Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors,olomoucine and isopentenyladenine

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(δ2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley-Liss, Inc.     

CDK 抑制剂在抗肿瘤领域的研发进展

细胞周期蛋白依赖性激酶（cyclin dependent kinase，CDK）为细胞周期调节的关键激酶，参与细胞增殖、转录、存活等生理过程。 CDK 在多种肿瘤中异常活化，是抗肿瘤药物研发的重要靶点之一。目前已有 1 个 CDK 抑制剂（palbociclib, CDK4/CDK6 抑制剂）被美国 食品药品监督管理局批准于 2015 年上市，数十个 CDK 抑制剂处于针对实体瘤和血液系统肿瘤的临床或临床前研究阶段。综述目前抗肿瘤 领域 CDK 抑制剂的研发现状、遇到的问题和可能的解决方案，并讨论其临床应用的可能。     

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.     

Circadian organization inAplysia: internal desynchronization and amplitude of locomotor rhythm

Summary We have tested the hypothesis that the circadian oscillators in the eyes ofAplysia are coequal driver oscillators for the circadian locomotor rhythm. Three predictions based on this hypothesis were tested. Prediction 1: at a time when the phase difference between the eye rhythms is small, the amplitude of the locomotor rhythm in two eyed animals will be as great or greater than the amplitude in one eyed animals. Prediction 2: the amplitude of the locomotor rhythm of two eyed animals will decline under conditions in which the two eye rhythms become out of phase with each other. Prediction 3: the form of the locomotor rhythm will broaden or become biphasic in two eyed animals when the two eye rhythms become out of phase with each other.None of the predictions was confirmed. One eyedAplysia had higher amplitude locomotor rhythms than two eyedAplysia, even under conditions in which the two eye rhythms were probably not far out of phase with each other. There was no tendency for the amplitude of the locomotor rhythm of two eyed animals to decline under circumstances in which the phase difference between the two eye rhythms changes from less than 4 h to as much as 11.5 h. There was no tendency in two eyed animals for the locomotor rhythm to broaden or become biphasic as the eye rhythms became more out of phase with each other.The results led us to reject the hypothesis that the eyes are co-equal drivers for the locomotor rhythm. The ocular influence on locomotion is more likely to be mediated via mechanisms in the central nervous system that do not faithfully conserve the phase of the eye rhythms. One possibility is that the driver is a third circadian oscillator that interacts with the two eye oscillators.Abbreviations CAP compound action potentials - CC constant conditions - CT circadian time - DO driver oscillator - EO eye oscillator - RSD relative standard deviations (see Methods)     

CDK inhibitors suppress apoptosis induced by chemicals and by excessive expression of a cell death gene, reaper, in Drosophila cells

The present study was aimed to investigate whether or not cyclin-dependent kinases (CDKs) participate in different cascades leading to apoptosis. We examined the effects of two CDK inhibitors, olomoucine (OLM) and buty-rolactone-I (BL-I), on apoptosis induced in two kinds of Drosophila cell lines. Increases of caspase activity induced by actinomycin D, cycloheximide, H-7 or A23187 in a Drosophila neuronal cell line, ML-DmBG2-c2, and induced by excessive expression of a Drosophila cell death gene, reaper, in Drosophila S2 cells were suppressed by 24-h pretreatment of each CDK inhibitor. Concomitant with the suppression of the caspase activity, fragmentations of cells and DNA, representatives of apoptosis, were also inhibited. These results suggest that CDK(s) participates in progression of apoptosis. However, these effects of the CDK inhibitors were also observed even at lower doses which did not affect cell proliferation. Therefore, it was shown that apoptosis is not always related to cell cycle in Drosophila cells. It was also suggested that the target(s) of the CDK inhibitors locates upstream of caspase in the cascade(s) of apoptosis.     

