The flavone acetic acid derivative DMXAA [5,6-dimethylXAA (xanthenone-4-acetic acid), Vadimezan, ASA404] is a drug that displayed vascular-disrupting activity and induced haemorrhagic necrosis and tumour regression in pre-clinical animal models. Both immune-mediated and non-immune-mediated effects contributed to the tumour regression. The vascular disruption was less in human tumours, with immune-mediated effects being less prominent, but nonetheless DMXAA showed promising effects in Phase II clinical trials in non-small-cell lung cancer. However, these effects were not replicated in Phase III clinical trials. It has been difficult to understand the differences between the pre-clinical findings and the later clinical trials as the molecular targets for the agent have never been clearly established. To investigate the mechanism of action, we sought to determine whether DMXAA might target protein kinases. We found that, at concentrations achieved in blood during clinical trials, DMXAA has inhibitory effects against several kinases, with most potent effects being on members of the VEGFR (vascular endothelial growth factor receptor) tyrosine kinase family. Some analogues of DMXAA were even more effective inhibitors of these kinases, in particular 2-MeXAA (2-methylXAA) and 6-MeXAA (6-methylXAA). The inhibitory effects were greatest against VEGFR2 and, consistent with this, we found that DMXAA, 2-MeXAA and 6-MeXAA were able to block angiogenesis in zebrafish embryos and also inhibit VEGFR2 signalling in HUVECs (human umbilical vein endothelial cells). Taken together, these results indicate that at least part of the effects of DMXAA are due to it acting as a multi-kinase inhibitor and that the anti-VEGFR activity in particular may contribute to the non-immune-mediated effects of DMXAA on the vasculature.

INTRODUCTION

DMXAA [5,6-dimethylXAA (xanthenone-4-acetic acid), ASA404, Vadimezan] is an analogue of flavone acetic acid that has antitumour activity [1]. In pre-clinical mouse tumour models it was demonstrated that administration of DMXAA rapidly leads to disruption of the existing vasculature in the tumour and consequent haemorrhagic necrosis of the tumour [28]. This was consistent with the finding that a single dose of DMXAA induced a prolonged reduction in the growth of xenografted tumours in animal models. The ability to disrupt the vasculature in these pre-clinical models has been attributed to a rapid induction of cytokines, particularly TNFα (tumour necrosis factor α) [1]. This mechanism seems to differ from other vascular disruptors such as combretastatin [9] or ZD6126 [10], which elicit their effects by directly binding to tubulin.

Despite the fact that the molecular targets for the drug remained unknown [11], the promising pre-clinical results led to DMXAA being selected for clinical development [1214]. Results of Phase I trials showed some restriction of tumour blood flow within 24 h of treatment, although this was not as dramatic as seen in pre-clinical models [15]. Unlike the animal models, there was also very little evidence for the rapid death of blood vessels or for increases in TNFα levels in human tumours [15,16]. No difference in antitumour activity, cytokine induction or toxicity was observed between two parallel Phase I trials, one dosed weekly [13] and the other dosed every 3 weeks [17]. Therefore the drug proceeded to Phase II clinical trials, dosed every 21 days in combination with chemotherapeutic agents [1820]. These trials indicated the drug had small benefits in the treatment of non-small-cell lung cancer and prostate cancer [1820]. However, a subsequent Phase III clinical trial was not able to reproduce this response and clinical development was halted [21]. The reasons for this are not clear, but it is unlikely that these problems can be resolved until appropriate biomarkers are developed to monitor efficacy or a patient population can be identified that would particularly benefit from DMXAA treatment [11]. However, this would require an understanding of the molecular targets of DMXAA.

To date the only molecular targets of DMXAA identified are members of the PDE (phosphodiesterase) family, which are partially inhibited at the concentrations achieved in clinical trials [22]. The inhibition of PDE6 explained the visual disturbances observed in clinical trials [22], but the inhibition of the members of the PDE family is unlikely to explain the antivascular effects of DMXAA [23,24].

