms due to the shift of a highly conserved residue, F 6.52 . Our study highlights the importance of GPCR dynamics to understand ligand binding and provides new opportunities for drug discovery.

Growing evidence indicates that disruption of our internal timing system contributes to the incidence and severity of metabolic diseases, including obesity and type 2 diabetes. This is perhaps not surprising since components of the circadian clockwork are tightly coupled to metabolic processes across the body. In the current study, we assessed the impact of obesity on the circadian system in mice at a behavioural and molecular level and determined whether pharmacological targeting of casein kinase 1δ and ε (CK1δ/ε), key regulators of the circadian clock, can confer metabolic benefit. We demonstrate that although behavioural rhythmicity was maintained in diet-induced obesity (DIO), gene expression profiling revealed tissue-specific alteration to the phase and amplitude of the molecular clockwork. Clock function was most significantly attenuated in visceral white adipose tissue (WAT) of DIO mice and was coincident with elevated tissue inflammation and dysregulation of clock-coupled metabolic regulators PPARα/γ. Further, we show that daily administration of a CK1δ/ε inhibitor (PF-5006739) improved glucose tolerance in both DIO and genetic ( ob/ob ) models of obesity. These data further implicate circadian clock disruption in obesity and associated metabolic disturbance and suggest that targeting of the clock represents a therapeutic avenue for the treatment of metabolic disorders.

Csnk1e, the gene encoding casein kinase 1-epsilon, has been implicated in sensitivity to amphetamines. Additionally, a polymorphism in CSNK1E was associated with heroin addiction, suggesting that this gene may also affect opioid sensitivity. In this study, we first conducted genome-wide quantitative trait locus (QTL) mapping of methamphetamine (MA)-induced locomotor activity in C57BL/6J (B6) × DBA/2J (D2)-F(2) mice and a more highly recombinant F(8) advanced intercross line. We identified a QTL on chromosome 15 that contained Csnk1e (63-86 Mb; Csnk1e=79.25 Mb). We replicated this result and further narrowed the locus using B6.D2(Csnk1e) and D2.B6(Csnk1e) reciprocal congenic lines (78-86.8 and 78.7-81.6 Mb, respectively). This locus also affected sensitivity to the μ-opioid receptor agonist fentanyl. Next, we directly tested the hypothesis that Csnk1e is a genetic regulator of sensitivity to psychostimulants and opioids. Mice harboring a null allele of Csnk1e showed an increase in locomotor activity following MA administration. Consistent with this result, coadministration of a selective pharmacological inhibitor of Csnk1e (PF-4800567) increased the locomotor stimulant response to both MA and fentanyl. These results show that a narrow genetic locus that contains Csnk1e is associated with differences in sensitivity to MA and fentanyl. Furthermore, gene knockout and selective pharmacological inhibition of Csnk1e define its role as a negative regulator of sensitivity to psychostimulants and opioids.

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The total syntheses of the naturally occurring Amaryllidaceae alkaloids (+)-lycoricidine, (+)-narciclasine, (+)-7-deoxypancratistatin, and a synthesis of unnatural lycoricidine are described. The key strategic element of the approach utilized in the synthesis of lycoricidine was an intermolecular radical addition-cyclization sequence. Thiyl radical addition to a disubstituted alkyne generated a vinyl radical that underwent a stereoselective 6-exo cyclization onto an oxime ether. This process was high yielding when thiyl radical was used, but fails when stannyl radical was used. Pivotal to the completion of this synthesis was the use of SmI2 in a reduction-cyclization sequence to form the lactam ring and reductive removal of the vinyl sulfide moiety. The synthesis described affords (−)-lycoricidine in 13 linear steps from D-lyxose in an 8% overall yield. The syntheses of (+)-lycoricidine and (+)-narciclasine both use a radical addition-cyclization sequence to construct the C-ring. The synthesis of (+)-lycoricidine proceeds in nine linear steps with a 41% overall yield from isopropylidine-D-gulonolactone. The aryl ring contained in narciclasine was synthesized from piperonal using an ortho metallation strategy. Removal of the N,N-dimethylamide ortho directing group was accomplished using trimethyloxonium tetrafluoroborate. The use of the electron withdrawing phenolic protecting group, tosylate, was critical to obtain high yields in the palladium-mediated coupling and in the radical addition-cyclization sequence. Pivotal to the completion of the synthesis was the use of SmI2 in the reduction of the N-O and reductive removal of the vinyl sulfide. High yields for these processes were obtained only after the closure of the lactam ring. The synthesis described is 12 steps from a D-gulonolactone derivative and proceeds in 22% overall yield. A tandem radical cyclization approach was used to construct 7-deoxypancratistan. The route described utilizes a 6-exo radical cyclization of an aryl radical onto an aziridinyl imine, followed by generation of a new radical that underwent a second 6-exo radical cyclization to an oxime ether to establish the trans-phenanthridone ring junction. The synthesis described is 13 linear steps from 6-iodopiperonol and affords a 21% overall yield.

