Kinetic and mechanistic characterisation of Choline Kinase-α.

Choline Kinase is a key component of the Kennedy pathway that converts choline into a number of structural and signalling lipids that are essential for cell growth and survival. One member of the family, Choline Kinase-α (ChoKα) is frequently up-regulated in human cancers, and expression of ChoKα is sufficient to transform cells. Consequently ChoKα has been studied as a potential target for therapeutic agents in cancer research. Despite great interest in the enzyme, mechanistic studies have not been reported. In this study, a combination of initial velocity and product inhibition studies, together with the kinetic and structural characterisation of a novel ChoKα inhibitor is used to support a mechanism of action for human ChoKα. Substrate and inhibition kinetics are consistent with an iso double displacement mechanism, in which the γ-phosphate from ATP is transferred to choline in two distinct steps via a phospho-enzyme intermediate. Co-crystal structures, and existing site-specific mutation studies, support an important role for Asp306, in stabilising the phospho-enzyme intermediate. The kinetics also indicate a distinct kinetic (isomerisation) step associated with product release, which may be attributed to a conformational change in the protein to disrupt an interaction between Asp306 and the phosphocholine product, facilitating product release. This study describes a mechanism for ChoKα that is unusual amongst kinases, and highlights the availability of different enzyme states that can be exploited for drug discovery.

Structure of human Bloom's syndrome helicase in complex with ADP and duplex DNA.

Bloom's syndrome is an autosomal recessive genome-instability disorder associated with a predisposition to cancer, premature aging and developmental abnormalities. It is caused by mutations that inactivate the DNA helicase activity of the BLM protein or nullify protein expression. The BLM helicase has been implicated in the alternative lengthening of telomeres (ALT) pathway, which is essential for the limitless replication of some cancer cells. This pathway is used by 10-15% of cancers, where inhibitors of BLM are expected to facilitate telomere shortening, leading to apoptosis or senescence. Here, the crystal structure of the human BLM helicase in complex with ADP and a 3'-overhang DNA duplex is reported. In addition to the helicase core, the BLM construct used for crystallization (residues 640-1298) includes the RecQ C-terminal (RQC) and the helicase and ribonuclease D C-terminal (HRDC) domains. Analysis of the structure provides detailed information on the interactions of the protein with DNA and helps to explain the mechanism coupling ATP hydrolysis and DNA unwinding. In addition, mapping of the missense mutations onto the structure provides insights into the molecular basis of Bloom's syndrome.

Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR.

Here we report a comprehensive biological characterization of a potent and selective small-molecule inhibitor of the DNA damage response (DDR) kinase ATR. We show a profound synthetic lethal interaction between ATR and the ATM-p53 tumor suppressor pathway in cells treated with DNA-damaging agents and establish ATR inhibition as a way to transform the outcome for patients with cancer treated with ionizing radiation or genotoxic drugs.

Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities.

ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy.

The ex vivo characterization of XR5944 (MLN944) against a panel of human clinical tumor samples.

XR5944 (MLN944) is a novel DNA targeting agent with potent antitumor activity, both in vitro and in vivo, against several murine and human tumor models. We have used an ATP-tumor chemosensitivity assay to assess the ex vivo sensitivity of a variety of solid tumors (n = 90) and a CCRF-CEM leukemia cell line selected with XR5944. Differences in gene expression between the parental CCRF-CEM and the resistant subline were investigated by quantitative reverse transcription-PCR. Immunohistochemistry for topoisomerases I and IIalpha and multidrug resistance (MDR1) protein was done on those tumors for which tissue was available (n = 32). The CCRF-CEM XR5944 line showed increased mRNA levels of MDR1, major vault protein, and MDR-associated protein 1 compared with the parental line, whereas the expression of topoisomerases I, IIalpha, and IIbeta was essentially unchanged, suggesting that XR5944 is susceptible to MDR mechanisms. The median IC90 and IC50 values for XR5944 in tumor-derived cells were 68 and 26 nmol/L, respectively, 6-fold greater than in resistant cell lines. XR5944 was 40- to 300-fold more potent than the other cytotoxics tested, such as doxorubicin, topotecan, and paclitaxel. Breast and gynecologic malignancies were most sensitive to XR5944, whereas gastrointestinal tumors showed greater resistance. A positive correlation (r = 0.68; P < 0.0001) was found between the IC50 values of XR5944 and P-glycoprotein/MDR1 staining but not with either topoisomerase I or IIalpha immunohistochemistry index. These data support the rapid introduction of XR5944 to clinical trials and suggest that it may be effective against a broad spectrum of tumor types, especially ovarian and breast cancer.

