The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.
Stereocontrolled total synthesis of (+)-vinblastine (1) has been achieved using a novel radical-mediated indole synthesis developed in our laboratories. The isothiocyanate 18, prepared readily from quinoline 17, underwent a facile addition of the malonate anion to give 19. The o-alkenylthioanilide 19 was then converted to indole 20 by radical cyclization and protection. (-)-Vindoline (2) was prepared from this key intermediate 20 in a highly efficient manner. The indole core of the 11-membered intermediate 3 was constructed similarly from quinoline. The critical coupling reaction between 2 and the chloroindolenine derived from 3 proceeded with complete control of stereochemistry to give the desired product 66 in 97 % yield, which could be successfully converted to (+)-vinblastine (1).Vinblastine (1), isolated from Catharanthus roseus [1], has been widely known as a prominent agent for cancer chemotherapy. Since chemical modifications of the natural product have been the major means for exploration of the more potent analogs, a limited number of its derivatives have so far been accessible [2a]. Total synthesis of vinblastine, on the other hand, has been the subject of intensive investigations in the area of alkaloid synthesis [2b]. Despite the endeavors spanning the past three decades, only four syntheses have been reported to date [3]. In all cases, however, the supply of the lower half of vinblastine (i.e., vindoline) relied upon the natural sources. The major challenges in the total synthesis of 1 include controlling the stereochemistry of C18′ as well as establishment of efficient routes to the two halves of the indole units.Our synthetic plan is shown in Scheme 1. Model studies by Schill [4] as well as our molecular modeling study strongly suggested that a stereocontrolled coupling with vindoline (2) could be per-*Lecture presented at the
Regulation of stomatal aperture is essential for plant growth and survival in response to environmental stimuli. Opening of stomata induces uptake of CO2 for photosynthesis and transpiration, which enhances uptake of nutrients from roots. Light is the most important stimulus for stomatal opening. Under drought stress, the plant hormone ABA induces stomatal closure to prevent water loss. However, the molecular mechanisms of stomatal movements are not fully understood. In this study, we screened chemical libraries to identify compounds that affect stomatal movements in Commelina benghalensis and characterize the underlying molecular mechanisms. We identified nine stomatal closing compounds (SCL1-SCL9) that suppress light-induced stomatal opening by >50%, and two compounds (temsirolimus and CP-100356) that induce stomatal opening in the dark. Further investigations revealed that SCL1 and SCL2 had no effect on autophosphorylation of phototropin or the activity of the inward-rectifying plasma membrane (PM) K+ channel, KAT1, but suppressed blue light-induced phosphorylation of the penultimate residue, threonine, in PM H+-ATPase, which is a key enzyme for stomatal opening. SCL1 and SCL2 had no effect on ABA-dependent responses, including seed germination and expression of ABA-induced genes. These results suggest that SCL1 and SCL2 suppress light-induced stomatal opening at least in part by inhibiting blue light-induced activation of PM H+-ATPase, but not by the ABA signaling pathway. Interestingly, spraying leaves onto dicot and monocot plants with SCL1 suppressed wilting of leaves, indicating that inhibition of stomatal opening by these compounds confers tolerance to drought stress in plants.
Chronic circadian disruption due to shift work or frequent travel across time zones leads to jet‐lag and an increased risk of diabetes, cardiovascular disease, and cancer. The development of new pharmaceuticals to treat circadian disorders, however, is costly and hugely time‐consuming. We therefore performed a high‐throughput chemical screen of existing drugs for circadian clock modulators in human U2OS cells, with the aim of repurposing known bioactive compounds. Approximately 5% of the drugs screened altered circadian period, including the period‐shortening compound dehydroepiandrosterone (DHEA; also known as prasterone). DHEA is one of the most abundant circulating steroid hormones in humans and is available as a dietary supplement in the USA. Dietary administration of DHEA to mice shortened free‐running circadian period and accelerated re‐entrainment to advanced light–dark (LD) cycles, thereby reducing jet‐lag. Our drug screen also revealed the involvement of tyrosine kinases, ABL1 and ABL2, and the BCR serine/threonine kinase in regulating circadian period. Thus, drug repurposing is a useful approach to identify new circadian clock modulators and potential therapies for circadian disorders.
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