Establishment of high-throughput screening HTRF assay for identification small molecule inhibitors of Skp2-Cks1.

S-phase kinase associated protein 2 (Skp2), a member of the F-box family that constitute the largest known class of ubiquitin E3 specificity components, is responsible for recognizing and recruiting cyclin-dependent kinase inhibitor p27 for its ubiquitination in the presence of the small accessory protein cyclin-dependent kinase regulatory subunit 1(Cks1). Skp2 is overexpressed in esophageal carcinoma tissues and closely related with tumor poor prognosis, and perturbation of the Skp2-Cks1 interaction by inhibitors or RNAi could inhibit the proliferation and metastasis of tumor cells. Therefore, inhibition of Skp2 function by small-molecule compounds targeting Skp2-Cks1 interaction is emerging as a promising and novel anti-cancer strategy. In this study, we establish an improved high-throughput screening platform to screen Skp2 inhibitors targeting Skp2-Cks1interaction, which may provide a new therapeutic approach for the clinic.

Targeted Inhibition of the E3 Ligase SCFSkp2/Cks1 Has Antitumor Activity in RB1-Deficient Human and Mouse Small-Cell Lung Cancer.

The RB1 tumor suppressor gene is mutated in highly aggressive tumors including small-cell lung cancer (SCLC), where its loss, along with TP53, is required and sufficient for tumorigenesis. While RB1-mutant cells fail to arrest at G1-S in response to cell-cycle restriction point signals, this information has not led to effective strategies to treat RB1-deficient tumors, as it is challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, which was confirmed as the mechanism of protection by using knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind/inhibit Skp2 have in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using genetic and pharmacologic approaches, antitumor activity was seen with Skp2 loss or inhibition in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. Our data highlight a downstream actionable target in RB1-deficient cancers, for which there are currently no targeted therapies available. SIGNIFICANCE: There are no effective therapies for SCLC. The identification of an actionable target downstream of RB1, inactivated in SCLC and other advanced tumors, could have a broad impact on its treatment.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Depletion of Cks1 and Cks2 expression compromises cell proliferation and enhance chemotherapy-induced apoptosis in HepG2 cells.

The present study explored the oncogenic roles of overexpressed Cks1 and Cks2 in human hepatocellular carcinoma cells. Gene expression of Cks1 and Cks2 in HepG2 cells was disrupted by siRNA or increased by cDNA transfection. Cell proliferation was assayed by CCK-8 analysis and cell counting. Cisplatin-induced apoptosis after transfection was measured by flow cytometry using Annexin V/propidium iodide (PI) double staining. Cell cycle changes after transfection were determined by flow cytometry with PI staining. Protein levels of Akt and GSK-3ß were measured after transfection. The results revealed that HepG2 proliferation was decreased by depletion of endogenous Cks1 or Cks2, and increased by overexpression of Cks1 or Cks2. HepG2 apoptosis increased concordantly with the decline of Cks1 or Cks2 expression. Overexpression of Cks1 or Cks2 prevented cell apoptosis. Protein levels of p­Akt and p­GSK-3ß were downregulated after RNA interference of Cks1 or Cks2. In conclusion, Cks1 and Cks2 promoted proliferation and prevented apoptosis of HepG2 cells. The Akt/GSK-3ß-related PI3K/Akt signaling pathway may be a key signaling pathway that is involved in the regulation of cell growth and cell death.

Developing structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 protein-protein interaction.

Structure-activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase complex. Disruption of this protein-protein interaction results in higher levels of CDK inhibitor p27, which can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two p27 sensitive tumor cell lines.

Tampering with cell division by using small-molecule inhibitors of CDK-CKS protein interactions.

Cyclin-dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small-molecule inhibitors of CDK-CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S-phase cell populations, and cause a decrease in CDK2, cyclin A, and p27(Kip1) levels. These molecules should help decipher the complex contributions of CDK-CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.

An inhibitor-driven study for enhancing the selectivity of indirubin derivatives towards leishmanial Glycogen Synthase Kinase-3 over leishmanial cdc2-related protein kinase 3.

BACKGROUND: In search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery. METHODS: A new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm. RESULTS: We have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 µΜ. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial kinases was subsequently undertaken to explain and rationalize the selectivity trend determined by the in vitro binding assays. Interestingly, the latter analysis showed that selectivity could be correlated with differences in kinase solvation thermo dynamics induced by minor sequence variations of the otherwise highly similar ATP binding pockets. CONCLUSIONS: In conclusion, 3'-bulky amino substituted 6-BIO derivatives, which demonstrate enhanced specificity towards LGSK-3, represent a new scaffold for targeted drug development to treat leishmaniasis.