Structure-activity relationships for pyrido-, imidazo-, pyrazolo-, pyrazino-, and pyrrolophenazinecarboxamides as topoisomerase-targeted anticancer agents.

Heterocyclic phenazinecarboxamides were prepared by condensation of aminoheterocycles and 2-halo-3-nitrobenzoic acids, followed by reductive ring closure and amidation. They showed similar inhibition of paired cell lines that underexpressed topo II or overexpressed P-glycoprotein, indicating a non topo II mechanism of cytotoxicity and indifference to P-glycoprotein mediated multidrug resistance. Compounds with a fused five-membered heterocyclic ring were generally less potent than the pyrido[4,3-a]phenazines. A 4-methoxypyrido[4,3-a]phenazine (IC(50)s 2.5-26 nM) gave modest (ca. 5 day) growth delays in H69/P xenografts with oral dosing.

Novel angular benzophenazines: dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents.

A series of substituted angular benzophenazines were prepared using a new synthetic route via a novel regiocontrolled condensation of 1,2-naphthoquinones and 2,3-diaminobenzoic acids. The synthesis and biological activity of this new series of substituted 8,9-benzo[a]phenazine carboxamide systems are described. The analogues were evaluated against the H69 parental human small cell lung carcinoma cell line and H69/LX4 resistant cell line which overexpresses P-glycoprotein. Selected analogues were evaluated against the COR-L23 parental human non small cell lung carcinoma cell line and the COR-L23/R resistant cell line which overexpresses multidrug resistance protein. This series of novel angular benzophenazines were potent cytotoxic agents in these cell lines and may be able to circumvent multidrug resistance mechanisms which result in the lack of efficacy of many drugs in cancer chemotherapy. These compounds show dual inhibition of topoisomerase I and topoisomerase II and thus target two key enzymes responsible for the topology of DNA that are active at different points in the cell cycle. The introduction of chirality into the carboxamide side chain of these novel benzophenazine carboxamides has resulted in the discovery of a potent enantiospecific series of cytotoxic agents, exemplified by 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576 ((R)-4j' '). In vivo activity has been demonstrated for 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576, after intravenous administration to female mice, and this compound has been selected as a development candidate for further evaluation.

Crystal structure of the Pseudomonas aeruginosa MurG: UDP-GlcNAc substrate complex.

MurG is an essential bacterial glycosyltransferase enzyme in Pseudomonas aeruginosa performing one of the key membrane steps of peptidoglycan synthesis catalyzing the transfer of N-acetyl glucosamine (GlcNAc) from its donor substrate, UDP-GlcNAc, to the acceptor substrate Lipid I. We have solved the crystal structure of the complex between Pseudomonas aeruginosa MurG and UDP-GlcNAc and compared it with the previously solved complex from E. coli. The structure reveals a large-scale conformational change in the relative orientations of the N- and C-terminal domains, which has the effect of widening the cofactor binding site and displacing the UDP-GlcNAc donor. These results suggest new opportunities to design potent inhibitors of peptidoglycan biosynthesis.

The novel ATR inhibitor VE-821 increases sensitivity of pancreatic cancer cells to radiation and chemotherapy.