In the present study, we have tested whether DMXAA might target protein kinases. Our enzyme screens show that, at pharmacologically relevant concentrations, DMXAA inhibits several protein kinases, including HIPK2 (homeodomain-interacting protein kinase 2), CK2 (casein kinase 2), Haspin, Aurora kinases, PIM kinases, c-FMS and TrKs (tropomyosin-receptor kinases), but the most potent inhibition was of VEGFR [VEGF (vascular endothelial growth factor) receptor] tyrosine kinases. We go on demonstrate that a range of DMXAA analogues have similar properties and to show that these compounds functionally inhibit VEGFR function in cells. Taken together, these findings provide evidence to help understand the molecular targets and mechanisms of action of DMXAA and related molecules, and suggest future directions for developing this drug.

MATERIALS AND METHODS

Chemical synthesis

Synthesis of XAA, DMXAA, α-MeXAA (methylXAA), XPA (xanthenone propionic acid) and 5-phenylXAA was performed as described previously [2528].

Enzyme screens

Enzyme screens were performed by two different service providers: the National Centre for Protein Kinase Profiling (Dundee, U.K.) using the method detailed previously [29], and the European Screening Centre (Invitrogen). The latter Centre used both the Z′-LYTE assay and the LanthaScreen® Eu Kinase Binding Assay [30]. All assays were performed using the apparent Km for ATP. The European Screening Centre also performed inhibitor IC50 determinations.

Cell culture experiments

Early passage (<8) HUVECs (human umbilical vein endothelial cells) were cultured in medium 200 supplemented with LSGS (low-serum growth supplement) (Invitrogen), according to the supplier's instructions. HUVECs (4×105 cells/well) were subcultured into six-well plates pre-coated with 1% gelatin (Sigma) and allowed to grow to approximately 80% confluence before starvation for 16 h in medium 200/0.5% FBS (fetal bovine serum). HUVECs were then pre-incubated for 1 h with medium 200/0.1% FBS in the presence/absence of inhibitors at the stated concentrations, before stimulation for 10 min with 50 ng/ml human VEGF165 (Symansis). Cells were lysed and the lysates were analysed by Western blotting using antibodies against phosphorylated ERK (extracellular-signal-regulated kinase) 1/2 (Thr202/Tyr204) or phosphorylated VEGFR2 (Tyr951) (Cell Signalling Technology). AV-951 (Tivozanib, KRN-951; Symansis) was used as a control inhibitor at a concentration of 50 nM.

Zebrafish experiments

Friend leukaemia integration 1a transgenic zebrafish (fli1a:EGFP), which express GFP (green fluorescent protein) in the developing vasculature [31], were used in our experiments. We used 15–20 embryos per experimental group, and each experiment was carried out in two independent replicates. Embryos were maintained in E3 solution at 28°C. A 4 hpf (h post-fertilization), zebrafish embryos were placed in 600 μl of E3 solution supplemented with penicillin/streptomycin (Invitrogen) and either AV951 or DMXAA/analogues or vehicle (DMSO) and maintained in the dark. At 24 hpf, 0.003% 1-phenyl-2-thiourea was added to maintain the optical transparency of the zebrafish embryos. At 48 hpf, zebrafish embryos were manually dechorinated, anaesthetized with 0.01% tricaine (3-aminobenzoic acid ethyl ester) and mounted in 3% methylcellulose for imaging on either a NikonD-Eclipse C1 confocal microscope or a Nikon SMZ1500 stereomicroscope equipped with a DS-U2/L2 camera. ISVs (intersegmental vessels) were counted manually and appraised as ‘complete’ when they had extended to the level of the DLAV (dorsal longitudinal anastomotic vessel). For simplicity, ISVs were only counted on one side of each embryo.

Molecular modelling

Modelling was performed as described recently [32]. The images were generated using PyMOL version 1.4 (http://www.pymol.org). The ATP-binding site surface was generated from the VEGFR2 structure (PDB code 2OH4) [33].

