Targeting radiation-resistant hypoxic tumour cells through ATR inhibition.

BACKGROUND: Most solid tumours contain regions of sub-optimal oxygen concentration (hypoxia). Hypoxic cancer cells are more resistant to radiotherapy and represent the most aggressive fraction of a tumour. It is therefore essential that strategies continue to be developed to target hypoxic cancer cells. Inhibition of the DNA damage response (DDR) might be an effective way of sensitising hypoxic tumour cells to radiotherapy. METHODS: Here, we describe the cellular effects of pharmacological inhibition of the apical DDR kinase ATR (Ataxia Telangiectasia and Rad 3 related) with a highly selective inhibitor, VE-821, in hypoxic conditions and its potential as a radiosensitiser. RESULTS: VE-821 was shown to inhibit ATR-mediated signalling in response to replication arrest induced by severe hypoxia. In these same conditions, VE-821 induced DNA damage and consequently increased Ataxia Telangiectasia Mutated-mediated phosphorylation of H2AX and KAP1. Consistently, ATR inhibition sensitised tumour cell lines to a range of oxygen tensions. Most importantly, VE-821 increased radiation-induced loss of viability in hypoxic conditions. Using this inhibitor we have also demonstrated for the first time a link between ATR and the key regulator of the hypoxic response, HIF-1. HIF-1 stabilisation and transcriptional activity were both decreased in response to ATR inhibition. CONCLUSION: These findings suggest that ATR inhibition represents a novel strategy to target tumour cells in conditions relevant to pathophysiology and enhance the efficacy of radiotherapy.

Structural basis for potent inhibition of the Aurora kinases and a T315I multi-drug resistant mutant form of Abl kinase by VX-680.

The small molecule inhibitor of the Aurora-family of protein kinases VX-680 or MK-0457, demonstrates potent anti-cancer activity in multiple in vivo models and has recently entered phase II clinical trials. Although VX-680 shows a high degree of enzyme selectivity against multiple kinases, it unexpectedly inhibits both Flt-3 and Abl kinases at low nanomolar concentrations. Furthermore VX-680 potently inhibits Abl and the Imatinib resistant mutant (T315I) that is commonly expressed in refractory CML and ALL. We describe here the crystal structure of VX-680 bound to Aurora-A and show that this inhibitor exploits a centrally located hydrophobic pocket in the active site that is only present in an inactive or "closed" kinase conformation. A tight association of VX-680 with this hydrophobic pocket explains its high affinity for the Aurora kinases and also provides an explanation for its selectivity profile, including its ability to inhibit Abl and the Imatinib-resistant mutant (T315I).

Antibodies to PAI-1 alter the invasive and migratory properties of human tumour cells in vitro.

Recent reports suggest that elevated levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to tumour progression. The studies reported here were designed to help elucidate PAI-1's contribution to the invasive and migratory phenotype. Antibodies to PA-1 dose-dependently, and significantly, inhibited the invasive and migratory potential of human HT1080 fibrosarcoma cells, as did an antibody to uPA and the plasmin inhibitor aprotinin. Invasion of the human melanoma cell line, BLM, was also attenuated by the anti-PAI-1 monoclonal antibody MAI-12. The non-invasive human melanoma cell line, IF6, which does not express uPA, provided further confirmation of PAI-1 and uPA's role as, upon transfection with uPA, this cell line attained an invasive phenotype, which was again attenuated by MAI-12. Although antibodies to PAI-1 did not affect the adhesion of HT1080 cells to vitronectin, the antibody to uPA reduced their attachment. Addition of exogenous PAI-1, however, prevented HT1080 cell adhesion (IC50 180 nM) and promoted cell detachment from vitronectin. Furthermore melanoma cells transfected with a uPA variant, which had an impaired interaction with PAI-1, were not invasive and had impaired binding to vitronectin. These data highlight the importance of a balanced proteolysis and suggest an additional role for PAI-1 distinct from its role in proteolysis. These data also suggest that uPA and PAI-1 may co-operate in the migratory process by respectively facilitating the attachment to, and subsequent detachment from, vitronectin in the extracellular matrix. These results support the clinical findings and indicate that modulation of PAI-1 activity may be of therapeutic benefit for the treatment of cancer.

Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-1 activity.

