Diurnal Changes in Capecitabine Clock-Controlled Metabolism Enzymes Are Responsible for Its Pharmacokinetics in Male Mice.

The circadian timing system controls absorption, distribution, metabolism, and elimination processes of drug pharmacokinetics over a 24-h period. Exposure of target tissues to the active form of the drug and cytotoxicity display variations depending on the chronopharmacokinetics. For anticancer drugs with narrow therapeutic ranges and dose-limiting side effects, it is particularly important to know the temporal changes in pharmacokinetics. A previous study indicated that pharmacokinetic profile of capecitabine was different depending on dosing time in rat. However, it is not known how such difference is attributed with respect to diurnal rhythm. Therefore, in this study, we evaluated capecitabine-metabolizing enzymes in a diurnal rhythm-dependent manner. To this end, C57BL/6J male mice were orally treated with 500 mg/kg capecitabine at ZT1, ZT7, ZT13, or ZT19. We then determined pharmacokinetics of capecitabine and its metabolites, 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'DFUR), 5-fluorouracil (5-FU), in plasma and liver. Results revealed that plasma Cmax and AUC0-6h (area under the plasma concentration-time curve from 0 to 6 h) values of capecitabine, 5'DFUR, and 5-FU were higher during the rest phase (ZT1 and ZT7) than the activity phase (ZT13 and ZT19) (p < 0.05). Similarly, Cmax and AUC0-6h values of 5'DFUR and 5-FU in liver were higher during the rest phase than activity phase (p < 0.05), while there was no significant difference in liver concentrations of capecitabine and 5'DFCR. We determined the level of the enzymes responsible for the conversion of capecitabine and its metabolites at each ZT. Results indicated the levels of carboxylesterase 1 and 2, cytidine deaminase, uridine phosphorylase 2, and dihydropyrimidine dehydrogenase (p < 0.05) are being rhythmically regulated and, in turn, attributed different pharmacokinetics profiles of capecitabine and its metabolism. This study highlights the importance of capecitabine administration time to increase the efficacy with minimum adverse effects.

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice.

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53-/- mice by ~25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation.

Structure-based design and classifications of small molecules regulating the circadian rhythm period.

Circadian rhythm is an important mechanism that controls behavior and biochemical events based on 24 h rhythmicity. Ample evidence indicates disturbance of this mechanism is associated with different diseases such as cancer, mood disorders, and familial delayed phase sleep disorder. Therefore, drug discovery studies have been initiated using high throughput screening. Recently the crystal structures of core clock proteins (CLOCK/BMAL1, Cryptochromes (CRY), Periods), responsible for generating circadian rhythm, have been solved. Availability of structures makes amenable core clock proteins to design molecules regulating their activity by using in silico approaches. In addition to that, the implementation of classification features of molecules based on their toxicity and activity will improve the accuracy of the drug discovery process. Here, we identified 171 molecules that target functional domains of a core clock protein, CRY1, using structure-based drug design methods. We experimentally determined that 115 molecules were nontoxic, and 21 molecules significantly lengthened the period of circadian rhythm in U2OS cells. We then performed a machine learning study to classify these molecules for identifying features that make them toxic and lengthen the circadian period. Decision tree classifiers (DTC) identified 13 molecular descriptors, which predict the toxicity of molecules with a mean accuracy of 79.53% using tenfold cross-validation. Gradient boosting classifiers (XGBC) identified 10 molecular descriptors that predict and increase in the circadian period length with a mean accuracy of 86.56% with tenfold cross-validation. Our results suggested that these features can be used in QSAR studies to design novel nontoxic molecules that exhibit period lengthening activity.

Transcriptome analysis of the circadian clock gene BMAL1 deletion with opposite carcinogenic effects.