RESULTS

To identify kinases that might be targeted by DMXAA we first performed screens at 50 μM DMXXA as this is within the range of concentrations at which DMXAA gives effects in cultured cells [2,3437]. The full results of our first screen against a panel of kinases are shown in Supplementary Table S1 (at http://www.clinsci.org/cs/122/cs1220449add.htm) with those compounds showing a greater than 50% inhibition being summarized in Table 1. To confirm and extend these results, we performed a second screen on a larger panel of kinases (see Supplementary Table S2 at http://www.clinsci.org/cs/122/cs1220449add.htm) with those compounds showing a greater than 50% inhibition also summarized in Table 1. These results demonstrate that DMXAA has the potential to inhibit a select range of kinases at the concentrations that are observed after therapeutic dosing [22,38].

Table 1
Summary of kinases showing greater than 50% inhibition by 50 μM DMXAA

The first eight kinase hits result from screening against 102 kinases in the National Centre for Protein Kinase Profiling screen (Dundee), and the remaining kinase hits result from screening against 267 kinases in the European Screening Centre screen (Invitrogen). JAK3, Janus kinase 3; FGFR4, fibroblast growth factor 4; IRAK1, interleukin-1-receptor-associated kinase 1; NTRK1 etc., neurotrophic tyrosine kinase receptor type 1 etc; NUAK1, sucrose-non-fermenting kinase-1-like kinase 1.

KinaseActivity remaining at 50 μM DMXAA (%)
Aurora B 48±8 
NUAK1 41±2 
CK2 29±6 
PIM1 25±1 
PIM3 12±3 
HIPK2 35±3 
TrkA 32±4 
VEGFR1 19±2 
AURKA (Aurora A) 33±1 
CSF1R (FMS) 34±1 
FGFR4 35±1 
FLT3 D835Y 44±0 
FLT4 (VEGFR3) 20±2 
IRAK1 41±0 
JAK3 48±0 
KDR (VEGFR2) 15±0 
NTRK1 (TrkA) 39±3 
NTRK2 (TrkB) 23±2 
NTRK3 (TrkC) 22±1 
GSG2 (Haspin) 47±7 
KinaseActivity remaining at 50 μM DMXAA (%)
Aurora B 48±8 
NUAK1 41±2 
CK2 29±6 
PIM1 25±1 
PIM3 12±3 
HIPK2 35±3 
TrkA 32±4 
VEGFR1 19±2 
AURKA (Aurora A) 33±1 
CSF1R (FMS) 34±1 
FGFR4 35±1 
FLT3 D835Y 44±0 
FLT4 (VEGFR3) 20±2 
IRAK1 41±0 
JAK3 48±0 
KDR (VEGFR2) 15±0 
NTRK1 (TrkA) 39±3 
NTRK2 (TrkB) 23±2 
NTRK3 (TrkC) 22±1 
GSG2 (Haspin) 47±7 

To understand more about the structure–activity relationship of these inhibitory effects, we screened 12 DMXAA analogues (Figure 1) against a range of kinases (Table 2 and Supplementary Table S3 at http://www.clinsci.org/cs/122/cs1220449add.htm). Broadly, the compounds displayed a similar target profile, but several of these compounds were consistently more potent than DMXAA against many of the targets, most notably XPA, 2-MeXAA and 6-MeXAA.

Structure of DMXAA and its analogues tested

Figure 1
Structure of DMXAA and its analogues tested
Figure 1
Structure of DMXAA and its analogues tested
Table 2
DMXAA analogues which inhibit targets by more than 50% in the initial screen from the National Centre for Protein Kinase Profiling Centre

Cl, chloro; Ph, phenyl.

