哺乳类细胞中周期蛋白依赖激酶抑制因子

近两年,人们相继发现了一组能结合性抑制周期蛋白依赖激酶（ＣＤＫｓ）活性的蛋白因子──ＣＤＫ抑制因子（ＣＫＩｓ）。它们分别是：ｐ２１、ｐ１６、ｐ１５、ｐ２７和ＣＤＩｌ。ｐ２１和ｐ２７有一定同源性,能抑制多种ＣＤＫｓ的活性;ｐ１６和ｐ１５则同源性更高,能特异地与ＣＤＫ４、ＣＤＫ６结合;ＣＤ１１的结合特异性还有待进一步的研究。ｐ２１的表达受ｐ５３的正调控;ＴＧＦ－β则上调ｐ１５的表达以及ｐ２７的抑制活性。以上表明ＣＫｌｓ不仅是ＣＤＫｓ活性的调控者,而且还是抑癌因子与细胞周期调控之间的直接联系者。     

Fragment based discovery of arginine isosteres through REPLACE: Towards non-ATP competitive CDK inhibitors

In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein–protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics.     

Synthesis of aminopyrazole analogs and their evaluation as CDK inhibitors for cancer therapy

We synthesized a library of aminopyrazole analogs to systematically explore the hydrophobic pocket adjacent to the hinge region and the solvent exposed region of cyclin dependent kinases. Structure-activity relationship studies identified an optimal substitution for the hydrophobic pocket and analog 24 as a potent and selective CDK2/5 inhibitor.     

Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.     

Synthesis and biological evaluation of novel 5,6-dihydropyrimido[4,5-f]quinazoline derivatives as potent CDK2 inhibitors

As serine/threonine kinase, the cyclin dependent kinase 2 (CDK2) is a promising target for various diseases such as cerebral hypoxia, cancer, and neurodegenerative diseases. Here we reported the structure-based synthesis and biological evaluation of novel 5,6-dihydropyrimido[4,5-f]quinazoline derivatives as CDK2 inhibitors, which exhibited potent CDK2 inhibitory activities, as well as anticancer activities in low concentration against two human cancer cell lines (MCF-7 and HCT116). In particular, compounds 11a and 11f (IC₅₀ values of 0.11 and 0.09?μM for CDK2, respectively) have demonstrated significantly inhibitory potency against CDK2 and have showed great inhibitory activities against MCF-7 and HCT116 cell lines.     

Modulating the interaction between CDK2 and cyclin A with a quinoline-based inhibitor

A new class of quinoline-based kinase inhibitors has been discovered that both disrupt cyclin dependent 2 (CDK2) interaction with its cyclin A subunit and act as ATP competitive inhibitors. The key strategy for discovering this class of protein–protein disrupter compounds was to screen the monomer CDK2 in an affinity-selection/mass spectrometry-based technique and to perform secondary assays that identified compounds that bound only to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (K_d) of the original hit improved from 1 to 0.005 μM.     

Fluorescent Biosensor of CDK5 Kinase Activity in Glioblastoma Cell Extracts and Living Cells

CDK5 plays a major role in neuronal functions, and is hyperactivated in neurodegenerative pathologies as well as in glioblastoma and neuroblastoma. Although this kinase constitutes an established biomarker and pharmacological target, there are few means of probing its activity in cell extracts or in living cells. To this aim a fluorescent peptide reporter of CDK5 kinase activity, derived from a library of CDK5‐specific substrates, is engineered and its ability to respond to recombinant CDK5/p25 is established and CDK5 activity in glioblastoma cell extracts is reported on through sensitive changes in fluorescence intensity. A cell‐penetrating variant of this biosensor which can be implemented to image CDK5 activation dynamics in space and in time is further implemented. This original biosensor constitutes a potent tool for quantifying differences in CDK5 activity following treatment with selective inhibitors and for monitoring CDK5 activation, following inhibition or stimulation, in a physiologically relevant environment. As such it offers attractive opportunities to develop a diagnostic assay for neuronal pathologies associated with hyperactivated CDK5, as well as a companion assay to evaluate response to new therapies targeting this kinase.     

Design, synthesis, and testing of an 6-O-linked series of benzimidazole based inhibitors of CDK5/p25

Alzheimer’s disease (AD) is a progressive neurodegenerative disease resulting in cognitive and behavioral impairment. The two classic pathological hallmarks of AD include extraneuronal deposition of amyloid ?? (A??) and intraneuronal formation of neurofibrillary tangles (NFTs). NFTs contain hyperphosphorylated tau. Tau is the major microtubule-associated protein in neurons and stabilizes microtubules (MTs). Cyclin dependent kinase 5 (CDK5), when activated by the regulatory binding protein p25, phosphorylates tau at a number of proline-directed serine/threonine residues, resulting in formation of phosphorylated tau as paired helical filaments (PHFs) then in subsequent deposition of PHFs as NFTs. Beginning with the structure of Roscovitine, a moderately selective CDK5 inhibitor, we sought to conduct structural modifications to increase inhibitory potency of CDK5 and increase selectivity over a similar enzyme, cyclin dependent kinase 2 (CDK2). The design, synthesis, and testing of a series of 1-isopropyl-4-aminobenzyl-6-ether-linked benzimidazoles is presented.     

