Month: July 2019

Forms hydrogen bonds with both the side chain hydroxyl of Ser470 and the hydrogen NH of Gly429 in PARP2, while one of the hydrogens on the primary amide forms a hydrogen bond with the main chain oxygen of Gly429 in PARP2. In addition, the imidazole of ABT-888 stacks with the side chain of Tyr472 of PARP2. Recently, tankyrases have gained increased attention as potential drug targets. They were first discovered as factors that regulate telomere homeostasis by modifying the negative regulator of telomere length, TRF1. Tankyrases also mark axin, the concentration-limiting component of the b-catenin destruction complex, for degradation, and tankyrase inhibition antagonizes the Wnt signal transduction pathway by stabilizing axin and promoting b-catenin degradation. In this TNKS2 structure, XAV939 cyclic amide behaves as an isosteres for ABT-888��s primary amide. There is also a Zelboraf stacking interaction between the pyrimidinone of XAV939 and the Tyr1071 side chain of TNKS2. IWR compounds, however, do not share these features for anchoring in the nicotinamide pocket. It is not clear how these IWR compounds bind to tankyrases and thus the structure-activity relationship for these compounds has been difficult to interpret. Herein, we report a high-resolution crystal structure of the Human TNKS1 catalytic domain in complex with IWR2 and describe the structural basis for its potency and selectivity over PARP1 and PARP2. Our structure reveals a novel binding mode for a tankyrase inhibitor and provides a clear explanation for the reported structure-activity relationship of the IWRs, and important clues for the further optimization of these compounds. The crystals of the TNKS1/2 complex diffracted to 1.9 A ? with synchrotron radiation. There are two crystallographically independent TNKS1/2 complexes in the crystal structure, highly similar to each other. The TNKS1/2 complex structure reveals that 2 does not bind to the nicotinamide pocket but instead occupies a different pocket, which is not present in either apo or XAV939 bound tankyrase structures. It only becomes available upon the binding of 2 and we thus refer to it as the induced pocket. This induced pocket is created by the movement of Phe1188 of the a3 helix and the D-loop, part of which is disordered in the present crystal structure, away from one BI-D1870 another. The binding of 2 to the induced pocket of TNKS1 suggests that IWR compounds are likely non-competitive inhibitors of tankyrases. In the crystal structure, 2 adopts a conformation in which the central phenyl is almost perpendicular to the norbornyl group and rotated by about 60u away from the plane of the amide group. There are three hydrogen bonds between 2 and TNKS1. One of the two carbonyl oxygens of the pyrrolidine dione group is hydrogen bonded to the main chain NH of Tyr1213 and the carbonyl oxygen of the amide group is hydrogen bonded to the main chain NH of Asp1198. The CH at the 6-position of the quinoline is also involved in a CH��O=C hydrogen bonding interaction with the main chain carbonyl oxygen of Gly1196. Moreover, the quinoline group in 2 engages in hydrophobic interaction with the side chain of Phe1188 and stacking interaction with the side chain of His1201 of the D-loop. The quinoline group is co-planar to the amide group as a result of the intra-molecular hydrogen bond between the quinoline nitrogen and the amide NH. Structure-activity relationship studies carried out previously with some of the analogs of 2 in a cellular luciferase-based reporter assay can now be interpreted with the hydrogen bonding and hydrophobic interactions identified from the TNKS1/2 crystal structure.