In mammals, the master circadian clock synchronizes daily rhythms of physiology and behavior with the day–night cycle. Failure of synchrony, which increases the risk for numerous chronic diseases, can be treated by phase adjustment of the circadian clock pharmacologically, for example, with melatonin, or a CK1δ/ε inhibitor. Here, using in silico experiments with a systems pharmacology model describing molecular interactions, and pharmacokinetic and behavioral experiments in cynomolgus monkeys, we find that the circadian phase delay caused by CK1δ/ε inhibition is more strongly attenuated by light in diurnal monkeys and humans than in nocturnal mice, which are common preclinical models. Furthermore, the effect of CK1δ/ε inhibition strongly depends on endogenous PER2 protein levels, which differs depending on both the molecular cause of the circadian disruption and the patient's lighting environment. To circumvent such large interindividual variations, we developed an adaptive chronotherapeutics to identify precise dosing regimens that could restore normal circadian phase under different conditions. Our results reveal the importance of photosensitivity in the clinical efficacy of clock‐modulating drugs, and enable precision medicine for circadian disruption. Synopsis The study identifies the sources of the inter‐ and intraspecies variability in the modulation of circadian phase by CK1δ/ε inhibition: photosensitivity and PER2 abundance, and proposes a personalized chronotherapy to treat circadian disruption. Light attenuates the effect of CK1δ/ε inhibition more strongly in diurnal monkeys than in nocturnal mice. The effect of CK1δ/ε inhibition becomes stronger as PER2 protein abundance increases, which leads to a large interindividual variability in circadian phase shift. To circumvent such large interindividual variability, an adaptive chronotherapeutic approach is developed, which identifies a personalized dosing time by tracking the patient's drug response. Graphical Abstract The study identifies the sources of the inter‐ and intraspecies variability in the modulation of circadian phase by CK1δ/ε inhibition: photosensitivity and PER2 abundance, and proposes a personalized chronotherapy to treat circadian disruption.

In mammals, the master circadian clock synchronizes daily rhythms of physiology and behavior with the day–night cycle. Failure of synchrony, which increases the risk for numerous chronic diseases, can be treated by phase adjustment of the circadian clock pharmacologically, for example, with melatonin, or a CK1δ/ε inhibitor. Here, using in silico experiments with a systems pharmacology model describing molecular interactions, and pharmacokinetic and behavioral experiments in cynomolgus monkeys, we find that the circadian phase delay caused by CK1δ/ε inhibition is more strongly attenuated by light in diurnal monkeys and humans than in nocturnal mice, which are common preclinical models. Furthermore, the effect of CK1δ/ε inhibition strongly depends on endogenous PER2 protein levels, which differs depending on both the molecular cause of the circadian disruption and the patient's lighting environment. To circumvent such large interindividual variations, we developed an adaptive chronotherapeutics to identify precise dosing regimens that could restore normal circadian phase under different conditions. Our results reveal the importance of photosensitivity in the clinical efficacy of clock‐modulating drugs, and enable precision medicine for circadian disruption.

Development, administration, activities, and evaluation of the Scientist-in-Residence program at the University of Wisconsin - Oshkosh.