A proteome-wide CDK/CRK-specific kinase inhibitor promotes tumor cell death in the absence of cell cycle progression

The identification of molecular determinants of tumor cell survival is an important objective in cancer research. Here, we describe a small-molecule kinase inhibitor (RGB-286147), which, besides inhibiting tumor cell cycle progression, exhibits potent cytotoxic activity toward noncycling tumor cells, but not nontransformed quiescent fibroblasts. Extensive yeast three-hybrid (Y3H)-based proteome/kinome scanning with chemical dimerizers revealed CDK1/2/3/5/7/9 and the less well-characterized CDK-related kinases (CRKs) p42/CCRK, PCTK1/3, and PFTK1 as its predominant targets. Thus, RGB-286147 is a proteome-wide CDK/CRK-specific kinase inhibitor whose further study could yield new insight into molecular determinants of tumor cell survival. Our results also suggest that the [1, 3, 6]-tri-substituted-pyrazolo[3,4-d]-pyrimidine-4-one kinase inhibitor scaffold is a promising template for the rational design of kinase inhibitors with potential applications to disease indications other than cancer, such as neurodegeneration, cardiac hypertrophic growth, and AIDS.     

A genome-wide overexpression screen in yeast for small-molecule target identification

We describe a multicopy gene suppression screen of drug sensitivity in Saccharomyces cerevisiae that facilitates the identification of cellular targets of small molecules. An array of yeast transformants harboring a multicopy yeast genomic library was screened for resistance to growth inhibitors. Comparison of array growth patterns for several such inhibitors allowed the differentiation of general and molecule-specific genetic suppressors. Specific resistance to phenylaminopyrimidine (1), an inhibitor identified from a kinase-directed library, was associated with the overexpression of Pkc1 and a subset of downstream kinases. Components of two other pathways (pheromone response/filamentous growth and Pho85 kinase) that genetically interact with the PKC1 MAPK signaling cascade were also identified. Consistent with the suppression screen, inhibitor 1 bound to Pkc1 in yeast cell lysate and inhibited its activity in vitro. These results demonstrate the utility of this approach for the rapid deconvolution of small-molecule targets.     

Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes

Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (DA-2) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase-drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found DA-2 was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates.     

Inverse in silico screening for identification of kinase inhibitor targets

Protein kinases are clinically relevant, attractive drug targets for cancer. One major problem with kinase inhibitors is broad promiscuity, causing off-target actions and side effects. In silico prediction of targets of a compound would immensely facilitate and accelerate drug development. Using a virtual "inverse" screening approach, where single compounds are docked into protein structures from a database, we identify among known targets of indirubin derivatives phosphoinositide-dependent kinase 1 (PDK1) as a target of one derivative (6BIO) in particular. This prediction is functionally supported by an in vitro kinase assay, inhibition of intracellular phosphorylation of PDK1-substrates, and inhibition of endothelial cell migration, which highly depends on PDK1. Virtual inverse screening combined with biological tests, thus, is proposed as a valuable tool for the drug discovery process and re-examination of already established kinase inhibitors.     

In Situ Proteome Profiling and Bioimaging Applications of Small‐Molecule Affinity‐Based Probes Derived From DOT1L Inhibitors

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small‐molecule inhibitors of DOT1L such as FED1 are potential anti‐cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo‐reactive and “clickable” affinity‐based probes (AfBPs), P1 and P2 , which were cell‐permeable and structural mimics of FED1 . The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub‐cellular localization properties of the probes were subsequently studied in live‐cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1 . Finally with P1 / P2 , large‐scale cell‐based proteome profiling, followed by quantitative LC‐MS/MS, was carried out to identify potential cellular off‐targets of FED1 . Amongst the more than 100 candidate off‐targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off‐target of FED1 by preliminary validation experiments including pull‐down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).     

PI3K/mTOR抑制剂的合成及生物活性

以NVP-BEZ235为先导化合物,设计合成了10个磷脂酰肌醇3-激酶/哺乳动物雷帕霉素靶蛋白(PI3K/mTOR)抑制剂;目标化合物的结构经核磁共振波谱(NMR)和液相色谱-质谱(LC-MS)分析确证.采用噻唑蓝(MTT)比色法在人急性单核细胞白血病细胞株(MV4-11)、人乳腺癌细胞株(BT474)和人前列腺癌细胞株(PC-3)中测定了目标化合物的抗肿瘤活性,并对其构效关系进行了初步的讨论.结果表明,化合物11(FP-189)对MV4-11细胞株表现出较强的抑制活性(IC_(50)=22.5 nmol/L),且具有良好的溶解性,可以作为白血病治疗的候选药物进行下一步开发.     