A critical component in the regulation of thrombus formation and clearance is the balance between tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1). An increase in the plasma concentration of PAI-1 has been proposed as a risk factor in thrombotic disease. Inhibition of PAI-1 activity may have utility in the treatment of thromboembolic disease. We report here the evaluation of three diketopiperazine-based low molecular weight inhibitors of PAI-1 activity (XR334, XR1853 and XR5082). In vitro these compounds reversed the inhibitory effects of PAI-1 against both tPA and urokinase (UK) (IC50: 5 to 80 muM). In contrast, other serpin-serine protease interactions, including alpha 1-antitrypsin-trypsin, alpha 2-antiplasmin- plasmin and antithrombin-thrombin, were not affected, neither did these inhibitors affect global tests of haemostasis. In the light of this promising in vitro profile these compounds were evaluated in a standard radioisotopic assay of clot lysis in whole rat blood following intravenous administration. In this assay these compounds dose-dependently enhanced fibrinolysis ex vivo. After intravenous bolus administration XR334, XR1853 and XR5082 at 5 mg/kg increased clot lysis by 32.0 +/- 5.1% SEM (n = 25, p < 0.01), 36.7 +/- 3.5% SEM (n = 36, p < 0.01) and 60.0 +/- 2.8% SEM (n = 17, p < 0.01) respectively compared to vehicle. Intravenous infusion of these compounds (1 mg/kg/min for 20 min) significantly prolonged (approximately twofold) the time to blood vessel occlusion in the rat electrically-stimulated carotid artery thrombosis model. Thus, these low molecular weight inhibitors of PAI-1 activity enhanced fibrinolysis ex vivo and protected against thrombus formation in the rat.

Acute and chronic effects of renin inhibitor GR70982 in the conscious marmoset.

The effects on plasma renin activity (PRA), mean arterial blood pressure (MABP) and heart rate (HR) of GR70982, a low molecular weight inhibitor of human renin, were studied in conscious marmosets. In vitro, GR70982 is a potent and selective inhibitor of human plasma renin (concentration producing 50% inhibition (IC50): human renin = 6.9 x 10(-9) M; porcine pepsin and bovine cathepin D = greater than 10(-3) M). In normotensive marmosets, i.v. GR70982 (0.001-0.1 mg kg-1) produced a dose-related inhibition of PRA. Larger oral doses (0.2, 1 and 5 mg kg-1) were required to achieve similar effects. In renin-dependent hypertensive marmosets, i.v. GR70982 (0.01 and 0.5 mg kg-1) produced dose-related decreases in MABP (-12 and -18 mm Hg) and PRA (-93 and -100%), with only minimal effects on HR. A 7-day continuous i.v. infusion of GR70982 (0.36 mg kg-1 day-1) in sodium-deplete marmosets produced a gradual decrease in MABP (-17 mm Hg at day 7, cf. control), accompanied by an inhibition of PRA (approximately 75%) and minimal HR effects.

Atrial natriuretic factor in the Langendorff perfused coronary vasculature of the rabbit isolated heart.

Removal of exogenously administered rat ANF (99-126) (rANF) from the rabbit coronary vasculature was investigated. Rabbit hearts were perfused using a modified Langendorff technique and ANF concentrations in the perfusate were measured by a radio-receptor assay. Under these conditions no major degradation of ANF was observed. On perfusion, however, the heart liberated large amounts of ANF. This release peaked 15 minutes after the initiation of perfusion, (685 + 220 pM) and then fell to a sustained basal level (305 + 80 pM) after 45 minutes. Although an increase in the perfusate flow rate reduced the ANF concentration, there was no significant difference in the rate of ANF release between the two flow rates used. After momentary cessation of flow ANF concentration fell to a significantly lower level, however, once again no significant change in rate of release occurred. These results suggest that the heart is not a major site of ANF degradation and that alterations in flow rate through the coronary vascular bed can cause changes in amounts of ANF released.

A non-catalytic role of choline kinase alpha is important in promoting cancer cell survival.

Choline kinase alpha (ChoKα) is regarded as an attractive cancer target. The enzyme catalyses the formation of phosphocholine(PCho), an important precursor in the generation of phospholipids essential for cell growth. ChoKα has oncogenic properties and is critical for the survival of cancer cells. Overexpression of the ChoKα protein can transform noncancer cells into cells with a cancerous phenotype, and depletion of the ChoKα protein can result in cancer cell death. However, the mechanisms underlying the tumourigenic properties of ChoKα are not fully understood. ChoKα was recently demonstrated to associate with other oncogenic proteins, raising the possibility that a non-catalytic protein scaffolding function drives the tumourigenic properties of ChoKα rather than a catalytic function. In order to differentiate these two roles, we compared the impact on cancer cell survival using two tools specific for ChoKα: (1) small interfering RNA (siRNA) to knockdown the ChoKα protein levels; and (2) compound V-11-0711, a novel potent and selective ChoKα inhibitor (ChoKα IC50 20 nM), to impede the catalytic activity. Both treatments targeted the endogenous ChoKα protein in HeLa cells, as demonstrated by a substantial reduction in the PCho levels. siRNA knockdown of the ChoKα protein in HeLa cells resulted in significant cell death through apoptosis. In contrast, compound V-11-0711 caused a reversible growth arrest. This suggests that inhibition of ChoKα catalytic activity alone is not sufficient to kill cancer cells, and leads us to conclude that there is a role for the ChoKα protein in promoting cancer cell survival that is independent of its catalytic activity.

Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pancreatic tumors to radiation.

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.

Antitumour activity of XR5944 in vitro and in vivo in combination with 5-fluorouracil and irinotecan in colon cancer cell lines.

XR5944 (MLN944), a novel bis-phenazine, has demonstrated potent cytotoxic activity against a variety of murine and human tumour models. In the present study, the antitumour activity of XR5944 was investigated in combination with 5-fluorouracil (5-FU) or irinotecan in human colon carcinoma cell lines and xenografts. In vitro cytotoxicity of the combinations following exposure to the drugs sequentially or simultaneously was evaluated by the sulphorhodamine-B assay and interactions were determined using median-effect analysis. Antagonism was observed (CI >1) following exposure of HT29 cells simultaneously to XR5944 and 5-FU or SN38 (active metabolite of irinotecan). In contrast, sequential exposure of either combination in either order demonstrated at least an additive response (CI< or =1). At least an additive response was also observed with these combinations in HCT116 cells regardless of schedule. Antitumour activity in HT29 xenografts in nude mice was enhanced by sequential administration of 5-FU (65 mg kg(-1)) or irinotecan (CPT-11) (35 mg kg(-1)) 48 h before XR5944 (5, 10, or 15 mg kg(-1)) compared to single agent treatment at the same or higher doses. Administration of irinotecan (35 mg kg(-1)) and XR5944 (15 mg kg(-1)) just 30 min apart yielded similar efficacy to sequential administration 48 h apart. All combinations were well tolerated. These data suggest that combinations of XR5944 with irinotecan or 5-FU are of significant interest in the treatment of colon cancer.

Ex vivo activity of XR5000 against solid tumors.

Topoisomerases I and II unravel DNA during transcription, DNA replication and DNA repair. Inhibitors of both enzymes are important anticancer drugs, but only now are combined inhibitors becoming available for clinical use. In this study we have used an ATP-based chemosensitivity assay to determine the activity of XR5000 and possible combinations against ovarian cancer, a tumor sensitive to current topoisomerase inhibitors, and melanoma, an insensitive tumor. A further six tumors of other types were also tested. The results from 20 ovarian cancer and 18 melanoma biopsies show remarkably little difference between the tumor types in terms of IC50, IC90 or two summary indices of chemosensitivity based on all of the concentrations tested. XR5000 on its own shows a steep concentration-response curve in most tumors, only achieving high reduction (above 95%) of ATP levels at 2440 ng/ml (6 microM). The results were often similar to the combination of etoposide and topotecan, particularly at the higher concentrations tested. The combinations with greatest activity in ovarian cancer were with paclitaxel or cisplatin, while melanoma showed greatest improvement with paclitaxel or treosulfan. The results are encouraging for the clinical introduction of this agent, and suggest that it will be effective in combination with currently available drugs for both ovarian cancer and melanoma.

Inhibition of aspartic proteinases by synthetic peptides derived from the propart region of human prorenin.

1. Five synthetic peptides which together spanned the propart segment of human prorenin were tested for their ability to interact with human renin, pepsin, gastricsin, cathepsin D, cathepsin E, calf chymosin and the aspartic proteinase from Endothia parasitica. 2. While two peptides showed no significant effect with any of the enzymes, a further two were cleaved by several enzymes. 3. Only one (corresponding to the 32P-43P residues in the propart sequence) acted as a weak competitive inhibitor of most of the enzymes.

The rapid purification and partial characterization of human serum angiotensinogen.

Human angiotensinogen has been purified 390-fold from serum by a rapid high-yielding procedure that involved chromatography on Blue Sepharose, phenyl-Sepharose, hydroxyapatite and immobilized 5-hydroxytryptamine (5-HT). Angiotensinogen was specifically bound to immobilized 5-HT, which effected a partial resolution into multiple forms, which were also evident when analysed by SDS/polyacrylamide-gel electrophoresis (Mr 59,400, 60,600, 62,600 and 63,800). This heterogeneity was confirmed by resolution into six main bands on isoelectric focusing, ranging from pI 4.40 to 4.82. N-terminal analysis, digestion with human renal renin and deglycosylation studies implied that the preparation comprised several forms of angiotensinogen, varying in their degree of glycosylation. The presence of sialic acid was shown to be a major factor in determining the heterogeneity.

Nutritional rickets in African American breast-fed infants.