DNA damaging agents such as radiotherapy and gemcitabine are frequently used for the treatment of pancreatic cancer. However, these treatments typically provide only modest benefit. Improving the low survival rate for pancreatic cancer patients therefore remains a major challenge in oncology. Inhibition of the key DNA damage response kinase ATR has been suggested as an attractive approach for sensitization of tumor cells to DNA damaging agents, but specific ATR inhibitors have remained elusive. Here we investigated the sensitization potential of the first highly selective and potent ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced phosphorylation of Chk1, confirming inhibition of ATR signaling. Consistently, VE-821 significantly enhanced the sensitivity of PSN-1, MiaPaCa-2 and primary PancM pancreatic cancer cells to radiation and gemcitabine under both normoxic and hypoxic conditions. ATR inhibition by VE-821 led to inhibition of radiation-induced G 2/M arrest in cancer cells. Reduced cancer cell radiosurvival following treatment with VE-821 was also accompanied by increased DNA damage and inhibition of homologous recombination repair, as evidenced by persistence of γH2AX and 53BP1 foci and inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a novel approach to improve the efficacy and therapeutic index of standard cancer treatments across a large proportion of pancreatic cancer patients.

Mode of action of the novel phenazine anticancer agents XR11576 and XR5944.

The substituted phenazines XR11576 and XR5944 were originally described as dual topoisomerase-I/II poisons. Subsequent reports, however, indicated that the association of their cytotoxicity with cellular topoisomerases was not clear. We set out to study this further using human tumour cell lines, PEO1 ovarian cancer, MDA-MB-231 breast cancer and variants with acquired resistance to VP-16 and XR11576: PEO1VPR, MB-231VPR, MB-231-11576R and camptothecin: PEO1CamR. Cytotoxicity testing [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay], DNA-protein crosslink formation, cell cycle analysis (flow cytometry) for DNA content, apoptosis (flow cytometry) for Annexin V and Western blotting for apoptotic factors. Cytotoxicity testing showed potent cytotoxicity with no cross-resistance to XR11576 or XR5944 in VP-16 or camptothecin-resistant lines. Importantly, we have shown for the first time that the activities of XR5944 and XR11576 are similar as MB-231-11576R cells were resistant to both agents and to a similar extent. XR5944 showed the greatest, albeit slower, interaction with DNA with high levels of DNA-protein crosslinks. Levels of apoptosis in XR5944-treated cells were significantly less than those in VP-16 or XR11576 treatments, suggestive of a more cytostatic rather than cytotoxic mode of action. Interestingly, XR5944 failed to give rise to a G2/M blockade, in contrast to VP-16 or XR11576. XR5944 and XR11576, in line with a dual topoisomerase-I/II-directed mechanism of action, retain potent activity in tumour cells with acquired resistance to VP-16 and camptothecin. Although these agents appear to behave differently from each other according to experimental conditions, this study suggests a substantial overlap in their mechanism(s) of action.

Phenazine-1-carboxamides: structure-cytotoxicity relationships for 9-substituents and changes in the H-bonding pattern of the cationic side chain.

A series of phenazine-1-carboxamides were prepared, including variations in both chromophore substituents and the nature of the cationic side chain. The novel side-chain analogues were prepared from the corresponding phenazine-1-carboxylic acids via Schmidt conversion to the 1-amines and from the corresponding 1-halides. Structure-cytotoxicity relationships for these compounds in a panel of tumor cell lines showed that there is very limited scope for variation of the structure of the 1-carboxamide side chain, consistent with the recent structural model of how tricyclic carboxamides bind to DNA. There was generally little difference in IC(50)s between parent and P-glycoprotein expressing cell lines, suggesting that most of the compounds are not affected by the presence of this efflux pump.

Preclinical anti-tumor activity of XR5944 in combination with carboplatin or doxorubicin in non-small-cell lung carcinoma.