Alterations of ciliate phosducin phosphorylation in Blepharisma japonicum cells

We have previously reported that motile photophobic response in ciliate Blepharisma japonicum correlates with dephosphorylation of a cytosolic 28 kDa phosphoprotein (PP28) exhibiting properties similar to those of phosducin. Here we demonstrate in in vivo phosphorylation assay that the light-elicited dephosphorylation of the PP28 is significantly modified by cell incubation with substances known to modulate protein phosphatase and kinase activities. Immunoblot analyses showed that incubation of ciliates with okadaic acid and calyculin A, potent inhibitors of type 1 or 2A protein phosphatases, distinctly increased phosphorylation of PP28 in dark-adapted cells and markedly weakened dephosphorylation of the ciliate phosducin following cell illumination. An enhancement of PP28 phosphorylation was also observed in dark-adapted ciliates exposed to 8-Br-cAMP and 8-Br-cGMP, slowly hydrolysable cyclic nucleotide analogs and 3-isobutyryl-1-methylxanthine (IBMX), a non-specific cyclic nucleotide phosphodiesterase (PDEs) inhibitor. Only slight changes in light-evoked dephosphorylation levels of PP28 were observed in cells treated with the cyclic nucleotide analogs and IBMX. Incubation of ciliates with H 89 or KT 5823, highly selective inhibitor of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, decreased PP28 phosphorylation levels in dark-adapted cells, whereas the extent of light-evoked dephosphorylation of the phosphoprotein was only slightly influenced. Cell treatment with higher Ca2+ concentration together with ionophore A23187 in culture medium resulted in marked increase in PP28 phosphorylation levels, while quite an opposite effect was observed in cells exposed to Ca2+ chelators, EGTA or BAPTA/AM as well as calmodulin antagonists, such as trifluoperazine (TFP), W-7 or calmidazolium. Light-dependent dephosphorylation was not considerably affected by these treatments. The experimental findings presented here suggest that an endogenous light-dependent protein kinase-phosphatase system may be engaged in the alteration of phosducin phosphorylation in ciliate B. japonicum thereby to modulate the cell motile photophobic behavior.     

Extraction and validation of substructure profiles for enriching compound libraries

Compounds known to be potent against a specific protein target may potentially contain a signature profile of common substructures that is highly correlated to their potency. These substructure profiles may be useful in enriching compound libraries or for prioritizing compounds against a specific protein target. With this objective in mind, a set of compounds with known potency against six selected kinases (2 each from 3 kinase families) was used to generate binary molecular fingerprints. Each fingerprint key represents a substructure that is found within a compound and the frequency with which the fingerprint occurs was then tabulated. Thereafter, a frequent pattern mining technique was applied with the aim of uncovering substructures that are not only well represented among known potent inhibitors but are also unrepresented among known inactive compounds and vice versa. Substructure profiles that are representative of potent inhibitors against each of the 3 kinase families were thus extracted. Based on our validation results, these substructure profiles demonstrated significant enrichment for highly potent compounds against their respective kinase targets. The advantages of using our approach over conventional methods in analyzing such datasets and its application in the mining of substructures for enriching compound libraries are presented.     

Simvastatin inhibits sphingosylphosphorylcholine-induced differentiation of human mesenchymal stem cells into smooth muscle cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle-like cells expressing α-smooth muscle actin (α-SMA) via transforming growth factor-β1/Smad2- and RhoA/Rho kinase-dependent mechanisms. 3-Hydroxy-3-methylglutaryl- coenzyme A reductase inhibitors (statins) have been known to have beneficial effects in the treatment of cardiovascular diseases. In the present study, we examined the effects of simvastatin on the SPC-induced α-SMA expression and Smad2 phosphorylation in hASCs. Simvastatin inhibited the SPC-induced α-SMA expression and sustained phosphorylation of Smad2 in hASCs. SPC treatment caused RhoA activation via a simvastatin-sensitive mechanism. The SPC-induced α-SMA expression and Smad2 phosphorylation were abrogated by pretreatment of the cells with the Rho kinase inhibitor Y27632 or overexpression of a dominant negative RhoA mutant. Furthermore, SPC induced secretion of TGF-β1 and pretreatment with either Y27632 or simvastatin inhibited the SPC-induced TGF-β1 secretion. These results suggest that simvastatin inhibits SPC-induced differentiation of hASCs into smooth muscle cells by attenuating the RhoA/Rho kinase-dependent activation of autocrine TGF-β1/Smad2 signaling pathway.     

Inverse screening of Simvastatin kinase targets from glioblastoma druggable kinome

The statin drug Simvastatin is a HMG-CoA reductase inhibitor that has been widely used to lower blood lipid. However, the drug is clinically observed to reposition a significant suppressing potency on glioblastoma (GBM) by unexpectedly targeting diverse kinase pathways involved in GBM tumorigensis. Here, an inverse screening strategy is described to discover potential kinase targets of Simvastatin. Various human protein kinases implicated in GBM are enriched to define a druggable kinome; the binding behavior of Simvastatin to the kinome is profiled systematically via an integrative computational approach, from which most kinases have only low or moderate binding potency to Simvastatin, while only few are identified as promising kinase hits. It is revealed that Simvastatin can potentially interact with certain known targets or key regulators of GBM such as ErbB, c-Src and FGFR signaling pathways, but exhibit low affinity to the well-established GBM target of PI3K/Akt/mTOR pathway. Further assays determine that Simvastatin can inhibit kinase hits EGFR, MET, SRC and HER2 at nanomolar level, which are comparable with those of cognate kinase inhibitors. Structural analyses reveal that the sophisticated T790 M gatekeeper mutation can considerably reduce Simvastatin sensitivity to EGFR by inducing the ligand change between different binding modes.     