Activity remaining at 50 μM of the drug (%)
KinaseDrug…XAA1-MeXAA2-MeXAA3-MeXAA5-MeXAA6-MeXAA7-MeXAA8-MeXAAα-MeXAAXPA5-ClXAA5-PhXAA
Aurora B  26±3 44±0 14±1 20±1 30±2 12±1 16±2 38±2 21±7 26±0 52±5 36±1 
NUAK1  24±1 34±4 17±1 26±2 18±1 21±1 12±1 66±17 9±2 39±2 30±1 16±1 
CK2  35±1 15±0 34±3 49±0 37±1 20±1 53±6 32±1 40±0 3±0 41±2 35±5 
PIM1  46±3 34±0 33±4 25±2 15±0 24±1 32±3 29±4 21±1 9±0 31±5 7±1 
PIM3  17±0 20±0 17±1 14±1 11±0 13±3 10±1 9±0 10±0 8±0 13±1 7±1 
HIPK2  13±1 17±1 22±1 18±0 10±0 11±1 19±0 14±1 10±0 4±0 12±1 10±0 
TrkA  63±5 95±18 23±3 31±5 37±4 26±4 34±1 77±10 40±1 29±1 49±5 14±2 
VEGFR1  18±2 67±6 7±3 13±0 29±3 9±1 24±2 33±3 19±1 15±1 28±4 19±2 
Activity remaining at 50 μM of the drug (%)
KinaseDrug…XAA1-MeXAA2-MeXAA3-MeXAA5-MeXAA6-MeXAA7-MeXAA8-MeXAAα-MeXAAXPA5-ClXAA5-PhXAA
Aurora B  26±3 44±0 14±1 20±1 30±2 12±1 16±2 38±2 21±7 26±0 52±5 36±1 
NUAK1  24±1 34±4 17±1 26±2 18±1 21±1 12±1 66±17 9±2 39±2 30±1 16±1 
CK2  35±1 15±0 34±3 49±0 37±1 20±1 53±6 32±1 40±0 3±0 41±2 35±5 
PIM1  46±3 34±0 33±4 25±2 15±0 24±1 32±3 29±4 21±1 9±0 31±5 7±1 
PIM3  17±0 20±0 17±1 14±1 11±0 13±3 10±1 9±0 10±0 8±0 13±1 7±1 
HIPK2  13±1 17±1 22±1 18±0 10±0 11±1 19±0 14±1 10±0 4±0 12±1 10±0 
TrkA  63±5 95±18 23±3 31±5 37±4 26±4 34±1 77±10 40±1 29±1 49±5 14±2 
VEGFR1  18±2 67±6 7±3 13±0 29±3 9±1 24±2 33±3 19±1 15±1 28±4 19±2 

The greatest degree of inhibition in these screens was of VEGFRs, which were strongly inhibited by 50 μM DMXAA and were also inhibited by most of the analogues (Table 1). Given the role these receptors play in angiogenesis, we determined the IC50 of these compounds against VEGFR1 and VEGFR2 (Table 3). These results show that a number of the compounds are able to inhibit VEGFR2 in the low-micromolar range, particularly 2-MeXAA and 6-MeXAA, although they were approximately 10-fold less potent as inhibitors of VEGFR1.

Table 3
IC50 values for the effects of the DMXAA analogues on VEGFR1 and VEGFR2 from the screen at the European Screening Centre

Cl, chlorinyl; Ph, phenyl.

Drug IC50 (μM)
KinaseXAADMXAA1-MeXAA2-MeXAA3-MeXAA5-MeXAA6-MeXAA7-MeXAA8-MeXAAα-MeXAAXPA5-ClXAA5-PhXAA
VEGFR1 172 119 2707 13 167 173 49 800 171 336 137 112 185 
VEGFR2 16 11 80 16 19 56 61 27 25 19 
Drug IC50 (μM)
KinaseXAADMXAA1-MeXAA2-MeXAA3-MeXAA5-MeXAA6-MeXAA7-MeXAA8-MeXAAα-MeXAAXPA5-ClXAA5-PhXAA
VEGFR1 172 119 2707 13 167 173 49 800 171 336 137 112 185 
VEGFR2 16 11 80 16 19 56 61 27 25 19 

