Structural basis for Chk1 inhibition by UCN-01

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G(2)/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G(2)/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01.     

Inhibition of checkpoint kinase 2 (CHK2) enhances sensitivity of pancreatic adenocarcinoma cells to gemcitabine

Checkpoint kinase 2 (CHK2) plays pivotal function as an effector of cell cycle checkpoint arrest following DNA damage. Recently, we found that co‐treatment of NSC109555 (a potent and selective CHK2 inhibitor) potentiated the cytotoxic effect of gemcitabine (GEM) in pancreatic cancer MIA PaCa‐2 cells. Here, we further examined whether NSC109555 could enhance the antitumour effect of GEM in pancreatic adenocarcinoma cell lines. In this study, the combination treatment of NSC109555 plus GEM demonstrated strong synergistic antitumour effect in four pancreatic cancer cells (MIA PaCa‐2, CFPAC‐1, Panc‐1 and BxPC‐3). In addition, the GEM/NSC109555 combination significantly increased the level of intracellular reactive oxygen species (ROS), accompanied by induction of apoptotic cell death. Inhibition of ROS generation by N‐acetyl cysteine (NAC) significantly reversed the effect of GEM/NSC109555 in apoptosis and cytotoxicity. Furthermore, genetic knockdown of CHK2 by siRNA enhanced GEM‐induced apoptotic cell death. These findings suggest that inhibition of CHK2 would be a beneficial therapeutic approach for pancreatic cancer therapy in clinical treatment.     

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.     

A Highly Selective Dual Insulin Receptor (IR)/Insulin-like Growth Factor 1 Receptor (IGF-1R) Inhibitor Derived from an Extracellular Signal-regulated Kinase (ERK) Inhibitor

Dual inhibitors of the closely related receptor tyrosine kinases insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) are promising therapeutic agents in cancer. Here, we report an unusually selective class of dual inhibitors of IGF-1R and IR identified in a parallel screen of known kinase inhibitors against a panel of 300 human protein kinases. Biochemical and structural studies indicate that this class achieves its high selectivity by binding to the ATP-binding pocket of inactive, unphosphorylated IGF-1R/IR and stabilizing the activation loop in a native-like inactive conformation. One member of this compound family was originally reported as an inhibitor of the serine/threonine kinase ERK, a kinase that is distinct in the structure of its unphosphorylated/inactive form from IR/IGF-1R. Remarkably, this compound binds to the ATP-binding pocket of ERK in an entirely different conformation to that of IGF-1R/IR, explaining the potency against these two structurally distinct kinase families. These findings suggest a novel approach to polypharmacology in which two or more unrelated kinases are inhibited by a single compound that targets different conformations of each target kinase.     

Alternative binding modes of an inhibitor to two different kinases.

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 micro m) (Sarno et al. (2001) FEBS Letts.496, 44-48). Three other kinases [cyclin-dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3beta] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2-cyclin A at 2.2 A resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.     

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both T_H1 and T_H2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.     

Development of thioquinazolinones,allosteric Chk1 kinase inhibitors

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K_m for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site ～13 Å from the ATP binding site. Preliminary data is presented for several of these compounds.     

Protein kinase A in complex with Rho-kinase inhibitors Y-27632, Fasudil, and H-1152P: structural basis of selectivity

Protein kinases require strict inactivation to prevent spurious cellular signaling; overactivity can cause cancer or other diseases and necessitates selective inhibition for therapy. Rho-kinase is involved in such processes as tumor invasion, cell adhesion, smooth muscle contraction, and formation of focal adhesion fibers, as revealed using inhibitor Y-27632. Another Rho-kinase inhibitor, HA-1077 or Fasudil, is currently used in the treatment of cerebral vasospasm; the related nanomolar inhibitor H-1152P improves on its selectivity and potency. We have determined the crystal structures of HA-1077, H-1152P, and Y-27632 in complexes with protein kinase A (PKA) as a surrogate kinase to analyze Rho-kinase inhibitor binding properties. Features conserved between PKA and Rho-kinase are involved in the key binding interactions, while a combination of residues at the ATP binding pocket that are unique to Rho-kinase may explain the inhibitors' Rho-kinase selectivity. Further, a second H-1152P binding site potentially points toward PKA regulatory domain interaction modulators.     

A novel mode of protein kinase inhibition exploiting hydrophobic motifs of autoinhibited kinases: discovery of ATP-independent inhibitors of fibroblast growth factor receptor

Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.     

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.     

The crystal structure of the phosphatidylinositol 4‐kinase IIα

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4‐kinase type IIα (PI4K IIα), in complex with ATP solved by X‐ray crystallography at 2.8 Å resolution. The structure revealed a non‐typical kinase fold that could be divided into N‐ and C‐lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C‐lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.     

DNA damage-induced S phase arrest in human breast cancer depends on Chk1, but G2 arrest can occur independently of Chk1, Chk2 or MAPKAPK2

Checkpoint kinases Chk1 and Chk2 are two key components in the DNA damage-activated checkpoint signaling pathways. To distinguish the roles of Chk1 and Chk2 in S and G₂ checkpoints after DNA damage, derivatives of the human breast cancer cell line MDA-MB-231 were established that express short hairpin RNAs to selectively suppress Chk1 or Chk2 expression. DNA damage was induced with the topoisomerase I inhibitor SN38 which arrests cells in S or G₂ phase depending on concentration. Depletion of Chk1 resulted in loss of S phase arrest upon incubation with SN38, but the cells still arrested in G₂. Suppression of Chk2 had no impact on cell cycle arrest, while cells concurrently suppressed for both Chk1 and Chk2 still arrested primarily in G₂ suggesting the presence of an alternate checkpoint regulator. One critical target for Chk1 is Cdc25A which is phosphorylated and degraded to prevent cell cycle progression. Cells arrested in G₂ in the absence of Chk1/Chk2 still showed regulation of Cdc25A consistent with the action of an alternate kinase. One candidate for an alternate checkpoint kinase is MAPKAPK2 (MK2), yet this kinase was minimally activated by DNA damage and its inhibition did not facilitate either S or G₂ progression. Furthermore, we were unable to substantiate the recent observation that the Chk1 inhibitor UCN-01 inhibits MK2. These results show that Chk1, but neither Chk2 nor MK2, is an important regulator of S phase arrest, and suggest that an additional kinase can contribute to the G2 arrest.     