The CEM/AKB4 cells were hypersensitive to the Aurora A inhibitor MLN8237. CEM/AKB4 cells were, however, cross resistant to a selective Aurora B inhibitor, AZD1152, indicating an Aurora B dependant mechanism of resistance. Although ZM447439 is known to inhibit Aurora A we excluded the possibility of an Aurora A dependent mechanism contributing to resistance to these cells by the lack of Aurora A gene and protein alterations in CEM/AKB4 cells and a lack of cross resistance to the selective Aurora A inhibitor MLN8237. This is in agreement with other reports that show the cytotoxic activity of ZM447439 is mediated through Aurora B, not Aurora A inhibition. Detection of a G160E point Trichostatin A mutation in the kinase domain of Aurora B suggested that resistance in CEM/ AKB4 cells is mediated through impaired binding of the drug to the target kinase. Genetic alterations to drug targets are common mechanisms mediating resistance to targeted therapies; point mutations in BCR-ABL conferring resistance to Imatinib in leukaemia is a classic example. Moreover, the G160E mutation in Aurora B has been reported in colorectal cells selected for resistance to ZM447439. Our findings in a leukaemia cell line further validate that the 160 position is particularly important for drug binding and that point mutations of this residue afford highly penetrant resistance. This mutation should be validated in a clinical setting as it may be important in the use of Aurora B inhibitors and resistance to therapy, much as the T315I BCR-ABL mutation is highly prognostic of outcome for Imatinib treatment in CML patients. As yet, the G160E mutation has not been reported in studies of Aurora B inhibitors in animal models or clinical studies. Although the Aurora B G160E substitution has been shown to independently confer resistance to Aurora B inhibitors it has not been conclusively shown how drug binding is affected. We therefore employed a molecular modelling approach to understand how the G160E substitution alters drug binding and to gain further insights into drug-target interactions of Aurora B inhibitors. Our docking results confirm that binding of ATP to Aurora B is unaltered in mutant Aurora B compared to the wildtype, thereby maintaining catalytic activity. We showed that hydrogen bonding of Aurora B inhibitors to the Ala173 and Lys122 residues are key interactions mediating drug activity by preventing catalytic binding of ATP. However, the presence of the G160E mutant hinders the ability of inhibitors to penetrate as far into the binding pocket as the wild-type enzyme precluding the formation of these hydrogen bonds. Presumably inhibitors are only able to bind to the mutant enzyme in modes that do not compete effectively with ATP and substrate binding, thereby allowing catalytic activity in the presence of the drug and a resistant phenotype. It would be Adriamycin expected that any Aurora B inhibitor that has a similar active binding motif would be affected, explaining the cross resistance of cells with this mutation to structurally related inhibitors in our studies and others. Our models could therefore be used as a screen to identify, or rationally design, inhibitors with novel binding modes that may abrogate Aurora B G160E mediated resistance. The progression of resistance with repeated or higher concentration drug exposure is an important consideration in the treatment of relapsed disease. Both CEM/AKB8 and CEM/AKB16 cells showed a dose dependent increase in transcriptional activity of MDR1, however P-glycoprotein was not functionally active in either case.

Resistance to kinase inhibitors may also be effected by MLN4924 aberrant activation of redundant signalling pathways to that of the target, an example being MET amplification inresistancetoEGFR kinase inhibitors. As CEM/ AKB16 cells were highly resistant to Aurora B inhibition it appears that sustained Aurora B activity in the presence of ZM447439 may still be driving resistance in these cells rather than activation of an alternative pathway. Previous work from our laboratory on drug resistance mediated by tubulin mutations showed that CEM cells acquire additional point mutations in tubulin at higher levels of resistance. Both CEM/AKB8 and CEM/AKB16 cells expressed the Aurora B G160E mutation described for CEM/ AKB4 cells, however no additional mutations in Aurora B were observed, further demonstrating theimportance of the160 residue in drug binding and high-level resistance. Our study of phosphorylated Histone H3 levels showed that CEM/AKB4 cells maintain resistance to Aurora B inhibition at 16 mM ZM, despite this drug concentration being sufficient to induce apoptosis and cell death. This is consistent with off-target kinase inhibition of ZM447439, where at high drug concentrations the contribution of targeting additional cytotoxic pathways to Aurora B inhibition becomes significant. Therefore the resistant phenotype in CEM/AKB16 cells may potentially be mediated LEE011 through alterations in these other targets of ZM447439. ZM447439 has been shown to potently inhibit Aurora A as well as Aurora B in biochemical assaysand we analysed CEM/AKB16 cells for alterations in Aurora A. We found no changes in gene or protein expression of Aurora A in CEM/AKB16 cells and no mutations in the Aurora A gene. Additionally, CEM/AKB16 cells were as equally sensitive as CEM cells to a selective Aurora A inhibitor MLN8237, suggesting that ZM447439 resistance in these cells is not mediated through an Aurora A dependent pathway. It is possible