Identification of chemically diverse Chk1 inhibitors by receptor-based virtual screening

Inhibition of the Chk1 kinase by small molecules is of great therapeutic interest for oncology and in understanding the cellular regulation of the G2/M checkpoint. We report how computational docking of a large electronic catalogue of compounds to an X-ray structure of the Chk1 ATP-binding site allowed prioritisation of a small subset of these compounds for assay. This led to the discovery of 10 novel Chk1 inhibitors, distributed among nine new and clearly different chemical scaffolds. Several of these scaffolds have promising lead-like properties. All these ligands act by competitive binding to the targeted ATP site. The crystal structures of four of these compounds bound to this site are presented, and reasonable modelled docking modes are suggested for the 5 other scaffolds. This structural context is used to assess the potential of these scaffolds for further medicinal chemistry efforts, suggesting that several of them could be elaborated to make additional interactions with the buried part of the ATP site. Some unusual interactions with the conserved kinase backbone motif are pointed out. The ligand-binding modes are also used to discuss their medicinal chemistry potential with respect to undesirable chemical functionalities, whether these functionalities bind directly to the protein or not. Overall, this work illustrates how virtual screening can identify a diverse set of ligands which bind to the targeted site. The structural models for these ligands in the Chk1 ATP-binding site will facilitate further medicinal chemistry efforts targeting this kinase.     

Identification of a buried pocket for potent and selective inhibition of Chk1: prediction and verification

Inhibition of the Chk1 kinase by small molecules binding to its active site is a strategy of great therapeutic interest for oncology. We report how computational modelling predicted the binding mode of ligands of special interest to the Chk1 ATP site, for representatives of an indazole series and debromohymenialdisine. These binding modes were subsequently confirmed by X-ray crystallography. The binding mode of a potent indazole derivative involves non-conventional C-H...O and N-H...pi-aromatic interactions with the protein. These interactions are formed in a buried pocket at the periphery of the ATP-binding site, the importance of which has previously been overlooked for ligand design against Chk1. It is demonstrated that filling this pocket can confer ligands with dramatically enhanced affinity for Chk1. Structural arguments in conjunction with assay data explain why targeting this pocket is also advantageous for selective binding to Chk1. Structural overlays of known inhibitors complexed with Chk1 show that only the indazole series utilizes the pocket of interest. Therefore, the analysis presented here should prove helpful in guiding future structure-based ligand design efforts against Chk1.     

A robust, target-driven, cell-based assay for checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z' factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery.     

Expression,purification, and biochemical characterization of Chk,a soluble protein tyrosine kinase

CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.     

A conserved proliferating cell nuclear antigen-interacting protein sequence in Chk1 is required for checkpoint function

Human checkpoint kinase 1 (Chk1) is an essential kinase required for cell cycle checkpoints and for coordination of DNA synthesis. To gain insight into the mechanisms by which Chk1 carries out these functions, we used mass spectrometry to identify previously uncharacterized interacting partners of Chk1. We describe a novel interaction between Chk1 and proliferating cell nuclear antigen (PCNA), an essential component of the replication machinery. Binding between Chk1 and PCNA was reduced in the presence of hydroxyurea, suggesting that the interaction is regulated by replication stress. A highly conserved PCNA-interacting protein (PIP) box motif was identified in Chk1. The intact PIP box is required for efficient DNA damage-induced phosphorylation and release of activated Chk1 from chromatin. We find that the PIP box of Chk1 is crucial for Chk1-mediated S-M and G(2)-M checkpoint responses. In addition, we show that mutations in the PIP box of Chk1 lead to decreased rates of replication fork progression and increased aberrant replication. These findings suggest an additional mechanism by which essential components of the DNA replication machinery interact with the replication checkpoint apparatus.     

Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM‐Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA‐damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC₅₀ = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP‐competitive inhibitor, as the electron density clearly reveals that it occupies the ATP‐binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.     