High-throughput screening AlphaScreen assay for identification of small-molecule inhibitors of ubiquitin E3 ligase SCFSkp2-Cks1.

Decreased levels of cell cycle inhibitor p27(Kip1) due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27(Kip1) expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27(Kip1) expression could improve prognosis and prevent excessive cell proliferation. SCF(Skp2) is one of the major ubiquitin E3 ligases responsible for degradation of p27(Kip1). Ubiquitination of p27(Kip1) also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27(Kip1). It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27(Kip1) degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27(Kip1) ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27(Kip1) ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.

Overexpression of Cks1 increases the radiotherapy resistance of esophageal squamous cell carcinoma.

PURPOSES: The Cks1 protein is a member of the highly conserved family of Cks/Suc1 proteins, which interact with Cdks, and was found to be an essential cofactor for efficient Skp2-dependent ubiquitination of p27. The present study was undertaken to examine the expression status of Cks1 in esophageal squamous cell carcinoma and its significance. MATERIALS AND METHODS: The expression of Cks1 in 140 esophageal squamous cell carcinoma patients was examined by immunohistochemistry. The correlations between Cks1 expression and tumor clinicopathologic features were analyzed. The effects of Cks1 expression on radiotherapy results were also examined. RESULTS: In the present study, we found that Cks1 is overexpressed in esophageal squamous cell carcinoma tissues. Elevated expression of Cks1 correlates significantly with tumor stage and positive lymph node metastasis (p < 0.05). Moreover, a significant negative correlation was found between Cks1 expression and the survival of patients who received radiotherapy (p < 0.05). At the molecular level, forced expression of Cks1 promotes the radio-resistance ability of EC9706 cells. Knockdown of Cks1 expression sensitizes cancer cells to radiation, and a wobble mutant of Cks1 that is resistant to Cks1 siRNA can rescue this effect. CONCLUSIONS: These results demonstrate for the first time that overexpression of Cks1 correlates with the increased radiotherapy resistance of esophageal squamous cell carcinoma.

A phase 1 study of SNS-032 (formerly BMS-387032), a potent inhibitor of cyclin-dependent kinases 2, 7 and 9 administered as a single oral dose and weekly infusion in patients with metastatic refractory solid tumors.

PURPOSE: SNS-032, (formerly BMS-387032) is a potent and selective inhibitor of cyclin-dependent kinases (CDK) 2, 7 and 9. The primary objective of the study was to establish the maximum tolerated dose (MTD), the maximum administered dose (MAD), dose limiting toxicity (DLT), and the recommended phase 2 dose for SNS-032 when administered as a weekly 1-h infusion. The secondary objective was to assess the safety and tolerability of SNS-032 and to evaluate its bioavailability as an oral solution. METHODS: Patients with metastatic solid tumors or refractory lymphoma were treated with a starting dose of 4 mg/m2 intravenously administered over 1-h with a cycle defined as 3 weekly doses of SNS-032 every 21 days. Three patient cohorts were utilized in the dose-escalation schema. Pharmacokinetic studies were performed. For the 13 and 16 mg/m2 dose cohorts, the first dose of cycle 2 was given as an oral solution to estimate the oral bioavailability of the drug in humans. RESULTS: A total of 21 patients were enrolled. Twenty treated patients received a total of 39 cycles of treatment. The most common treatment-related adverse events occurring with greater than 20% incidence were fatigue (25%) and nausea (20%). Following intravenous administration, plasma concentrations declined in a biphasic manner, resulting in mean terminal half-lives between 5 and 10 hours. The mean Cmax and AUC0-inf increased nearly linearly with dose, ranging from 0.067 to 0.287 microg/ml and 0.103 to 0.553 microg h/ml, respectively. The CL and Vss remained unchanged with increasing dose levels, averaging 38 l/h/m2 and 212 l/m2, respectively. Average oral bioavailability was 19% (range: 4-33%). Three (15%) patients experienced a best response of stable disease. Study enrollment was terminated during dose-escalation due to a change in the development strategy for the study drug. CONCLUSIONS: SNS-032 administered as a weekly 1-h infusion was well tolerated, although study enrollment was terminated during dose-escalation and the MTD of SNS-032 administered intravenously on days 1, 8, and 15 of each treatment cycle was not reached. Tumor progression or stable disease was determined to be the best response in all evaluable patients. At the dose levels tested, the oral bioavailability of SNS-032 ranged from 4-33%. The data suggest that oral administration of SNS-032 may be feasible, though the tolerability and bioavailability of the oral formulation would have to be formally assessed.