that alterations in other unknown targets of ZM447439 may be responsible, and ultimately, an understanding of the precise mechanisms underpinning resistance in the more highly resistant CEM/AKB8 and CEM/AKB16 cells will shed further light on the mode of action of this drug. Aurora B inhibitors remain a promising area for targeted anticancer therapy, yet a fuller understanding of drug response and resistance mechanisms will aid their clinical implementation. Our findings have confirmed that resistance to these agents is likely across a variety of malignancies and that point mutations in Aurora B, particularly of the 160 residue, may be highly significant markers of treatment outcome. Moreover, our analysis of highly resistant cells suggests that sustained or high-level drug treatment may give rise to an evolution of multiple mechanisms of resistance in patients. Accordingly, our models provide a basis for designing and testing alternative Aurora B inhibitors, and for screening agents that may be employed in combination therapeutic approaches. The two highly homologous human tankyrase isoforms, TNKS1 and TNKS2, are members of the poly ADP-ribose polymerasefamily of 17 proteins that share a catalytic PARP domain. These PARP proteins cleave NAD+into ADP-ribose and nicotinamide and transfer the ADP-ribose units onto their substrates, resulting in a post-translational modification referred to as PARsylation. Cellular functions of many PARP proteins remain unknown. PARP1 and PARP2, the two best characterized family members, are key players in homologous recombination DNA damage response and have been pursued as cancer targets for over a decade. Structural studies of PARP inhibitor complexes reveal that these compounds are anchored in the nicotinamide pocket in a very similar manner.

Additionally, recent reports revealed that upregulated myc expression sensitizes cells for therapeutics targeting CK1e. In our studies, IC261 induced a transient full G2/M arrest at a cell type dependent concentration. We have also shown that at half of this concentration ) the subG1 population increases. This increase of apoptotic cells cannot be due to G2/ M arrest, because at IC50 the population of 4N cells is not significantly increased. It was shown, that CK1 phosphorylates Bid and thereby prevents cleavage by caspase 8. Therefore an inhibition of CK1 at low concentrations of IC261 could lead to activation of pro-apoptotic protein Bid and thereby to increased apoptosis indicated by increased subG1 population. However, it remains unclear why at higher concentrations of IC261 the cell cycle arrest at G2/M is dominant over the pro-apoptotic effect. In summary this study provides data that extends the knowledge of IC261 induced effects in cells. We demonstrate that the CK1 kinase inhibitor IC261 mediates off-CK1-target effects by depolymerizing MTs in a dose-dependent and reversible manner. Therefore, results of previous studies using IC261 as a CK1 inhibitor should be interpreted carefully. Here, we also present evidence that CK1 is neither localized at the TGN nor at the GA, but co-localizes with the COPI protein b-COP. Opportunistic pathogens secrete multiple virulence factors to modulate interactions with the host, to acquire nutrients from the environment and to facilitate adhesion and colonization to a variety of substrates. Pseudomonas aeruginosa secretes a series of proteases that target host proteins to modulate the immune response and to facilitate colonization in infected tissues. Bacterial adherence and colonization may be facilitated by the degradation of host immune and signaling proteins that would otherwise initiate or potentiate the host response. Alternatively, remodeling the local environment of a bacterium may promote its adherence or growth. While the pathophysiological mechanisms in patients have not been fully elucidated, AP has been shown to cleave bacterial flagellin, host signaling molecules and the epithelial ARRY-142886 sodium channel. Cleavage of flagellin and cytokines would putatively alter the host response to the pathogen, while ENaC cleavage would be predicted to remodel the airway surface hydration state, reduce muco-cilliary clearance, and facilitate bacterial adherence and colonization. The combined effects of blunting the host immune response and altering ion channel activity would putatively contribute to an increase in bacterial load within the airway and the apparent virulence of the pathogen. To evaluate the potential use of the aprI inhibitor as a modulator of AP activity in airway epithelial cells, AP and AP Inh were purified. Tight association and protease inhibition were CUDC-907 HDAC inhibitor measured in vitro and demonstrated that near stoichiometric addition of the inhibitor completely bound the protease and inhibited its activity. This inhibition was blocked with N-terminal fusions to the inhibitor, consistent with the known structures of the proteaseinhibitor complexes. ENaC-mediated sodium transport in a model cell line and primary airway cultures confirmed that AP addition to the apical bathing surface activated ENaC and that near stoichiometric addition of AP Inh blocked the observed ENaC activation. Similarly, ENaC activation was observed in response to apical addition of serralysin from S. marcescens. This activation was blocked by the addition of the purified AP Inh protein. These data show that multiple M10/serralysin family members can activate ENaC and more broadly implicate the M10 protease family as modulators of ENaC activity.