Structure-based and shape-complemented pharmacophore modeling for the discovery of novel checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a member of the serine/threonine kinase family, is an attractive therapeutic target for anticancer combination therapy. A structure-based modeling approach complemented with shape components was pursued to develop a reliable pharmacophore model for ATP-competitive Chk1 inhibitors. Common chemical features of the pharmacophore model were derived by clustering multiple structure-based pharmacophore features from different Chk1-ligand complexes in comparable binding modes. The final model consisted of one hydrogen bond acceptor (HBA), one hydrogen bond donor (HBD), two hydrophobic (HY) features, several excluded volumes and shape constraints. In the validation study, this feature-shape query yielded an enrichment factor of 9.196 and performed fairly well at distinguishing active from inactive compounds, suggesting that the pharmacophore model can serve as a reliable tool for virtual screening to facilitate the discovery of novel Chk1 inhibitors. Besides, these pharmacophore features were assumed to be essential for Chk1 inhibitors, which might be useful for the identification of potential Chk1 inhibitors.     

Discovery of a novel class of triazolones as Checkpoint Kinase inhibitors—Hit to lead exploration

Checkpoint Kinase-1 (Chk1, CHK1, CHEK1) is a Ser/Thr protein kinase that mediates cellular responses to DNA-damage. A novel class of Chk1 inhibitors, triazoloquinolones/triazolones (TZ’s) was identified by high throughput screening. The optimization of these hits to provide a lead series is described.     

Chemical screening by mass spectrometry to identify inhibitors of anthrax lethal factor

Mass spectrometry (MS) analysis is applicable to a broad range of biological analytes and has the important advantage that it does not require analytes to be labeled. A drawback of MS methods, however, is the need for chromatographic steps to prepare the analyte, precluding MS from being used in chemical screening and rapid analysis. Here, we report that surfaces that are chemically tailored for characterization by matrix-assisted laser-desorption ionization time-of-flight MS eliminate the need for sample processing and make this technique adaptable to parallel screening experiments. The tailored substrates are based on self-assembled monolayers that present ligands that interact with target proteins and enzymes. We apply this method to screen a chemical library against protease activity of anthrax lethal factor, and report a compound that inhibits lethal factor activity with a K(i) of 1.1 microM and blocks the cleavage of MEK1 in 293 cells.     

Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones

Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.     

Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1

The mediator protein Claspin is critical for the activation of the checkpoint kinase Chk1 during checkpoint responses to stalled replication forks. This function involves the Chk1-activating domain (CKAD) of Claspin, which undergoes phosphorylation on multiple conserved sites. These phosphorylations promote binding of Chk1 to Claspin and ensuing activation of Chk1 by ATR. However, despite the importance of this regulatory process, the kinase responsible for these phosphorylations has remained unknown. By using a multifaceted approach, we have found that casein kinase 1 gamma 1 (CK1γ1) carries out this function. CK1γ1 phosphorylates the CKAD of Claspin efficiently in vitro, and depletion of CK1γ1 from human cells by small interfering RNA (siRNA) results in dramatically diminished phosphorylation of Claspin. Consequently, the siRNA-treated cells display impaired activation of Chk1 and resultant checkpoint defects. These results indicate that CK1γ1 is a novel component of checkpoint responses that controls the interaction of a key checkpoint effector kinase with its cognate mediator protein.     

DNA-damage response in the basidiomycete fungus Ustilago maydis relies in a sole Chk1-like kinase

Chk1 is a protein kinase that acts as a key signal transducer within the complex network responsible of the cellular response to different DNA damages. It is a conserved element along the eukaryotic kingdom, together with a second checkpoint kinase, called Chk2/Rad53. In fact, all organisms studied so far carried at least one copy of each kind of checkpoint kinase. Since the relative contribution to the DNA-damage response of each type of kinase varies from one organism to other, the current view about the roles of Chk1 and Chk2/Rad53 during DNA-damage response is one of mutual complementation and intimate cooperation. However, in this work it is reported that Ustilago maydis – a phytopathogenic fungus exhibiting extreme resistance to UV and ionizing radiation – have a single kinase belonging to the Chk1 family but strikingly no kinases related to Chk2/Rad53 family are apparent. The U. maydis Chk1 kinase is able to respond to different classes of DNA damages and its activity is required for the cellular adaptation to such damages. As other described components of the Chk1 family of kinases, U. maydis Chk1 is phosphorylated and translocated to nucleus in response to DNA-damage signals. Interestingly subtle differences in this response depending on the kind of DNA damage are apparent, suggesting that in U. maydis the sole Chk1 kinase recapitulates the roles that in other organisms are shared by Chk1 and the Chk2/Rad53 family of protein kinases.     