These findings suggest that these compounds could have anti-angiogenic effects, so to test this we used an embryonic zebrafish model in which blood vessels were labelled with GFP. Representative results are shown in Figure 2(A) and a quantitative analysis of all embryos is shown in Figure 2(B). In these studies, 25 μM 1-MeXAA and 5-phenyl-XAA were toxic. However, 25 μM DMXAA was not toxic and had some anti-angiogenic effect. 2-MeXAA and 6-MeXAA were also non-toxic and were very effective inhibitors of angiogenesis, and, like the control compound AV-951, caused complete inhibition of ISV formation. Overall, the anti-angiogenic effects of the different drugs correlated very well with the degree to which they inhibited VEGFR1 and VEGFR2 (Figure 2B and Table 3). These studies clearly demonstrate that DMXAA and some of its analogues have the ability to block embryonic angiogenesis in this zebrafish model.

Effect of DMXAA and its analogues on angiogenesis in zebrafish

Figure 2
Effect of DMXAA and its analogues on angiogenesis in zebrafish

Friend leukaemia integration 1a transgenic zebrafish (fli1a:EGFP) embryos were incubated with the indicated drug concentration and analysed as described in the Materials and methods section. (A) Representative results for the controls, XAA, DMXAA and the methyl (Me) analogues of XAA are shown. Scale bar, 200 μm. (B) Quantification of all of the compounds tested; # ISV, total number of ISVs counted; # complete ISV, number of ISVs which extend to the DLAV.

Figure 2
Effect of DMXAA and its analogues on angiogenesis in zebrafish

Friend leukaemia integration 1a transgenic zebrafish (fli1a:EGFP) embryos were incubated with the indicated drug concentration and analysed as described in the Materials and methods section. (A) Representative results for the controls, XAA, DMXAA and the methyl (Me) analogues of XAA are shown. Scale bar, 200 μm. (B) Quantification of all of the compounds tested; # ISV, total number of ISVs counted; # complete ISV, number of ISVs which extend to the DLAV.

To further directly test whether these compounds were blocking VEGFRs in mammalian cells, we examined the effects of the drugs on VEGF165-induced signalling in HUVECs (Figure 3). VEGFRs were immunoprecipitated from VEGF165-treated HUVECs and then analysed by Western blotting using a VEGFR2-specific phosphotyrosine antibody (Figure 3A). In these experiments, the VEGFR2 inhibitor AV951 [39], 2-MeXAA and 6-MeXAA blocked receptor phosphorylation. Further evidence that these drugs were inhibiting VEGFR signalling was provided by the finding that DMXAA and its analogues were able to block signalling pathways activated by VEGF165. VEGF potently stimulates ERK signalling in these cells and the small-molecule VEGFR inhibitor AV-951 blocked this, as expected, but the effect was also blocked by DMXAA, 2-MeXAA and 6-MeXAA (Figures 3A and 3B). It is notable that, in these same experiments, the total level of VEGFR2 extracted in the lysis buffer was reduced by VEGF165 treatment (Figure 3B), which is consistent with ligand-induced receptor internalization and degradation [40]. This was reversed by AV951, 2-MeXAA and 6-MeXAA, which provides further evidence that these compounds can directly inhibit VEGFR2 signalling in mammalian cells. We next investigated the potency of these effects and found that by 30 μM DMXAA had begun to significantly reduce the VEGF-induced activation of ERK in cells, despite the fact that some serum (and hence binding proteins) was present in the cell culture medium (Figure 3C). This is consistent with the potency with which DMXAA inhibits VEGFR2 in vitro. 2-MeXAA and 6-MeXAA were even more potent inhibitors of ERK activation (Figure 3C), consistent with their greater potency against the VEGFRs in vitro.