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 novel protein with similarities to Rb binding protein 2 compensates for loss of Chk1 function and affects histone modification in fission yeast

The conserved protein kinase Chk1 mediates cell cycle progression and consequently the ability of cells to survive when exposed to DNA damaging agents. Cells deficient in Chk1 are hypersensitive to such agents and enter mitosis in the presence of damaged DNA, whereas checkpoint-proficient cells delay mitotic entry to permit time for DNA repair. In a search for proteins that can improve the survival of Chk1-deficient cells exposed to DNA damage, we identified fission yeast Msc1, which is homologous to a mammalian protein that binds to the tumor suppressor Rb (RBP2). Msc1 and RBP2 each possess three PHD fingers, domains commonly found in proteins that influence the structure of chromatin. Msc1 is chromatin associated and coprecipitates a histone deacetylase activity, a property that requires the PHD fingers. Cells lacking Msc1 have a dramatically altered histone acetylation pattern, exhibit a 20-fold increase in global acetylation of histone H3 tails, and are readily killed by trichostatin A, an inhibitor of histone deacetylases. We postulate that Msc1 plays an important role in regulating chromatin structure and that this function modulates the cellular response to DNA damage.     

Crystal structures of the Mnk2 kinase domain reveal an inhibitory conformation and a zinc binding site

Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) target the translational machinery by phosphorylation of the eukaryotic initiation factor 4E (eIF4E). Here, we present the 2.1 A crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity.     

Chk1 has an essential role in the survival of differentiated cortical neurons in the absence of DNA damage

Neuronal death in the central nervous system contributes to the development of age-related neurodegeneration. The ATR/Chk1 pathway appears to function neuroprotectively to prevent DNA damage induced by cytotoxic agents. Here, we examine the function of Chk1 on cell viability of cortical neurons in the absence of additional DNA damaging stimuli. The Chk1-specific inhibitor, UCN-01, and the ATR inhibitor, Caffeine, cause neuronal apoptosis in differentiated neurons in the absence of additional treatment, whereas inhibition of ATM or Chk2, does not. UCN-01 treatment increased the detection of γ-H2AX phosphorylation, DNA strand breaks, and an activated p53-dependent DNA damage response (DDR), suggesting that Chk1 normally helps to maintain genomic stability. UCN-01 treatment also enhanced the apoptosis seen in neurons treated with DNA damaging agents, such as camptothecin (CPT). Our results indicate that Chk1 is essential for neuronal survival, and perturbation of this pathway increases a cell’s sensitivity to naturally accumulating DNA damage.     

Regulation of CHK2 by DNA-dependent protein kinase

Chk2 is a critical mediator of diverse cellular responses to DNA damage. Activation of Chk2 by DNA damage requires phosphorylation at sites including Thr68. In earlier work, we found that an activity present in rabbit reticulocyte lysates phosphorylates and activates Chk2. We now find that hypophosphorylated Chk2 can be phosphorylated at Thr68 by various subcellular fractions of HEK293 cells. This activity is sensitive to the phosphatidylinositol 3'-kinase-like kinase inhibitor wortmannin, but not to caffeine. DNA enhances the Chk2 phosphorylation by cellular fractions in vitro. The wortmannin-sensitive Chk2 kinase activity is present in fractions from ATM-deficient cells. In contrast, Chk2 was not efficiently phosphorylated at Thr68 in vitro by fractions from cells with a defective DNA-dependent protein kinase (DNA-PK) catalytic subunit. Chk2 is phosphorylated by purified DNA-PK in vitro. Endogenous Chk2 coimmunoprecipitates Ku70 and Ku80. In a series of matched cell lines having and lacking functional DNA-PK, Chk2 activation by exposure of cells to ionizing radiation, or to camptothecin was consistently diminished in the absence of DNA-PK. Down-regulation of DNA-PK(cs) by either siRNA or a chemical inhibitor attenuated radiation-induced Chk2 phosphorylation. Ionizing radiation-induced Chk2 phosphorylation was wortmannin-sensitive in ATM-defective cells with depleted ATR. These results suggest that DNA-PK augments ATM and ATR in activation of Chk2 by DNA damage.     

Synthesis and biological evaluation of 3-ethylidene-1,3-dihydro-indol-2-ones as novel checkpoint 1 inhibitors

Chk1 inhibitors have emerged as a novel class of neoplastic agents for abrogating the G2 DNA damage checkpoint arrest. Analogs of the Chk1 inhibitor, 3-ethylidene-1,3-dihydro-indol-2-one, were synthesized and tested in vitro for their inhibitory activities. The most promising compound identified from this series is analog 28, which possesses potent enzymatic and cellular activities.     

Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.     

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling

RIPK2 mediates inflammatory signaling by the bacteria‐sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD‐mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP‐binding and XIAP‐mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N‐lobe of the kinase, which is in close proximity to the ATP‐binding pocket. Through characterization of a new series of ATP pocket‐binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2‐XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP‐binding pocket in RIPK2 can be exploited to interfere with the RIPK2‐XIAP interaction for modulation of NOD signaling.