We have previously reported that the deletion of BMAL1 gene has opposite effects in respect to its contribution to the pathways that are effective in the multistage carcinogenesis process. BMAL1 deletion sensitized nearly normal breast epithelial (MCF10A) and invasive breast cancer cells (MDA-MB-231) to cisplatin- and doxorubicin-induced apoptosis, while this deletion also aggravated the invasive potential of MDA-MB-231 cells. However, the mechanistic relationship of the seemingly opposite contribution of BMAL1 deletion to carcinogenesis process is not known at genome-wide level. In this study, an RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways after treating BMAL1 knockout (KO) or wild-type (WT) MDA-MB-231 cells with cisplatin and doxorubicin to initiate apoptosis. Gene set enrichment analysis with the DEGs demonstrated that enrichment in multiple genes/pathways contributes to sensitization to cisplatin- or doxorubicin-induced apoptosis in BMAL1-dependent manner. Additionally, our DEG analysis suggested that non-coding transcript RNA (such as lncRNA and processed pseudogenes) may have role in cisplatin- or doxorubicin-induced apoptosis. Protein-protein interaction network obtained from common DEGs in cisplatin and doxorubicin treatments revealed that GSK3ß, NACC1, and EGFR are the principal genes regulating the response of the KO cells. Moreover, the analysis of DEGs among untreated BMAL1 KO and WT cells revealed that epithelial-mesenchymal transition genes are up-regulated in KO cells. As a negative control, we have also analyzed the DEGs following treatment with an endoplasmic reticulum (ER) stress-inducing agent, tunicamycin, which was affected by BMAL1 deletion minimally. Collectively, the present study suggests that BMAL1 regulates many genes/pathways of which the alteration in BMAL1 KO cells may shed light on pleotropic phenotype observed.

CRY1-CBS binding regulates circadian clock function and metabolism.

Circadian disruption influences metabolic health. Metabolism modulates circadian function. However, the mechanisms coupling circadian rhythms and metabolism remain poorly understood. Here, we report that cystathionine ß-synthase (CBS), a central enzyme in one-carbon metabolism, functionally interacts with the core circadian protein cryptochrome 1 (CRY1). In cells, CBS augments CRY1-mediated repression of the CLOCK/BMAL1 complex and shortens circadian period. Notably, we find that mutant CBS-I278T protein, the most common cause of homocystinuria, does not bind CRY1 or regulate its repressor activity. Transgenic CbsZn/Zn  mice, while maintaining circadian locomotor activity period, exhibit reduced circadian power and increased expression of E-BOX outputs. CBS function is reciprocally influenced by CRY1 binding. CRY1 modulates enzymatic activity of the CBS. Liver extracts from Cry1-/- mice show reduced CBS activity that normalizes after the addition of exogenous wild-type (WT) CRY1. Metabolomic analysis of WT, CbsZn/Zn , Cry1-/- , and Cry2-/- samples highlights the metabolic importance of endogenous CRY1. We observed temporal variation in one-carbon and transsulfuration pathways attributable to CRY1-induced CBS activation. CBS-CRY1 binding provides a post-translational switch to modulate cellular circadian physiology and metabolic control.

Organic Photovoltaic Pseudocapacitors for Neurostimulation.

Neural interfaces are the fundamental tools to understand the brain and cure many nervous-system diseases. For proper interfacing, seamless integration, efficient and safe digital-to-biological signal transduction, and long operational lifetime are required. Here, we devised a wireless optoelectronic pseudocapacitor converting the optical energy to safe capacitive currents by dissociating the photogenerated excitons in the photovoltaic unit and effectively routing the holes to the supercapacitor electrode and the pseudocapacitive electrode-electrolyte interfacial layer of PEDOT:PSS for reversible faradic reactions. The biointerface showed high peak capacitive currents of ∼3 mA·cm-2 with total charge injection of ∼1 µC·cm-2 at responsivity of 30 mA·W-1, generating high photovoltages over 400 mV for the main eye photoreception colors of blue, green, and red. Moreover, modification of PEDOT:PSS controls the charging/discharging phases leading to rapid capacitive photoresponse of 50 µs and effective membrane depolarization at the single-cell level. The neural interface has a device lifetime of over 1.5 years in the aqueous environment and showed stability without significant performance decrease after sterilization steps. Our results demonstrate that adopting the pseudocapacitance phenomenon on organic photovoltaics paves an ultraefficient, safe, and robust way toward communicating with biological systems.