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.     

SARS冠状病毒主蛋白酶抑制剂的筛选及抑制动力学研究

对SARS冠状病毒主蛋白酶(SARS-CoV M^pro)进行异源重组表达与提纯,并以其为靶点,利用基于荧光共振能量转移(FRET)技术的体外药物筛选模型,对蛋白酶抑制剂聚焦库96种化合物进行了体外抑制活性的评价,并从动力学的角度探讨筛选出的阳性化合物对SARS-CoV M^pro的抑制能力与机制。结果表明:通过筛选获得抑制率>80%、淬灭率<20%的化合物5种,为P-1-08、P-1-19、P-2-24、P-2-28、P-2-54,其半数有效抑制浓度(IC₅₀)分别为:0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L。其中化合物P-1-08、P-1-19、P-2-24、P-2-54对SARS冠状病毒主蛋白酶的抑制作用为不可逆抑制,化合物P-2-28的抑制作用为可逆抑制。根据Lineweaver-Burk图和Dixon图的研究,发现化合物P-2-28对SARS冠状病毒主蛋白酶呈竞争性抑制,抑制常数Ki为0.81μmol/L。通过对底物浓度,IC₅₀值及Ki值关系的研究,进一步验证了P-2-28的抑制作用为竞争性抑制。该抑制剂的发现为SARS冠状病毒主蛋白酶抑制剂的研究打下基础,为抗SARS病毒药物开发提供了先导化合物。     

Establishment of screening system toward discovery of kinase inhibitors using label-free on-chip phosphorylation assays

We describe a label-free method for the kinase inhibition assay toward discovery of kinase inhibitors. The surface plasmon resonance (SPR) imaging analysis using zinc(II) compound was adopted on the on-chip phosphorylation analysis. In this study, following three subjects were focused: (1) to monitor the inhibition of three inhibitors supporting by their specific inhibition mechanisms, (2) to quantify the inhibitory activities, and (3) to prove the reliability of the obtained 50% inhibition concentration (IC₅₀) value. First, the inhibitory activities of Amide 5-24, H-89 and Gö6983 on PKA and PKCδ were determined, and specific inhibitions for two kinases could be observed quantitatively. Second, the inhibition curves of Amide 5-24, Amide 14-22 and H-89 were obtained, and the results supported the two previous reports: (1) the inhibition efficiency of Amide 5-24 was much higher than that of Amide 14-22, and (2) the inhibitory activity of H-89 followed ATP-binding site blocking mechanism. Last, the obtained IC₅₀ values by the SPR imaging were almost corresponded to those by the solution assay, although on-chip phosphorylation efficiency was low (approximately 12%). In conclusion, validation of the on-chip phosphorylation analysis for kinase inhibitors was achieved. This label-free method might be applied for discovery of kinase inhibitors.     

High-Throughput Selection of Transmembrane Sequences That Enhance Receptor Tyrosine Kinase Activation

Dimerization is a critical requirement for the activation of the intracellular kinase domains of receptor tyrosine kinases (RTKs). The single transmembrane (TM) helices of RTKs contribute to dimerization, but the details are not well understood. Work with TM helices in various model systems has revealed a small number of specific dimerization sequence motifs, and it has been suggested that RTK dimerization is modulated by such motifs. Yet questions remain about the universality of these sequence motifs for RTK dimerization and about how TM domain dimerization in model systems relates to RTK activation in mammalian membranes. To investigate these questions, we designed a 3888-member combinatorial peptide library based on the TM domain of Neu (ErbB2) as a model RTK. The library contains many closely related, Neu-like sequences, including thousands of sequences with known dimerization motifs. We used an SDS-PAGE-based screen to select peptides that dimerize better than the native Neu sequence, and we assayed the activation of chimeric Neu receptors in mammalian cells with TM sequences selected in the screen. Despite the very high abundance of known dimerization motifs in the library, only a very few dimerizing sequences were identified by SDS-PAGE. About half of those sequences activated the Neu kinase significantly more than did the wild-type TM sequence. This work furthers our knowledge about the requirements for membrane protein interactions and the requirements for RTK activation in cells.     