XR5944 (MLN944) is a novel bis-phenazine currently in phase I clinical trials that has demonstrated potent cytotoxic activity against a variety of tumor models. The combinations of XR5944 with carboplatin or doxorubicin were investigated in COR-L23/P human non-small-cell lung carcinoma (NSCLC) cells in vitro and the corresponding xenografts in vivo. In vitro cytotoxicity was evaluated by the sulforhodamine B assay and the drug interactions following simultaneous or sequential exposure were determined using median-effect analysis to calculate combination indices (CIs). XR5944 demonstrated potent cytotoxicity compared to either carboplatin or doxorubicin in COR-L23/P cells. Simultaneous or sequential exposure of XR5944 followed by carboplatin led to a synergistic response (CI<1), whereas the reverse order of addition showed an additive or antagonistic response (CI< or =1). Sequential administration of doxorubicin followed by XR5944 demonstrated marginally improved cytotoxicity (CI=1.31-0.77) than other schedules (CI=1.50-1.22) relative to individual drugs. Anti-tumor activity against COR-L23/P xenografts in nude mice was enhanced by administration of XR5944 (2 or 5 mg/kg) immediately before carboplatin (50 mg/kg) compared to single-agent treatment at the same doses. Improved efficacy was also observed by sequential administration of 7 mg/kg doxorubicin 48 h before 2.5 or 5 mg/kg XR5944. No additional toxicity was observed with combinations compared to single-agent treatment alone as determined by body weights. These data suggest that combinations of XR5944 with carboplatin or doxorubicin are of significant interest for clinical use, and that the schedule of administration may be important for achieving clinical efficacy over single-agent therapy.

XR5967, a novel modulator of plasminogen activator inhibitor-1 activity, suppresses tumor cell invasion and angiogenesis in vitro.

Recent reports suggest that elevated levels of plasminogen activator inhibitor (PAI)-1 may contribute to tumor progression. We have recently shown that antibodies to PAI-1 block the invasive and migratory potential of human fibrosarcoma cells and suppress angiogenesis in vitro. Here we report the in vitro evaluation of a low-molecular-weight modulator of PAI-1, XR5967, on invasion, migration and angiogenesis. XR5967, a diketopiperazine, dose-dependently inhibited the activity of human and murine PAI-1, towards urokinase plasminogen activator (uPA), with IC50 values of 800 nM and 8.3 microM, respectively. This was confirmed by SDS-PAGE, revealing that XR5967 inhibited complex formation between PAI-1 and uPA. This suppression may be caused by XR5967 promoting insertion of the reactive center loop within PAI-1. XR5967 dose-dependently inhibited the invasion of human HT1080 fibrosarcoma cells through Matrigel. Their invasion was reduced by 57% (p<0.001) at 5 microM. HT1080 cell migration was inhibited in a similar manner, indicating that PAI-1 may play an additional role in invasion, which is distinct to its role in the regulation of proteolysis. The potential of XR5967 to inhibit the invasion/migration of human endothelial cells was investigated in an in vitro model of angiogenesis. In this model XR5967 reduced tubule formation by 77% at 5 microM (p<0.001), highlighting a crucial role for PAI-1 in angiogenesis. These data stress the importance of a balanced proteolysis in the processes of invasion, migration and angiogenesis. Our results support the clinical findings and indicate that modulation of PAI-1 activity, with low-molecular-weight inhibitor of PAI-1 activity, may be of therapeutic benefit for the treatment of cancer.

Ex vivo characterization of XR11576 (MLN576) against ovarian cancer and other solid tumors.

XR11576 (MLN576) is a novel monophenazine with a mechanism of action that includes interaction with both topoisomerase (Topo) I and II. The aim of this study was to evaluate its cytotoxicity against fresh tumor cells taken from patients with a variety of solid tumors. Cells were obtained from 89 patients and exposed for 6 days to XR11576 alone, or in combination with doxorubicin, cisplatin, treosulfan, paclitaxel or vinorelbine. Cell survival was measured using the ATP-Tumor Chemosensitivity Assay (ATP-TCA). Immunohistochemical staining of Topo I, Topo IIalpha and MDR1 was performed on paraffin-embedded blocks in those tumors for which tissue was available (n = 49). Overall, the median IC90 and IC50 values of XR11576 in tumor-derived cells were 242 and 110 nM, respectively. In all samples XR11576 was more potent than the other cytotoxics tested. Breast and gynecological malignancies were most sensitive to XR11576, while the potency of this compound was slightly attenuated in gastrointestinal tumors, in which the median IC90 and IC50 values were 308 and 212 nM, respectively. Cases of synergism were identified when combining XR11576 with vinorelbine (nine of 30 samples) and doxorubicin (12 of 38 samples), while the addition of paclitaxel resulted in an antagonistic effect (CI50>1.2) in 38 of 42 tumors. A very modest correlation by linear regression analysis was found between the intensity of MDR1 staining and the IC50 of XR11576 (r = 0.311, p = 0.0312), but not with the IC90 (r = 0.247, NS). These data support the rapid introduction of XR11576 to clinical trials and suggest that it may be effective against a broad spectrum of tumor types.