A CLOCK-binding small molecule disrupts the interaction between CLOCK and BMAL1 and enhances circadian rhythm amplitude.

Proper function of many physiological processes requires a robust circadian clock. Disruptions of the circadian clock can result in metabolic diseases, mood disorders, and accelerated aging. Therefore, identifying small molecules that specifically modulate regulatory core clock proteins may potentially enable better management of these disorders. In this study, we applied a structure-based molecular-docking approach to find small molecules that specifically bind to the core circadian regulator, the transcription factor circadian locomotor output cycles kaput (CLOCK). We identified 100 candidate molecules by virtual screening of ∼2 million small molecules for those predicted to bind closely to the interface in CLOCK that interacts with its transcriptional co-regulator, Brain and muscle Arnt-like protein-1 (BMAL1). Using a mammalian two-hybrid system, real-time monitoring of circadian rhythm in U2OS cells, and various biochemical assays, we tested these compounds experimentally and found one, named CLK8, that specifically bound to and interfered with CLOCK activity. We show that CLK8 disrupts the interaction between CLOCK and BMAL1 and interferes with nuclear translocation of CLOCK both in vivo and in vitro Results from further experiments indicated that CLK8 enhances the amplitude of the cellular circadian rhythm by stabilizing the negative arm of the transcription/translation feedback loop without affecting period length. Our results reveal CLK8 as a tool for further studies of CLOCK's role in circadian rhythm amplitude regulation and as a potential candidate for therapeutic development to manage disorders associated with dampened circadian rhythms.

Oncogenic K-Ras4B Dimerization Enhances Downstream Mitogen-activated Protein Kinase Signaling.

Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) ß-interface, mapped to Switch I and effector-binding regions, (ii) α-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the α- and ß-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. α-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While ß-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked K-Ras4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the α-interface in K-Ras4B homodimerization and the ß-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both α- and ß-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling.

Cryptochrome deletion in p53 mutant mice enhances apoptotic and anti-tumorigenic responses to UV damage at the transcriptome level.

Previous studies have demonstrated that deletion of cryptochrome (Cry) genes protects p53-/- mutant mice from the early onset of cancer and extends their median life-span by about 1.5-fold. Subsequent in vitro studies had revealed that deletion of Crys enhances apoptosis in response to UV damage through activation of p73 and inactivation of GSK3ß. However, it was not known at the transcriptome-wide level how deletion of Crys delays the onset of cancer in p53-/- mutant mice. In this study, the RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways following UV-induced DNA damage in p53-/- and p53-/-Cry1-/-Cry2-/- mouse skin fibroblasts. Gene set enrichment analysis with the DEGs demonstrated enrichment in immune surveillance-associated genes regulated by IFN-γ and genes involved in TNFα signaling via NF-κB. Furthermore, protein network analysis enabled identification of DEGs p21, Sirt1, and Jun as key players, along with their interacting partners. It was also observed that the DEGs contained a high ratio of non-coding transcripts. Collectively, the present study suggests new genes in NF-κB regulation and IFN-γ response, as well as non-coding RNAs, may contribute to delaying the onset of cancer in p53-/-Cry1-/-Cry2-/- mice and increasing the life-span of these animals compared to p53-/- mice.

Effective Neural Photostimulation Using Indium-Based Type-II Quantum Dots.

Light-induced stimulation of neurons via photoactive surfaces offers rich opportunities for the development of therapeutic methods and high-resolution retinal prosthetic devices. Quantum dots serve as an attractive building block for such surfaces, as they can be easily functionalized to match the biocompatibility and charge transport requirements of cell stimulation. Although indium-based colloidal quantum dots with type-I band alignment have attracted significant attention as a nontoxic alternative to cadmium-based ones, little attention has been paid to their photovoltaic potential as type-II heterostructures. Herein, we demonstrate type-II indium phosphide/zinc oxide core/shell quantum dots that are incorporated into a photoelectrode structure for neural photostimulation. This induces a hyperpolarizing bioelectrical current that triggers the firing of a single neural cell at 4 µW mm-2, 26-fold lower than the ocular safety limit for continuous exposure to visible light. These findings show that nanomaterials can induce a biocompatible and effective biological junction and can introduce a route in the use of quantum dots in photoelectrode architectures for artificial retinal prostheses.

