ONIOM study of the nonbonding interaction of the 2PU inhibitor with the CDK2 and CDK4 cyclin‐dependant kinases

We discuss in this article, the applicability of hybrid techniques [especially the our‐own N‐layered integrated molecular orbital and molecular mechanics (ONIOM) method] to weak chemical interactions in large systems, such as the interaction of cyclin‐dependant kinases, CDK4, and CDK2, with a specific ligand (2PU) showing selectivity for CDK4. Our results show that the energies from the ONIOM calculations perfectly match our former molecular dynamics results, both for determining the amino acids which have strongest interactions with the ligand and for explaining the selectivity of 2PU towards CDK4, as compared with CDK2. We show that the ONIOM method is a good candidate for studying such interactions in large systems, even though there are still some technical and theoretical problems to solve. The calculation details will be presented together with the methodology we devised for using the ONIOM approach in such a context. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

Synthesis and CDK2 kinase inhibitory activity of 7/7′-azaindirubin derivatives

A series of novel 7′-azaindirubin (1a-g) and 7-azaindirubin (2a, 2c, 2e and 2f) derivatives were designed and synthesized. Their structures were characterized by 1H NMR and MS spectroscopy as well as by elemental analysis. Their inhibitory properties against CDK2/cylinA were evaluated in vitro. In contrast to indirubin, some of the described azaindirubins emerged as potent inhibitors of CDK2/cylinA and compound 2b had more potent activity. Biological tests also showed that nitrogen atom at 7-position of azaindirubin was more beneficial to enhance the kinase inhibitory activity.     

基于结构的新型CDK7抑制剂的设计、合成及其抗肿瘤活性

根据细胞周期依赖性激酶7(CDK7)的蛋白结构, 利用Discovery Studio 2.1程序包中的LigandFit模块建立了CDK7抑制剂的分子对接模型, 采用受试者工作特征曲线(ROC)方法选择LigScore2为最佳打分函数(ROC曲线下的面积为0.95), 并验证了该模型的准确性. 利用该模型对设计的化合物与CDK7蛋白进行对接分析, 得到了2个打分最高的化合物16、17, 进而通过13步的合成路线, 以中等至高的收率得到目标化合物, 并测定其体外抗肿瘤活性. 结果表明, 所合成的化合物对急性前髓细胞性白血病细胞(HL60)、鼻咽癌细胞(KB)、肝肿瘤细胞(SMMC-7721)、结肠腺癌细胞(HCT-116)、肺癌细胞(A549)细胞株均有抑制作用(IC50值为0.84-19.70 μmol·L-1), 其中化合物16对HL60细胞株的IC50值最低, 为0.84 μmol·L-1.     

靛玉红类CDK1抑制剂的同源模建、分子对接及3D-QSAR研究

细胞周期蛋白依赖性激酶1的异常表达会导致G2期的停滞及多种肿瘤的发生,故CDK1近年来已成为一个理想的治疗靶点. 本文以细胞分裂调控蛋白2的同源体为模板,同源模建了CDK1的结构,并与靛玉红类小分子抑制剂进行分子对接. 分别运用三种叠合方法进行分子叠合,并在此基础上采用Sybyl 7.1中的比较分子场分析(CoMFA)模块及Discovery Studio 3.0中的三维定量构效关系(3D-QSAR)模块(以下简称为DS)分别建立了3D-QSAR模型. 其中,将分子对接叠合与公共骨架叠合联合运用的叠合方法所得3D-QSAR模型的评价参数是最佳的(CoMFA：q²=0.681,r²=0.909,r_pred.²=0.836; DS：q²=0.579,r²=0.971,r_pred.²=0.795,其中q²为交叉验证系数,r²为非交叉验证系数). 本文的研究结果在对靛玉红类小分子进行结构修饰设计出新的CDK1抑制剂方面,可提供重要的理论基础.     

New fascaplysin-based CDK4-specific inhibitors: design, synthesis and biological activity

The first biologically active non-planar analogues of the toxic anti-cancer agent, fascaplysin, have been produced; we present the design, synthesis and biological activity of three tryptamine derivatives.     

Design, synthesis and biological activity of new CDK4-specific inhibitors, based on fascaplysin

We present the design, synthesis, and biological activity of three classes of tryptamine derivatives, which are non-planar analogues of the toxic anti-cancer agent fascaplysin. We show these compounds to be selective inhibitors of CDK4 over CDK2, the most active compound has an IC50 for the inhibition of CDK4 of 6 microM.     

Biochemical characterizations reveal different properties between CDK4/cyclin D1 and CDK2/cyclin A

CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 microM, a value unusually high whereas CDK2/cyclin A was 23 microM, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1) respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1) min(-1) and 170 pM(-1) min(-1) respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.     