OBJECTIVE: To analyze the characteristics of infants and children diagnosed with nutritional rickets at two medical centers in North Carolina in the 1990s. STUDY DESIGN: The physical and radiographic findings, calcium, phosphorus, alkaline phosphatase, and 25-hydroxyvitamin D levels of infants and children diagnosed with nutritional rickets at two medical centers were reviewed. Breast-feeding data were obtained from the North Carolina Women, Infants and Children Program (WIC). RESULTS: Thirty patients with nutritional rickets were first seen between 1990 and June of 1999. Over half of the cases occurred in 1998 and the first half of 1999. All patients were African American children who were breast fed without receiving supplemental vitamin D. The average duration of breast-feeding was 12.5 months. The age at diagnosis was 5 to 25 months, with a median age of 15.5 months. Growth failure was common: length was <5th percentile in 65% of cases, and weight was <5th percentile in 43%. CONCLUSION: Factors that may have contributed to the increase in referrals of children with nutritional rickets include more African American women breast-feeding, fewer infants receiving vitamin D supplements, and mothers and children exposed to less sunlight. We recommend that all dark-skinned breast-fed infants and children receive vitamin D supplementation.

Analysis of latent forms of renin using antibodies raised against the propart segment of human prorenin: validation with representative samples of ovarian cyst and follicular fluids.

Antisera were raised against synthetic peptides from the prosegment of human prorenin. The use of each of these for detection of the appropriate prosegment region of prorenin was validated by development of an ELISA protocol standardised with recombinant prorenin present in culture medium conditioned by myeloma cells transfected with a prorenin expression plasmid. Detection of the respective epitopes in the prosegment required prior exposure of the prorenin in the medium to acid pH in order to partially unfold the prorenin molecule by dislodging the prosegment from the main body of the protein. By these ELISA protocols, the form of latent renin present in representative samples from ovarian cyst and follicular fluids was analysed; one follicular cyst fluid was found to contain full-length prorenin whereas the fluid from a benign cyst and ovarian follicular fluid samples contained the precursor in truncated form.

Hydrogen-1 nuclear magnetic resonance study of the complexes of two diastereoisomers of folinic acid with dihydrofolate reductase.

The 1H chemical shifts for the formyl and benzoyl protons of the individual diastereoisomeres of folinic acid bound to dihydrofolate reductase have been measured. For the tightly bound biologically active 6S, alpha S isomer, the "bound" signals were assigned by using transfer of saturation methods. In this case, only one of the two rotameric states of the formyl group in folinic acid (form I) is bound to the enzyme. The H3' and H5' benzoyl protons have identical shifts in the bound state (as do the H2' and H6' protons). This equivalence is attributed to flipping of the benzoyl ring about the N10-C4' and C1'-CO bonds in the bound state. In the case of the biologically inactive 6R, alpha S isomer, both rotameric forms (I and II) bind to the enzyme. The "bound" shifts for the formyl and aromatic protons are different in the complexes with the 6S, alpha S and 6R, alpha S isomers, indicating that the pteridine ring and benzoyl moiety are binding in different environments in their enzyme complexes. The glutamic acid moiety is probably binding at the same site in the two complexes.

Antitumor activity of XR5944, a novel and potent topoisomerase poison.

Inhibitors of topoisomerases are widely used in the treatment of cancer, including inhibitors of topoisomerase I (camptothecin analogs such as irinotecan and topotecan) and topoisomerase II (etoposide and doxorubicin). The novel bis-phenazine, XR5944, is a joint inhibitor of topoisomerase I and II as shown by the stabilization of topoisomerase-dependent cleavable complexes. XR5944 demonstrated exceptional activity against human and murine tumor cells in vitro and in vivo. In a range of cell lines XR5944 (IC50 0.04-0.4 nM) was significantly more potent than TAS-103, originally proposed as a joint topoisomerase I and II inhibitor, as well as agents specific for topoisomerase I or II (topotecan, doxorubicin and etoposide). In addition, XR5944 was unaffected by atypical drug resistance and retained significant activity in cells overexpressing P-glycoprotein or multidrug resistance-associated protein. Antitumor efficacy of XR5944 was demonstrated in human carcinoma xenograft models (H69 small cell lung cancer and HT29 colon). In the HT29 model, which is relatively unresponsive to chemotherapy, XR5944 (15 mg/kg i.v., q4dx3) induced tumor regression in the majority of animals (six of eight), whereas TAS-103, dosed at its maximum tolerated dose (45 mg/kg i.v., q7dx3), only induced a delay in tumor growth compared with control animals. In the H69 model, low doses of XR5944 (5 mg/kg i.v., qdx5/week for 2 weeks or 10-15 mg/kg i.v., q4dx3), induced complete tumor regression in the majority of animals. In contrast, topotecan (20 mg/kg i.v., q4dx3) or etoposide (30 mg/kg i.v., q5dx5) only slowed the tumor growth rate. These studies show that XR5944 is a highly active novel anticancer agent that is well tolerated at efficacious doses.