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.     

Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining

A method for detecting carbonylated proteins in two-dimensional electrophoresis (2-DE) was developed using biotinylation and avidin-fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal-catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4 degrees C in a lysis buffer containing 5 mM biotin-hydrazide. Biotin-hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2-DE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotinylated species were detected using avidin-FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin-FITC staining approached 0.64 pmol of protein-associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose-bisphosphate aldorase, and glyceraldehyde-3-phosphate dehydrogenase were among the major targets of oxidative stress.     

Approach for the Design of Covalent Protein Kinase Inhibitors via Focused Deep Generative Modeling

Deep machine learning is expanding the conceptual framework and capacity of computational compound design, enabling new applications through generative modeling. We have explored the systematic design of covalent protein kinase inhibitors by learning from kinome-relevant chemical space, followed by focusing on an exemplary kinase of interest. Covalent inhibitors experience a renaissance in drug discovery, especially for targeting protein kinases. However, computational design of this class of inhibitors has thus far only been little investigated. To this end, we have devised a computational approach combining fragment-based design and deep generative modeling augmented by three-dimensional pharmacophore screening. This approach is thought to be particularly relevant for medicinal chemistry applications because it combines knowledge-based elements with deep learning and is chemically intuitive. As an exemplary application, we report for Bruton’s tyrosine kinase (BTK), a major drug target for the treatment of inflammatory diseases and leukemia, the generation of novel candidate inhibitors with a specific chemically reactive group for covalent modification, requiring only little target-specific compound information to guide the design efforts. Newly generated compounds include known inhibitors and characteristic substructures and many novel candidates, thus lending credence to the computational approach, which is readily applicable to other targets.     

Synthesis of a beta-estradiol-biotin chimera that potently heterodimerizes estrogen receptor and streptavidin proteins in a yeast three-hybrid system

Small molecules that dimerize proteins in living cells provide powerful probes of biological processes and have potential as tools for the identification of protein targets of natural products. We synthesized 7-alpha-substituted derivatives of beta-estradiol tethered to the natural product biotin to regulate heterodimerization of estrogen receptor (ER) and streptavidin (SA) proteins expressed as components of a yeast three-hybrid system. Addition of an estradiol-biotin chimera bearing a 19-atom linker to yeast expressing DNA-bound ER-alpha or ER-beta LexA fusion proteins and wild-type SA protein fused to the B42 activation domain activated reporter gene expression by as much as 450-fold in vivo (10 muM ligand). Comparative analysis of lower affinity Y43A (biotin Kd approximately 100 pM) and W120A (biotin Kd approximately 100 nM) mutants of SA indicated that moderate affinity interactions can be readily detected with this system. Comparison of a 7-alpha-substituted estradiol-biotin chimera with a structurally similar dexamethasone-biotin chimera revealed that yeast expressing ER proteins can detect cognate ligands with up to 5-fold greater potency and 70-fold higher activity than yeast expressing analogous glucocorticoid receptor (GR) proteins. This approach may facilitate the identification of protein targets of biologically active small molecules screened against genetically encoded libraries of proteins expressed in yeast three-hybrid systems.     

New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design,Synthesis and Biological Evaluation

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure–activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.     

Identification and evaluation of small molecule pan-caspase inhibitors in Huntington's disease models

Huntington's Disease (HD) is characterized by?a mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats on the N terminus of the Htt protein. Numerous studies have identified Htt proteolysis as a critical pathological event in HD postmortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD therapeutic targets. We report the use?of the substrate activity screening method against caspase-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models these irreversible inhibitors suppressed Hdh(111Q/111Q)-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study, the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development.     

In situ kinase profiling reveals functionally relevant properties of native kinases

Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in?vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.     

BaCe0.9Y0.1O3-α固体电解质的离子导电性

用交流复阴抗谱法测定了混合离子(质子+氧离子)导电性固体电解质BaCe0.9Y0.1O3-α在600～1000℃下不同气氛(干燥空气、湿润空气及湿润氢气)中的电导率;通过测定总电导率(离子电导率+电子电导率)随气氛中氧分压po2变化,求得离子电导率和离子迁移数;用氢浓差电池方法测得氢气中的质子迁移数。结果表明,BaCe0.9Y0.1O3-α固体电解质在氧分压<10Pa的气氛(如氢气)中几乎为纯离子导体,而在氧分压为10～10^5Pa的气氛(如空气)中为离子和电子空穴混合导体;样品在各气氛中的离子电导率均高于10^-2S·cm^-1。