This publication raises questions about the specificity of IC261 and the interpretation of the reported effects. The situation is complicated by the fact that several studies have suggested that CK1d/e could be directly involved in microtubule dynamics. CK1d co-localizes with spindle microtubules and phosphorylates a- and b-tubulin in vitro. Furthermore, direct interactions between CK1d and microtubule associated proteins, such as MAP1A, MAP4 and end binding SCH772984 moa protein 1 have been reported. In the present study, re-investigation of the subcellular localization of CK1d using high resolution confocal microscopy revealed that CK1d is located in the perinuclear region close to the TGN and Golgi apparatus, but does not co-localize with these compartments. Instead, CK1d partly co-localizes with COPI positive membranes and b-COP. Further studies of the IC261mediated effects on microtubules showed that high concentrations of IC261 disrupt interphase microtubules, finally leading to a dispersed phenotype of perinuclear membranes compartments. This effect of IC261 can be blocked by pretreatment of cells with taxol. Low concentrations of IC261 disrupt spindle microtubules leading to mitotic arrest, post-mitotic arrest or DAPT apoptosis. The effect of IC261 on microtubules is reversible. These results are in line with the recent finding that IC261 can act as a microtubule depolymerizing agent. Therefore, the effects on cells induced by IC261 should be interpreted carefully as such effects may be due to either inhibition of CK1 or the depolymerization of microtubules, or a combination of the two. The evolutionary conserved serine/threonine-specific kinase family CK1 is involved in a broad range of intracellular processes and can be regulated by intracellular compartmentalization. We here provide evidence that CK1d is localized at perinuclear membrane compartments and co-localizes with b-COP, a subunit of the coatomer protein complex coating COPI vesicles. Treatment of cells with the CK1-inhibitor IC261 induces changes in CK1d localization as well as changes of other membrane compartments such as the TGN and Golgi apparatus, most likely due to depolymerization of microtubules. The aim of the present study was to unravel the various effects of IC261 described in recent years on CK1d, on microtubule dynamics, and on membrane transport processes. Since it has been reported that CK1d is localized on several intracellular membrane compartments, e.g. TGN or GA, we investigated the subcellular localization of CK1d by fluorescence microscopy at high resolution and found that CK1d neither co-localizes with the TGN nor GA structures, but is in close proximity to both compartments. This finding was confirmed by using multiple antibodies for CK1d and for typical TGN and GA markers in two rat cell lines. Whereas the GA and TGN compartments looked like the well-known stack of cisternae, CK1d-positive structures appeared more vesicular and in close proximity to the TGN and GA. Furthermore CK1d seemed to be closer to the GA markers than the TGN marker. Interestingly, CK1d showed partial co-localization with bCOP positive vesicles. b-COP is a subunit of the coatomer complex coating COPI vesicles, which are responsible for retrograde GA-to-ER or intra-GA membrane transport processes. The hypothesis that CK1d could be involved in GA-ER transport is supported by CK1d co-localizes with another coatomer protein b’-COP, and by the report of CK1d regulating membrane binding of ARF GAP1 – a protein stimulating GTPase activity of ARF1, which is required for the uncoating of COPI vesicles. However, in the latter report IC261 was used at high concentration for experiments in cells. The authors argue that in vitro experiments use a lower ATP concentration, whereas intracellular ATP concentrations in vivo are higher.