Effect of DMXAA and its analogues on VEGFR signalling in HUVECs

Figure 3
Effect of DMXAA and its analogues on VEGFR signalling in HUVECs

HUVECs were serum-starved overnight in medium 200 with 0.5% FBS. Cells were then incubated fresh medium with 0.1% FBS. (A) Cells were treated with 50 nM AV951, or 100 μM of DMXAA, 2-MeXAA or 6-MeXAA for 60 min before the addition of 50 ng/ml VEGF165 for a further 10 min. Cells were lysed and the lysates were immunoprecipitated (IP) with an anti-VEGFR2-specific antibody and then Western blots were performed with an antibody specific for the tyrosine-phosphorylated VEGFR2. (B) The indicated DMXAA analogue (100 μM) was added for 60 min before the addition of 50 ng/ml VEGF165 for a further 10 min. Cells were lysed and the resulting lysates were analysed by Western blotting with the indicated antibody. Results are representative of at least three independent experiments. (C) The indicated concentrations of DMXAA, 2-MeXAA or 6-MeXAA were added 60 min before the stimulation with 50 ng/ml VEGF165 for 10 min. Cells were lysed and the lysates were analysed by Western blotting with the indicated antibodies. Results are representative of at least three independent experiments.

Figure 3
Effect of DMXAA and its analogues on VEGFR signalling in HUVECs

HUVECs were serum-starved overnight in medium 200 with 0.5% FBS. Cells were then incubated fresh medium with 0.1% FBS. (A) Cells were treated with 50 nM AV951, or 100 μM of DMXAA, 2-MeXAA or 6-MeXAA for 60 min before the addition of 50 ng/ml VEGF165 for a further 10 min. Cells were lysed and the lysates were immunoprecipitated (IP) with an anti-VEGFR2-specific antibody and then Western blots were performed with an antibody specific for the tyrosine-phosphorylated VEGFR2. (B) The indicated DMXAA analogue (100 μM) was added for 60 min before the addition of 50 ng/ml VEGF165 for a further 10 min. Cells were lysed and the resulting lysates were analysed by Western blotting with the indicated antibody. Results are representative of at least three independent experiments. (C) The indicated concentrations of DMXAA, 2-MeXAA or 6-MeXAA were added 60 min before the stimulation with 50 ng/ml VEGF165 for 10 min. Cells were lysed and the lysates were analysed by Western blotting with the indicated antibodies. Results are representative of at least three independent experiments.

DISCUSSION

The findings of the present study demonstrate that DMXAA and its analogues inhibit a range of kinases in the low-to-mid-micromolar concentrations. For most drugs this would not be relevant to their therapeutic effects, but levels of free DMXAA found in the circulation are relatively high at therapeutic dosing levels in humans. The Cmax following doses used in clinical trials (1200 mg/m2) have been reported to be up to 20 μM (reviewed in [41]) and free DMXAA levels of 240 μM were achieved at only a four times higher dose (4800 mg/m2) in pharmacology studies [38]. Together, this suggests that DMXAA and some of its analogues could act as multi-kinase inhibitors at the dosing levels used in humans.

The important question is how these inhibitory effects on kinases might relate to the therapeutic effects of DMXAA and its ability to disrupt the vasculature. None of the inhibitory effects on the kinases that we have observed are likely to explain the dramatic immune-mediated antivascular effects of DMXAA observed in some mouse tumour models [2,4,5,7,8,42]. In fact, the results with the DMXAA analogues support this, for example the inhibition of VEGFR2 is not sufficient to induce the vascular disruption, as 2-MeXAA and 6-MeXAA were even more potent inhibitors of VEGFR2 signalling, but both of these have were less effective than DMXAA in disrupting the vasculature in colon-38 xenograft models [27,43]. Further studies will be required to determine the molecular targets of DMXAA responsible for the immune-modulated vascular-disrupting effects seen in animal models. However, DMXAA also has non-immune-mediated effects and a number of the kinases inhibited by DMXAA, and its analogues have the potential to contribute to such antitumour activity. Kinases targeted by DMXAA that have been implicated in cancer include the serine/threonine kinases CK2 [44,45], Haspin [46], Aurora kinase [47,48] and PIM kinases [49]. They also include a number of receptor tyrosine kinases, including c-FMS [50], VEGFRs [51] and TrKs [52]. The activity against VEGFRs was of particular interest given the role these play in angiogenesis [51]. In the present study, we have focussed on VEGFR2 as this is the most important VEGFR controlling angiogenesis [51], and DMXAA was a more potent inhibitor of this than other kinases in our screen. Our studies provide evidence for inhibition of VEGFR2 signalling in HUVECs by DMXAA and its analogues. This indicates that DMXAA will attenuate VEGF signalling in vivo. This is supported by the finding that DMXAA and its analogues have anti-angiogenic effects in zebrafish, the potency of which correlates reasonably well with the potency with which these compounds inhibit VEGFR2. Several of the other kinases inhibited by DMXAA also have roles in blood vessel formation, including CK2 [44,45], Haspin [46], PIM-1 [53] and various isoforms of VEGFR [51]. The results in the present study have focussed only on VEGFR2 and provide strong evidence that the anti-VEGFR2 activity of DMXAA can contribute to anti-angiogenic activity. Further studies will be required to determine whether the inhibition of the other kinases might also contribute to the anti-angiogenic activity observed.