The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation

Calmodulin (CaM)-dependent eukaryotic elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of eukaryotic elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca²⁺/CaM binds eEF-2K with high affinity (K_d(CaM)^app = 24 ± 5 nm) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 10⁴-fold (k_auto = 2.6 ± 0.3 s⁻¹). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca²⁺/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing k_cat^app for peptide substrate phosphorylation by 10³-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (k_cat^app/K_m(Pep-S)^app), with equal contributions from k_cat^app and K_m(Pep-S)^app, suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). Because upstream signaling events have the potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output.     

Analysis of CDK2 active-site hydration: a method to design new inhibitors

The interactions between the protein and the solvent were analyzed, and protein regions with a high density of water molecules, as well as structural water molecules, were determined by using molecular dynamics (MD) simulations. A number of water molecules that were in contact with the protein for the whole trajectory were determined. Their interaction energies and hydrogen bonds with protein residues were analyzed. Altogether, 39, 27, 49, and 32 water molecules bound to the protein were found for trajectories of the free CDK2, CDK2/ATP, CDK2/roscovitine, and CDK2/isopentenyladenine complexes, respectively. Positions of observed water molecules were compared with X-ray crystallography data. Special attention was paid to water molecules in the active site of the enzyme, and especially to the deep pocket, where the N9 roscovitine side-chain is buried. Exchange of active-site water molecules with bulk water through the tunnel from the pocket was observed. In the CDK2/isopentenyladenine complex simulation, two water molecules that arrange interaction between the inhibitor and the enzyme via an H-bond were observed. Two stable water molecules in the trajectory of the free CDK2 were found that occupy the same position as the nitrogens N3 and N9 of the isopentenyladenine or N1 and N6 nitrogens of the adenosine triphosphate (ATP). The positions of structural water molecules were compared with the positions of substrate polar groups and crystallographic water molecules found in the Brookhaven Protein Data Bank for various CDK2 complexes. It was concluded that tracing tightly bound water molecules may substantially help in designing new inhibitors.     

Activation of Cdc2 kinase during meiotic maturation of axolotl oocyte

Activity of Cdc2, the universal inducer of mitosis, is regulated by phosphorylation and binding to cyclin B. Comparative studies using oocytes from several amphibian species have shown that different mechanisms allow Cdc2 activation and entry into first meiotic division. In Xenopus, immature oocytes stockpile pre-M-phase promoting factor (MPF) composed of Cdc2-cyclin B complexes maintained inactive by Thr14 and Tyr15 phosphorylation of Cdc2. Activation of MPF relies on the conversion of pre-MPF into MPF by Cdc2 dephosphorylation, implying a positive feedback loop known as MPF auto-amplification. On the contrary, it has been proposed that pre-MPF is absent in immature oocyte and that MPF activation depends on cyclin synthesis in some fishes and other amphibians. We demonstrate here that MPF activation in the axolotl oocyte, an urodele amphibian, is achieved through mechanisms resembling partly those found in Xenopus oocyte. Pre-MPF is present in axolotl immature oocyte and is activated during meiotic maturation. However, monomeric Cdc2 is expressed in large excess over pre-MPF, and pre-MPF activation by Cdc2 dephosphorylation takes place progressively and not abruptly as in Xenopus oocyte. The intracellular compartmentalization as well as the low level of pre-MPF in axolotl oocyte could account for the differences in oocyte MPF activation in both species.     

Homology Model of the CDK1/cyclin B Complex

Abstract

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.     

Investigating the neuroprotective mechanism of action of a CDK5 inhibitor by phosphoproteome analysis

Small molecule inhibitors of cyclin-dependent kinase 5 (CDK5) protect neurons from cell death following various insults. To elucidate the cellular mechanism of action we investigated changes in protein phosphorylation in cultured rat cerebellar granule neurons after administration of the CDK5 inhibitor Indolinone A. By immunoblot analysis we detected enhanced phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) and the Jun N-terminal kinase (JNK) substrate c-Jun. Co-administration of U0126, an inhibitor of ERK1/2, or SP600125, an inhibitor of JNK, blocked phosphorylation of ERK1/2 or c-Jun, but did not affect neuroprotection by the CDK5 inhibitor. By metal affinity chromatography, two-dimensional (2D) gel electrophoresis, and MALDI-TOF mass spectrometry we identified several phosphoproteins that accumulated in neurons treated with Indolinone A. Among them were proteins involved in neurotransmitter release, which is consistent with a physiological function of CDK5 in synaptic signaling. Moreover, we identified proteins acting in energy metabolism, protein folding, and oxidative stress response. Similar findings have been reported in yeast following inhibition of Pho85 kinase, which is homologous to mammalian CDK5 and acts in environmental stress signaling. These results suggest that inhibition of CDK5 activates stress responsive proteins that may protect neurons against subsequent injurious stimuli.