Fit-for-Purpose Synthesis of CDK2 Inhibitor GNE-140

A six-step, stereoselective synthesis of cyclin-dependent kinase 2 (CDK2) inhibitor GNE-140 was developed. The key bonds were assembled through palladium-catalyzed Negishi cross-coupling and Buchwald–Hartwig C−N coupling steps. The stereodiad was established through a single-step, CuH-catalyzed enone reduction proceeding in >99 : 1 er and 89 : 11 dr. GNE-140 was obtained in 25 % overall yield with 98.6 A% HPLC purity.     

CDK2-抑制剂结合自由能计算

细胞周期蛋白依赖性激酶Ⅱ(cyclin-dependent kinase 2,CDK2)是一种重要的治疗癌症的靶标.本文中采用分子动力学取样,运用MM-PBSA/GBSA两种方法计算了CDK2-NU6102复合物的绝对结合自由能.通过能量分解的方法考察了CDK2大分子主要残基与配体NU6102之间的相互作用和识别.     

含磷嘧啶类CDK9抑制剂的分子对接、3D-QSAR和分子动力学模拟

选取64个具有潜力的含磷嘧啶类细胞周期依赖性蛋白激酶(CDK9)小分子抑制剂,采用分子对接方法研究了该类小分子与CDK9的结合作用,结果表明,分子构象、氢键形成、疏水性和氨基酸残基Cys106在此类抑制剂与CDK9的结合过程中具有重要作用.在配体叠合的基础上,运用比较分子力场分析(Co MFA)、比较分子相似性指数分析(Co MSIA)和Topomer Co MFA(T-COMFA)研究了分子结构与抑制活性的关系,发现由训练集立体场、静电场和疏水场组合的Co MSIA模型为最优模型,其内部交叉验证相关系数(Q2=0.557)、非交叉验证相关系数(R2=0.959)和外部预测相关系数(r2=0.863)具有统计学意义,该模型的三维等值线图直观显示了化合物的活性与其三维结构的关系.根据这些结果设计了10个具有新结构的含磷嘧啶类化合物,分子对接和分子动力学模拟结果表明,新化合物和CDK9的结合模式与原化合物64相同,自由能分析从理论上证明了新化合物64d的CDK9抑制活性优于化合物64,并且显示含磷基团与残基Asp109的静电场能在化合物与CDK9作用过程中有重要作用.     

Computational Analysis for Residue-Speci c CDK2-Inhibitor Bindings

Cyclin-dependent kinase 2 (CDK2) is a key macromolecule in cell cycle regulation. In cancer cells, CDK2 is often overexpressed and its inhibition is an effective therapy of many cancers including breast carcinomas, leukemia, and lymphomas. Quantitative characterization of the interactions between CDK2 and its inhibitors at atomic level may provide a deep understanding of protein-inhibitor interactions and clues for more effective drug discovery. In this study, we have used the computational alanine scanning approach in combination with an efficient interaction entropy method to study the microscopic mechanism of binding between CDK2 and its 13 inhibitors. The total binding free energy from the method shows a correlation of 0.76?0.83 with the experimental values. The free energy component reveals two binding mode in the 13 complexes, namely van der Waals dominant, and electrostatic dominant. Decomposition of the total energy to per-residue contribution allows us to identify five hydrophobic residues as hot spots during the binding. Residues that are responsible for determining the strength of the binding were also analyzed.     

Cross-docking of inhibitors into CDK2 structures. 1

Predicting protein/ligand binding affinity is one of the most challenging computational chemistry tasks. Numerous methods have been developed to address this challenge, but they all have limitations. Failure to account for protein flexibility has been a shortcoming of many methods. In this cross-docking study the data set comprised 150 inhibitor complexes of the protein kinase CDK2. Gold and Glide performed well in terms of docking accuracy. The chance of cross-docking a ligand within a 2 A RMSD of its experimental pose was found to be 50%. Relative binding potency was not properly predicted from scoring functions, even though cross-docking of each inhibitor into each protein structure was performed and only scores of correctly docked ligands were considered. An accompanying paper (Voigt, J. H.; Elkin, C.; Madison, V. S. Duca, J. S. J. Chem. Inf. Model. 2008, 48, 669-678) covers cross-docking and docking accuracy from the perspective of using multiple protein structures.     

CDK9 inhibitors push cancer cells over the edge

The trouble with CDK active-site inhibitors is their tendency to have off-target effects. This is not surprising, as the ATP binding sites of most protein kinases are very similar. Wang et?al. (2010) have used some clever screening approaches to identify selective CDK9 inhibitors that drive cancer cells into apoptosis.     