Identification of potential binding sites for the FHA domain of human Chk2 by in vitro binding studies

Human Chk2 is a newly identified tumor suppressor protein involved in signaling pathways in response to DNA damage. The protein consists of a forkhead-associated (FHA) domain and a kinase domain. Identification of binding partners of the Chk2FHA domain is important in understanding the roles of Chk2 in signaling. We report development of an approach involving the use of combinatorial libraries, pull-down assays, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) methods to identify possible candidates for the binding sites of Chk2FHA. The approach has been used to identify Thr329 of p53 and Thr1852 of breast cancer type 1 susceptibility protein (BRCA1) as very likely biological binding sites of Chk2FHA. The results provide useful leads for further biological analyses of cell signaling involving the FHA domain of Chk2 protein.     

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.     

Context-dependent cell cycle checkpoint abrogation by a novel kinase inhibitor

Background

Checkpoint kinase 1 and 2 (Chk1/Chk2), and the Aurora kinases play a critical role in the activation of the DNA damage response and mitotic spindle checkpoints. We have identified a novel inhibitor of these kinases and utilized this molecule to probe the functional interplay between these two checkpoints.

Principal Findings

Fragment screening, structure guided design, and kinase cross screening resulted in the identification of a novel, potent small molecule kinase inhibitor (VER-150548) of Chk1 and Chk2 kinases with IC₅₀s of 35 and 34 nM as well as the Aurora A and Aurora B kinases with IC₅₀s of 101 and 38 nM. The structural rationale for this kinase specificity could be clearly elucidated through the X-ray crystal structure. In human carcinoma cells, VER-150548 induced reduplication and the accumulation of cells with >4N DNA content, inhibited histone H3 phosphorylation and ultimately gave way to cell death after 120 hour exposure; a phenotype consistent with cellular Aurora inhibition. In the presence of DNA damage induced by cytotoxic chemotherapeutic drugs, VER-150548 abrogated DNA damage induced cell cycle checkpoints. Abrogation of these checkpoints correlated with increased DNA damage and rapid cell death in p53 defective HT29 cells. In the presence of DNA damage, reduplication could not be observed. These observations are consistent with the Chk1 and Chk2 inhibitory activity of this molecule.

Conclusions

In the presence of DNA damage, we suggest that VER-150548 abrogates the DNA damage induced checkpoints forcing cells to undergo a lethal mitosis. The timing of this premature cell death induced by Chk1 inhibition negates Aurora inhibition thereby preventing re-entry into the cell cycle and subsequent DNA reduplication. This novel kinase inhibitor therefore serves as a useful chemical probe to further understand the temporal relationship between cell cycle checkpoint pathways, chemotherapeutic agent induced DNA damage and cell death.     

Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation

We have purified and characterized human Chk2 both from baculovirus-infected insect cells and from either untreated or DNA damage-stressed human HCT116 cells. Chk2 from unstressed human cells is largely monomeric and inactive in phosphorylating its substrate, Cdc25C. It is also unphosphorylated at Thr-68, a site that is the target of the ataxia telangiectasia-mutated protein kinase. After treatment of HCT116 cells with a radiomimetic compound neocarzinostatin, active Chk2 exists as stable Thr-68-phosphorylated dimers as well as interconvertable Thr-68-unphosphorylated monomers and dimers. Interestingly, Chk2 from insect cells behaves by all criteria tested like active Chk2 from neocarzinostatin-treated HCT116 cells. Based on Stokes radius and sedimentation coefficient values, Chk2 monomers and dimers have asymmetric rather than globular shapes. Both Thr-68-phosphorylated and Thr-68-unphosphorylated forms of active Chk2 are capable of phosphorylating Cdc25C. Thus, although phosphorylation of Thr-68 may be required for initial oligomerization and activation of Chk2, it is not needed for maintenance of dimerization or kinase activity.     

The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation

The checkpoint kinase Chk1 is an important mediator of cell cycle arrest following DNA damage. The 1.7 A resolution crystal structures of the human Chk1 kinase domain and its binary complex with an ATP analog has revealed an identical open kinase conformation. The secondary structure and side chain interactions stabilize the activation loop of Chk1 and enable kinase activity without phosphorylation of the catalytic domain. Molecular modeling of the interaction of a Cdc25C peptide with Chk1 has uncovered several conserved residues that are important for substrate selectivity. In addition, we found that the less conserved C-terminal region negatively impacts Chk1 kinase activity.     

Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345

Checkpoints are biochemical pathways that provide the cell with mechanisms to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase Chk1 regulates mitotic progression in response to DNA damage and replication interference by blocking the activation of Cdk1/cyclin B. Chk1 is phosphorylated on Ser-317 and Ser-345 following a checkpoint signal, a process that is regulated by Atr, and by the sensor complexes containing Rad17 and Hus1. We show that Chk1 is associated with chromatin in cycling cells and that the chromatin-associated Chk1 is phosphorylated in the absence of exogenous DNA damage. The UV-induced Ser-345-phosphorylated forms of Chk1 that appear minutes after treatment are predominantly associated with chromatin. The Ser-345 site is in a 14-3-3 consensus binding motif and is required for nuclear retention of Chk1 following an hydroxyurea-induced checkpoint signal; nonetheless, Ser-345 or Ser-317 are not required for the chromatin association of Chk1. Hus1, a member of the proliferating cell nuclear antigen-like damage recognition complex plays a role in the phosphorylation of Chk1 on Ser-345, however, Hus1 is not required for phosphorylation on Ser-317 or for Chk1 localization to chromatin. These results indicate that there is more than one step in Chk1 activation and that the regulation of this checkpoint signaling is achieved at least in part through phosphorylation of Ser-345, which serves to localize Chk1 in the nucleus presumably by blocking Crm1-dependent nuclear export.     

P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

The ataxia telangiectasia mutated- and rad3-related kinase (ATR)/Chk1 pathway is a sentinel of cell cycle progression. On the other hand, the Ras/mitogen-activated protein kinase/90-kDa ribosomal S6 kinase (p90 RSK) pathway is a central node in cell signaling downstream of growth factors. These pathways are closely correlated in cell proliferation, but their interaction is largely unknown. Here we show that Chk1 is phosphorylated predominantly at Ser-280 and translocated from cytoplasm to nucleus in response to serum stimulation. Nonphosphorylated Chk1-Ser-280 mutation attenuates nuclear Chk1 accumulation, whereas the phosphomimic mutation has a reverse effect on the localization. Treatment with p90 RSK inhibitor impairs Chk1 phosphorylation at Ser-280 and accumulation at the nucleus after serum stimulation, whereas these two phenomena are induced by the expression of the constitutively active mutant of p90 RSK in serum-starved cells. In vitro analyses indicate that p90 RSK stoichiometrically phosphorylates Ser-280 on Chk1. Together with Chk1 phosphorylation at Ser-345 by ATR and its autophosphorylation at Ser-296, which are critical for checkpoint signaling, Chk1-Ser-280 phosphorylation is elevated in a p90 RSK-dependent manner after UV irradiation. In addition, Chk1 phosphorylation at Ser-345 and Ser-296 after UV irradiation is also attenuated by the treatment with p90 RSK inhibitor or by Ser-280 mutation to Ala. These results suggest that p90 RSK facilitates nuclear Chk1 accumulation through Chk1-Ser-280 phosphorylation and that this pathway plays an important role in the preparation for monitoring genetic stability during cell proliferation.     

Combined inhibition of Chk1 and Wee1: In vitro synergistic effect translates to tumor growth inhibition in vivo

Targeting Chk1 protein kinase can enhance the antitumor effects of radio- and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. To identify genes in synthetic lethality with Chk1, we did a high-throughput screening using a siRNA library directed against 719 human protein kinases in the human ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibitors. Wee1 tyrosine kinase was the most significant gene in synthetic lethality with Chk1. Treatment with non-toxic concentrations of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) confirmed the marked synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate), independently of the p53 status. Detailed molecular analysis showed that the combination caused cancer cells to undergo premature mitosis before the end of DNA replication, with damaged DNA leading to cell death partly by apoptosis. In vivo treatment of mice bearing OVCAR-5 xenografts with the combination of Chk1 and Wee1 inhibitors led to greater tumor growth inhibition than with the inhibitors used as single agents with no toxicity. These data provide a strong rationale for the clinical investigation of the combination of a Chk1 and a Wee1 inhibitor.