Molecular Aspects of Circadian Pharmacology and Relevance for Cancer Chronotherapy.

The circadian timing system (CTS) controls various biological functions in mammals including xenobiotic metabolism and detoxification, immune functions, cell cycle events, apoptosis and angiogenesis. Although the importance of the CTS is well known in the pharmacology of drugs, it is less appreciated at the clinical level. Genome-wide studies highlighted that the majority of drug target genes are controlled by CTS. This suggests that chronotherapeutic approaches should be taken for many drugs to enhance their effectiveness. Currently chronotherapeutic approaches are successfully applied in the treatment of different types of cancers. The chronotherapy approach has improved the tolerability and antitumor efficacy of anticancer drugs both in experimental animals and in cancer patients. Thus, chronobiological studies have been of importance in determining the most appropriate time of administration of anticancer agents to minimize their side effects or toxicity and enhance treatment efficacy, so as to optimize the therapeutic ratio. This review focuses on the underlying mechanisms of the circadian pharmacology i.e., chronopharmacokinetics and chronopharmacodynamics of anticancer agents with the molecular aspects, and provides an overview of chronotherapy in cancer and some of the recent advances in the development of chronopharmaceutics.

Glu-370 in the large subunit influences the substrate binding, allosteric, and heat stability properties of potato ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWTAGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of potato the AGPase.

Transcriptional regulation of the ADP-glucose pyrophosphorylase isoforms in the leaf and the stem under long and short photoperiod in lentil.

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme in plant starch biosynthesis. It contains large (LS) and small (SS) subunits encoded by two different genes. In this study, we explored the transcriptional regulation of both the LS and SS subunits of AGPase in stem and leaf under different photoperiods length in lentil. To this end, we first isolated and characterized different isoforms of the LS and SS of lentil AGPase and then we performed quantitative real time PCR (qPCR) to see the effect of photoperiod length on the transcription of the AGPase isforms under the different photoperiod regimes in lentil. Analysis of the qPCR results revealed that the transcription of different isoforms of the LSs and the SSs of lentil AGPase are differentially regulated when photoperiod shifted from long-day to short-day in stem and leaves. While transcript levels of LS1 and SS2 in leaf significantly decreased, overall transcript levels of SS1 increased in short-day regime. Our results indicated that day length affects the transcription of lentil AGPase isoforms differentially in stems and leaves most likely to supply carbon from the stem to other tissues to regulate carbon metabolism under short-day conditions.

Investigation of the interaction between the large and small subunits of potato ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield.

Insights into subunit interactions in the heterotetrameric structure of potato ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. The LS is involved in mainly allosteric regulation through its interaction with the catalytic SS. Recently the crystal structure of the SS homotetramer has been solved, but no crystal structure of the native heterotetrameric enzyme is currently available. In this study, we first modeled the three-dimensional structure of the LS to construct the heterotetrameric enzyme. Because the enzyme has a 2-fold symmetry, six different dimeric (either up-down or side-by-side) interactions were possible. Molecular dynamics simulations were carried out for each of these possible dimers. Trajectories obtained from molecular dynamics simulations of each dimer were then analyzed by the molecular mechanics/Poisson-Boltzmann surface area method to identify the most favorable dimers, one for up-down and the other for side-by-side. Computational results combined with site directed mutagenesis and yeast two hybrid experiments suggested that the most favorable heterotetramer is formed by LS-SS (side-by-side), and LS-SS (up-down). We further determined the order of assembly during the heterotetrameric structure formation. First, side-by-side LS-SS dimers form followed by the up-down tetramerization based on the relative binding free energies.