Design,Synthesis, and Molecular Docking of Paracyclophanyl-Thiazole Hybrids as Novel CDK1 Inhibitors and Apoptosis Inducing Anti-Melanoma Agents

Three new series of paracyclophanyl-dihydronaphtho[2,3-d]thiazoles and paracyclophanyl-thiazolium bromides were designed, synthesized, and characterized by their spectroscopic data, along with X-ray analysis. One-dose assay results of anticancer activity indicated that 3a–e had the highest ability to inhibit the proliferation of different cancer cell lines. Moreover, the hybrids 3c–e were selected for five-dose analyses to demonstrate a broad spectrum of antitumor activity without apparent selectivity. Interestingly, series I compounds (Z)-N-substituted-4,9-dihydronaphtho[2,3-d]thiazol-3(2H)-yl)-4′-[2.2]paracyclophanylamide) that are carrying 1,4-dihydronaphthoquinone were more active as antiproliferative agents than their naphthalene-containing congeners (series II: substituted 2-(4′-[2.2]paracyclophanyl)hydrazinyl)-4-(naphth-2-yl)-thiazol-3-ium bromide hybrids) and (series III: 3-(4′-[2.2]paracyclophanyl)amido-2-(cyclopropylamino)-4-(naphth-2-yl)thiazol-3-ium bromide) toward the SK-MEL-5 melanoma cell line. Further antiproliferation investigations of 3c and 3e on the healthy, normal unaffected SK-MEL-5 cell line indicated their relative safety. Compound 3c showed an inhibition of eight isoforms of cyclin-dependent kinases (CDK); however, it exhibited the lowest IC₅₀ of 54.8 nM on CDK1 in comparison to Dinaciclib as a reference. Additionally, compound 3c revealed a remarkable downregulation of phospho-Tyr15 with a level (7.45 pg/mL) close to the reference. 3c mainly showed cell cycle arrest in the pre-G1 and G2/M phases upon analysis of the SK-MEL-5 cell line. The sequential caspase-3 assay for 3c indicated a remarkable overexpression level. Finally, a molecular docking study was adopted to elucidate the binding mode and interactions of the target compounds with CDK1.     

Discovery of New Pyrazolopyridine,Furopyridine, and Pyridine Derivatives as CDK2 Inhibitors: Design,Synthesis, Docking Studies,and Anti-Proliferative Activity

New pyridine, pyrazoloyridine, and furopyridine derivatives substituted with naphthyl and thienyl moieties were designed and synthesized starting from 6-(naphthalen-2-yl)-2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (1). The chloro, methoxy, cholroacetoxy, imidazolyl, azide, and arylamino derivatives were prepared to obtain the pyridine-⁻C² functionalized derivatives. The derived pyrazolpyridine-N-glycosides were synthesized via heterocyclization of the C²-thioxopyridine derivative followed by glycosylation using glucose and galactose. The furopyridine derivative 14 and the tricyclic pyrido[3′,2′:4,5]furo[3,2-d]pyrimidine 15 were prepared via heterocyclization of the ester derivative followed by a reaction with formamide. The newly synthesized compounds were evaluated for their ability to in vitro inhibit the CDK2 enzyme. In addition, the cytotoxicity of the compounds was tested against four different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549). The CDK2/cyclin A2 enzyme inhibitory results revealed that pyridone 1, 2-chloro-6-(naphthalen-2-yl)-4-(thiophen-2-yl)nicotinonitrile (4), 6-(naphthalen-2-yl)-4-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridin-3-amine (8), S-(3-cyano-6-(naphthaen-2-yl)-4-(thiophen-2-yl)pyridin-2-yl) 2-chloroethanethioate (11), and ethyl 3-amino-6-(naphthalen-2-yl)-4-(thiophen-2-yl)furo[2,3-b]pyridine-2-carboxylate (14) are among the most active inhibitors with IC₅₀ values of 0.57, 0.24, 0.65, 0.50, and 0.93 µM, respectively, compared to roscovitine (IC₅₀ 0.394 μM). Most compounds showed significant inhibition on different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549) with IC₅₀ ranges of 31.3–49.0, 19.3–55.5, 22.7–44.8, and 36.8–70.7 μM, respectively compared to doxorubicin (IC₅₀ 40.0, 64.8, 24.7 and 58.1 µM, respectively). Furthermore, a molecular docking study suggests that most of the target compounds have a similar binding mode as a reference compound in the active site of the CDK2 enzyme. The structural requirements controlling the CDK2 inhibitory activity were determined through the generation of a statistically significant 2D-QSAR model.     

Cross-docking of inhibitors into CDK2 structures. 2

In the preceding paper (Duca, J. S.; Madison, V. S.; Voigt, J. H. J. Chem. Inf. Model. 2008, 48, 659-668), the accuracy of docking and affinity predictions of the Gold and Glide programs were investigated using single protein conformations spanning 150 CDK2/inhibitor crystallographic complexes. High docking accuracy was observed with both methods; furthermore, Glide showed modest log(IC50)/score correlations. In this part of the study, the effect of combining docking results from multiple protein conformations in a consensus fashion was probed. This approach enhanced docking accuracy only for Glide, which was attributed to the nature of its scoring function. For log(IC50)/score correlations, particular emphasis was placed on considering only scores from correctly docked poses. Using multiple instead of single protein structures showed an improvement in the correlations. Validation sets and scrambling experiments were used to examine the statistical significance and predictivity of these correlations. Rather than actual improvements in scoring accuracy, docking to multiple protein conformations produced overfitting artifacts.