Identification of an Annexin-Like Protein and Its Possible Role in the Aplysia Eye Circadian System

Abstract: Light and serotonin regulate the phase of the circadian rhythm of the isolated eye of Aplysia . To screen for possible protein components of the eye circadian oscillator, we identified a number of proteins whose synthesis was altered in opposite ways by light and serotonin. The cellular function of one of these proteins was investigated by obtaining a partial amino acid sequence of it and by examining its immunoreactivity. A 38-amino acid sequence was obtained from a 40-kDa (isoelectric point 5.6) protein. A greater than 60% amino acid identity existed between this sequence and sequences of a family of calcium/phospholipid-binding proteins called annexins. Furthermore, the 40-kDa protein reacted with antibodies generated against a conserved amino acid sequence of annexins and with antibodies raised against human annexin I. The identification of the 40-kDa, light- and serotonin-regulated protein as an annexin led us to hypothesize that arachidonic acid metabolism plays a role in the Aplysia eye circadian system. To test this hypothesis, we examined the ability of an inhibitor of the arachidonic acid metabolic pathway to perturb the eye rhythm. Pulse treatments of isolated eyes with a lipoxygenase inhibitor, nordihydroguaiaretic acid, phase shifted the rhythm. The phase-shifting ability of nordihydroguaiaretic acid suggests that arachidonic acid and some of its metabolites may play a role in the eye circadian system. The results of our studies raise the possibility that links may exist between the 40-kDa annexin-like protein, arachidonic acid metabolism, and the circadian oscillator.     

A new, unexpected action of olomoucine, a CDK inhibitor, on normal human cells: up-regulation of CLIMP-63, a cytoskeleton-linking membrane protein

Inhibition of cyclin-dependent kinases (CDKs) is a novel strategy in the therapy of human malignancies. The pharmacological CDK inhibitors representing a few distinct classes of compounds exert different target specificity. Considering the fact that dividing and quiescent cells differ in their CDK activity and in the pattern of their expression, one might expect that anti-proliferative efficiency of the pharmacological CDK inhibitors would depend on the mitotic index of treated cells. The present article shows that olomoucine (OLO), a weak CDK2 inhibitor has new, unexpected activity. At concentrations up to 100 microM OLO did not inhibit proliferation of normal human cells, but arrested growth of human HL-60 leukemia cells. The anti-proliferative effect of OLO was clearly weaker than that of roscovitine (ROSC). Surprisingly, OLO at low doses strongly up-regulated a cellular protein with approximately 65 kDa in normal, but not in immortalized and cancer cells. By mass spectrometric analysis CLIMP-63, a cytoskeleton-linking membrane protein was identified as the major component of the up-regulated protein band. These results were subsequently confirmed by immunoblotting. Further experiments revealed that OLO, but not ROSC, strongly up-regulates CLIMP-63 in a dose- and time-dependent manner solely in senescent cells.     

Characterization of Plasmodium falciparum cdc2-related kinase and the effects of a CDK inhibitor on the parasites in erythrocytic schizogony

The cell cycle of Plasmodium is unique among major eukaryotic cell cycle models. Cyclin-dependent kinases (CDKs) are thought to be the key molecular switches that regulate cell cycle progression in the parasite. However, little information is available about Plasmodium CDKs. The present study was performed to investigate the effects of a CDK inhibitor, olomoucine, on the erythrocytic growth of Plasmodium falciparum. This agent inhibited the growth of the parasite at the trophozoite/schizont stage. Furthermore, we characterized the Plasmodium CDK homolog, P. falciparum cdc2-related kinase-1 (Pfcrk-1), which is a potential target of olomoucine. We synthesized a functional kinase domain of Pfcrk-1 as a GST fusion protein using a wheat germ protein expression system, and examined its phosphorylation activity. The activity of this catalytic domain was higher than that of GST-GFP control, but the same as that of a kinase-negative mutant of Pfcrk-1. After the phosphatase treatment, the labeling of [γ-³²P]ATP was abolished. Recombinant human cyclin proteins were added to these kinase reactions, but there were no differences in activity. This report provides important information for the future investigation of Plasmodium CDKs.     

Light and serotonin interact in affecting the circadian system of Aplysia

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt rhythm of spontaneous compound action potentials. The current study evaluated the influence of serotonin on light-induced phase shifts of this ocular rhythm. The application of serotonin in combination with light was found to have profound and interactive effects on the magnitude of the resulting phase shifts. Further, the phase shifts that resulted from the interaction between light and serotonin appeared to be phase dependent, i.e., the application of serotonin inhibited the phase shifting effects of light during one part of the circadian cycle but enhanced them during another. Finally, the results show that the interaction between light and serotonin is dependent upon the sequence in which these two treatments are paired. These data, coupled with previous findings, suggest that serotonin may act to modulate light's phase shifting effects on the ocular pacemaker in Aplysia.Abbreviations CAP compound action potential - ASW artificial sea water - CT circadian time - 5-HT serotonin