Because of the dramatic vascular-disrupting effects of DMXAA in animal models [2,4,5,7,8,42], subsequent studies have mostly focussed on its potential to act as a vascular-disrupting agent. The rapid disruption of blood vessels leading to haemorrhagic necrosis of tumours seen in mouse models is linked with a rapid induction of high levels of cytokines, particularly TNFα [54]. The effects of DMXAA on the vasculature in human tumours are not as dramatic as those seen in mouse xenograft models and this may be due to the fact that DMXAA does not induce such large increases in tumour TNFα [15,16] or other cytokines [12] in humans. The reason for this is not clear, but may explain why the clinical efficacy was lower in humans when DMXAA was used as a single agent. However, although often overlooked, DMXAA also had anti-angiogenic effects in pre-clinical models [55]. Compounds specifically designed as VEGFR inhibitors have similar anti-angiogenic effects in both pre-clinical models [5658] and in human tumours [58,59]. This suggests that the anti-VEGFR activity of DMXAA has the potential to contribute to the therapeutic antitumour effects of DMXAA. However, this would require continual suppression of the VEGFR and so would require constant presence of drug at micromolar concentrations in blood. This would not have been achieved in the Phase II and Phase III clinical trials as dosing was only once every 3 weeks [41]. The pharmacology suggests that once or twice daily dosing would be required to achieve a level of drug exposure to allow effective inhibition of angiogenesis [41]. This dosing regime has not yet been tested in humans.

Our findings that, in addition to its vascular-disrupting activity, DMXAA inhibits VEGFR2 may have other implications for efficacy. The literature suggests that the combinations of these two effects can synergize in blocking tumour growth. In one such study, the antivascular effects of TNFα are potentiated by the VEGFR inhibitor ZD6474 (Vandetinib) in animal models [60]. In another study, the effects of a vascular-disrupting agent targeting microtubules and ZD6474 were also found to synergize [10]. This could help explain the dramatic effects of DMXAA on the vasculature in animals. Such combinations have not yet been tried in humans.

The present findings also provide some direction for the future development of this drug family, in particular they suggest that further investigations of analogues of DMXAA such as 2- and 6-MeXAA are warranted. To gain some insight into how DMXAA might inhibit the VEGFR2 kinase activity, the compound series was modelled into the VEGFR2 kinase domain (Figure 4). Binding modes were predicted that indicated a possible hydrogen bond between the central carbonyl group of the DMXAA analogues and the backbone amide of Cys919 in the inter-lobe linker region of the kinase domain, an interaction seen in ligand-bound VEGFR2 protein structures used in the present study. This is consistent with the results presented in Table 1, which illustrate a negative effect of substitutions adjacent to the carbonyl group at positions 1 and 8. These may sterically hinder the essential ligand linker region interaction. The best binding mode predicted for the most active analogue (2-MeXAA) was found in the active site of PDB code 2XIR, and indicated that the carboxylate group is directed toward the mouth of the ATP-binding site, whereas the 2-Me group is in close proximity to the surface created by Leu840 and Phe918. In this orientation the 5 and 6 positions of DMXAA (in PDB code 2OH4) are orientated toward the back of the pocket, with the 6 position directed toward a site occupied by aromatic groups in other VEGFR inhibitors [6163], as is shown in Figure 4. This suggests that addition of bulk at the 6 position would be tolerated and that such compounds may be more selective and potent inhibitors of VEGFR2.