In vitro and in vivo characterization of XR11576, a novel, orally active, dual inhibitor of topoisomerase I and II.

XR11576, a novel phenazine, was developed as an inhibitor of both topoisomerase I and II. This study characterized the ability of XR11576 to inhibit both enzymes, and determined its in vitro and in vivo antitumor efficacy against a number of murine and human tumor models. XR11576 was a potent inhibitor of purified topoisomerase I and IIalpha, and exhibited similar potency for both enzymes. The compound stabilized enzyme-DNA cleavable complexes indicating that it acted as a topoisomerase poison. The DNA cleavage patterns obtained with XR11576 were different from those induced by camptothecin and etoposide, which are topoisomerase I and II poisons, respectively. XR11576 demonstrated potent cytotoxic activity against a variety of human and murine tumor cell lines (IC50=6-47 nM). Its activity profile was comparable to or better than that of many widely used anticancer drugs. Moreover, XR11576 was unaffected by multidrug resistance (MDR) mediated by overexpression of either P-glycoprotein or MDR-associated protein, or by down-regulation of topoisomerase II. The latter property supports the dual inhibitory mechanism of action of the compound. XR11576 exhibited a similar pharmacokinetic profile in mice and rats after either i.v. or p.o. administration. In vivo XR11576 showed marked efficacy against a number of tumors including sensitive (H69/P) and multidrug-resistant (H69/LX4) small cell lung cancer and the relatively refractory MC26 and HT29 colon carcinomas following i.v. and p.o. administration. The efficacy of XR11576 was at least comparable to that of TAS-103, originally proposed as a dual inhibitor of topoisomerase I and II. These results suggest that XR11576 is a promising new antitumor agent with oral and i.v. activity, and warrants further development.

Ex vivo reversal of chemoresistance by tariquidar (XR9576).

The expression of P-glycoprotein (P-gp) has been demonstrated to confer resistance to several anticancer drugs, including anthracyclines, taxanes and vinca alkaloids. Tariquidar is a novel inhibitor of P-gp that has been shown to reverse resistance to cytotoxic drugs in tumor cell lines and mouse xenografts. We have used an ATP-based chemosensitivity assay (ATP-TCA) to compare the activity of cytotoxic drugs in combination with tariquidar against a variety of solid tumors (n = 37). The expression of P-gp was determined in a subset of solid tumor samples by immunohistochemistry (n = 16). Resistance was seen in 20 of 37 (54%) tumors tested with doxorubicin, in 27 of 34 (79%) samples tested with paclitaxel and 17 of 31 (55%) with vinorelbine. Tariquidar alone showed no activity over a wide range of concentrations up to 2 microM (n = 14). The median IC90s for doxorubicin, paclitaxel and vinorelbine, alone were 2.57, 27.4 and 15.5 microM. These decreased to 1.67 (p<0.0005), 20.6 (p<0.05) and 9.5 microM (p<0.001), respectively, in combination with tariquidar. Tariquidar also significantly decreased resistance in 14 of 20 (70%), six of 27 (22%) and six of 17 (35%) samples tested with doxorubicin, paclitaxel and vinorelbine, respectively. Immunohistochemical staining for P-gp was positive in nine of 16 (56%) samples and in all of these cases addition of tariquidar improved the activity of the cytotoxic. The results show that tariquidar is able to decrease resistance in a number of solid tumors resistant to cytotoxic drugs known to be P-gp substrates. These data support the introduction of tariquidar in combination with chemotherapy to clinical trials of patients expressing P-gp.