Structure-based drug design of a highly potent CDK1,2,4,6 inhibitor with novel macrocyclic quinoxalin-2-one structure.

The design of a novel series of cyclin-dependent kinase (CDK) inhibitors containing a macrocyclic quinoxaline-2-one is reported. Structure-based drug design and optimization from the starting point of diarylurea 2, which we previously reported as a moderate CDK1,2,4,6 inhibitor [J. Biol.Chem.2001, 276, 27548], led to the discovery of potent CDK1,2,4,6 inhibitor that were suitable for iv administration for in vivo study.

Synthesis of novel 5-substituted indirubins as protein kinases inhibitors.

In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we synthesized and evaluated new 5-substituted indirubins. The effects of 34 indirubin derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3, as well as on SH-SY5Y human neuroblastoma cell survival, were investigated.

The Evi1 proto-oncoprotein blocks endomitosis in megakaryocytes by inhibiting sustained cyclin-dependent kinase 2 catalytic activity.

The 3q21q26 syndrome leukaemias are characterised by dystrophic megakaryocytes, elevated platelet counts, ectopic EVI1 protein production and poor prognosis. To investigate the molecular basis of this disease, we developed a model system to examine the biological activity of EVI1 in a megakaryocyte progenitor cell line. For this purpose, Evi1 was conditionally expressed in human erythroleukaemia cells (HEL) that progress along the megakaryocyte lineage in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA-stimulated HEL cells normally undergo: (1) growth arrest; (2) altered morphology; (3) endomitosis and (4) characteristic changes in gene expression, including reduction of the erythroid-specific glycophoryn A and elevation of the specific glycoproteins GPIIIa and GPVI. Enforced Evi1 expression alone had no effect upon HEL cell proliferation or differentiation but a phenotype was manifest upon stimulation to differentiate. Evi1-expressing, TPA-treated HEL cells still showed growth arrest, had reduced and enhanced glycophoryn A and GPIIIa mRNA's, respectively, but failed to significantly elevate GPVI mRNA. This was accompanied by inhibition of endomitosis and altered cell morphology. Sustained CDK2 catalytic activity, typically associated with megakaryocyte endomitosis, was dramatically decreased in TPA-stimulated Evi1-expressing HEL cells because of significantly reduced levels of cyclin A. Therefore, enforced Evi1 expression could inhibit megakaryocyte differentiation although retention of some characteristic molecular changes, in combination with a block in endomitosis and altered morphology, suggest a defect in lineage progression. These results suggest that ectopic Evi1 expression contributes to a defective megakaryocyte differentiation programme and is likely to contribute to the phenotype observed in 3q21q26 syndrome leukaemias.

An analysis of the binding modes of ATP-competitive CDK2 inhibitors as revealed by X-ray structures of protein-inhibitor complexes.

CDK2 is an attractive target for the design of new therapeutic antitumor agent. Numerous CDK2 inhibitors have been discovered and their crystallographic structures either in complex with CDK2 or CDK2/Cyclin A have been published. This review aims to summarize the publicly available structural characterization of CDK2/(Cyclin A) -- ligand complexes and to highlight the similarities among the binding modes of structurally diverse inhibitors.

Perifosine inhibits multiple signaling pathways in glial progenitors and cooperates with temozolomide to arrest cell proliferation in gliomas in vivo.

Perifosine is an oral Akt inhibitor which exerts a marked cytotoxic effect on human tumor cell lines, and is currently being tested in several phase II trials for treatment of major human cancers. However, the efficacy of perifosine in human gliomas has not been established. As Akt is activated in approximately 70% of human glioblastomas, we investigated the impact of perifosine on glia in culture and on a mouse glioma model in vivo. Here we show that perifosine strongly reduces phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk) 1/2, induces cell cycle arrest in G1 and G2, and causes dose-dependent growth inhibition of mouse glial progenitors in which Akt and/or Ras-Erk 1/2 pathways are activated. Furthermore, because temozolomide is a common oral alkylating agent used in the treatment of gliomas, we investigated the effect of perifosine in combination with temozolomide. We observed an enhanced effect when both were used in culture. With these results, we combined perifosine and temozolomide as treatment of platelet-derived growth factor B-driven gliomas in mice. Animal studies showed that perifosine and temozolomide combination therapy was more effective than temozolomide treatment alone (P < 0.01). These results indicate that perifosine is an effective drug in gliomas in which Akt and Ras-Erk 1/2 pathways are frequently activated, and may be a new candidate for glioma treatment in the clinic.

Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors.

Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.

Antiproliferative activity of olomoucine II, a novel 2,6,9-trisubstituted purine cyclin-dependent kinase inhibitor.

The study describes the protein kinase selectivity profile, as well as the binding mode of olomoucine II in the catalytic cleft of CDK2, as determined from cocrystal analysis. Apart from the main cell cycle-regulating kinase CDK2, olomoucine II exerts specificity for CDK7 and CDK9, with important functions in the regulation of RNA transcription. In vitro anticancer activity of the inhibitor in a panel of tumor cell lines shows a wide potency range with a slight preference for cells harboring a wild-type p53 gene. Cell-based assays confirmed activation of p53 protein levels and events leading to accumulation of p21(WAF1). Additionally, in olomoucine II-treated cells, Mdm2 was found to form a complex with the ribosomal protein L11, which inhibits Mdm2 ubiquitin ligase function. We conclude that perturbations in RNA synthesis may lead to activation of p53 and that this contributes to the antiproliferative potency of cyclindependent kinase inhibitors.

8-Azapurines as new inhibitors of cyclin-dependent kinases.

Purine inhibitors of cyclin-dependent kinases (CDK) seem to be a potential anticancer drug candidate as one of the first representatives, roscovitine, is passing Phase II clinical trials for cancer and glomerulonephritis. In this article, we describe a novel modification of the purine scaffold influencing CDK2 inhibitory activities as well as anticancer properties in cell lines of different histopathological origin. The introduced N at position 8 of the purine ring generally lowered CDK2 inhibitory activity of new 8-azapurines (1,2,3-triazolo[4,5-d]pyrimidines) in comparison to the model trisubstituted purines, whereas the antiproliferative potential of some derivatives remained very high, reflecting their ability to activate p53 tumor suppressor.

Boosting: an ensemble learning tool for compound classification and QSAR modeling.

A classification and regression tool, J. H. Friedman's Stochastic Gradient Boosting (SGB), is applied to predicting a compound's quantitative or categorical biological activity based on a quantitative description of the compound's molecular structure. Stochastic Gradient Boosting is a procedure for building a sequence of models, for instance regression trees (as in this paper), whose outputs are combined to form a predicted quantity, either an estimate of the biological activity, or a class label to which a molecule belongs. In particular, the SGB procedure builds a model in a stage-wise manner by fitting each tree to the gradient of a loss function: e.g., squared error for regression and binomial log-likelihood for classification. The values of the gradient are computed for each sample in the training set, but only a random sample of these gradients is used at each stage. (Friedman showed that the well-known boosting algorithm, AdaBoost of Freund and Schapire, could be considered as a particular case of SGB.) The SGB method is used to analyze 10 cheminformatics data sets, most of which are publicly available. The results show that SGB's performance is comparable to that of Random Forest, another ensemble learning method, and are generally competitive with or superior to those of other QSAR methods. The use of SGB's variable importance with partial dependence plots for model interpretation is also illustrated.

Signal transduction of phorbol 12-myristate 13-acetate (PMA)-induced growth inhibition of human monocytic leukemia THP-1 cells is reactive oxygen dependent.

Human monocytic THP-1 cells can be induced to differentiate to macrophages when treated with phorbol 12-myristate 13-acetate (PMA). It is understood that before initiating cell differentiation, PMA treatment must first induce an inhibition of cell growth. Since the initial biochemical and molecular events that are associated with this growth inhibition have not been characterized, the present study was carried out to elucidate the molecular mechanisms associated with the PMA-induced growth arrest of THP-1 cells. Our results indicate that PMA inhibits THP-1 cells at G1-phase of the cell cycle, via a complex mechanism associated with the modulation of the expression of several cell cycle regulators, initiated by the cellular generation of reactive oxygen species (ROS). Both p21WAF1/CIP1 mRNA and protein were upregulated 24 h post PMA treatment as demonstrated by ribonuclease protection assay and Western blotting, respectively. Because these cells lack functional p53, this effect was independent of p53 activity. Electrophoretic mobility shift assay showed that the PMA-induced activation of the p21WAF1/CIP1 promoter was driven by the specific protein 1 (Sp1) transcription factor through Sp1-binding sites. Additionally, our study demonstrates that PMA-induces the upregulation of p21 through a protein kinase C (PKC)-mediated ROS-dependent signaling mechanism involving MAP kinase activation.