Models of DMXAA and its analogues in the VEGFR2 structure

Figure 4
Models of DMXAA and its analogues in the VEGFR2 structure

Models of DMXAA (yellow ball and stick), 2-MeXAA (cyan ball and stick) and 6-MeXAA (magenta ball and stick) bound in the ATP-binding site of the VEGFR2 kinase domain. A possible interaction with the backbone amide of Cys919 (green ball and stick) is shown as a broken line. The ATP-binding site surface was generated from the VEGFR2 structure (PDB code 2OH4) in which a DMXAA-binding mode similar to that best ranked for 2-MeXAA was predicted. The carboxylate group is orientated toward solvent, whereas the 6 position is orientated toward the rear of the active site and a pocket occupied by other VEGFR kinase inhibitors. A fragment of the benimidazole inhibitor present in the VEGFR2 structure (PDB code 2OH4) is shown to indicate the pocket location (shown by white sticks).

Figure 4
Models of DMXAA and its analogues in the VEGFR2 structure

Models of DMXAA (yellow ball and stick), 2-MeXAA (cyan ball and stick) and 6-MeXAA (magenta ball and stick) bound in the ATP-binding site of the VEGFR2 kinase domain. A possible interaction with the backbone amide of Cys919 (green ball and stick) is shown as a broken line. The ATP-binding site surface was generated from the VEGFR2 structure (PDB code 2OH4) in which a DMXAA-binding mode similar to that best ranked for 2-MeXAA was predicted. The carboxylate group is orientated toward solvent, whereas the 6 position is orientated toward the rear of the active site and a pocket occupied by other VEGFR kinase inhibitors. A fragment of the benimidazole inhibitor present in the VEGFR2 structure (PDB code 2OH4) is shown to indicate the pocket location (shown by white sticks).

In summary, the results of the present study have identified new potential molecular targets for DMXAA which will help in understanding how this drug works in vivo and how it might be optimized for clinical use.

FUNDING

This work was supported by the New Zealand Foundation for Research, Science and Technology [contract number UOAX1005]. The zebrafish work was funded by the Ministry of Science & Technology (to P.S.C.).

We thank Dr Mark McKeage and Professor Bruce Baguley for comments on the paper prior to submission.

Abbreviations

     
  • CK2

    casein kinase 2

  •  
  • DLAV

    dorsal longitudinal anastomotic vessel

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • FBS

    fetal bovine serum

  •  
  • GFP

    green fluorescent protein

  •  
  • hpf

    h post-fertilization

  •  
  • HIPK2

    homeodomain-interacting protein kinase 2

  •  
  • HUVEC

    human umbilical vein endothelial cell

  •  
  • ISV

    intersegmental vessel

  •  
  • PDE

    phosphodiesterase

  •  
  • TNFα

    tumour necrosis factor α

  •  
  • TrK

    tropomyosin-receptor kinase

  •  
  • VEGF

    vascular endothelial growth

  •  
  • VEGFR

    VEGF receptor

  •  
  • XAA

    xanthenone 4-acetic acid

  •  
  • DMXAA

    5,6-dimethylXAA

  •  
  • MeXAA

    methyl-XAA

AUTHOR CONTRIBUTION

Christina Buchanan, Jen-Hsing Shih, Jonathan Astin, Gordon Rewcastle, Jack Flanagan, Philip Crosier and Peter Shepherd all participated in planning the experiments. Christina Buchanan, Jen-Hsing Shih, Jonathan Astin, Gordon Rewcastle and Jack Flanagan all performed the experiments. Christina Buchanan, Jonathan Astin, Philip Crosier and Peter Shepherd interpreted the results. Christina Buchanan, Jack Flanagan and Peter Shepherd wrote the paper.

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Supplementary data